The rapid localization of phagocytes to sites of microbial invasion or trauma represents a first-line defense mechanism of particular importance in nonimmune hosts. Quantitative or qualitative aberrations of either the cellular or humoral contributions to these adaptive responses may impair inflammatory defenses, and thus, increase infectious susceptibility. Early studies210 demonstrated a critical 2- to 4-h period after cutaneous invasion by bacterial pathogens during which phagocytic cells must arrive at a site of invasion in order to prevent the establishment of an infectious process. Recurrent bacterial or fungal infections of the skin or mucous membranes are prominent in patients with quantitative deficiencies of peripheral blood leukocytes.222 Such infections are also evident in patients with qualitative disorders resulting in insufficient accumulation of phagocytes at inflammatory sites, despite normal numbers of leukocytes in the peripheral blood.223,224 Among both patient groups, common pathogens such as Staphylococcus aureus, Pseudomonas, other gram-negative enteric species, or Candida albicans account for most infectious complications. Infected tissues in these patients are characteristically gangrenous or necrotic, devoid of pus, and contain few granulocytes when examined microscopically. Local inflammatory signs or symptoms in such patients may be minimal, even though chronic infections may eventually lead to the destruction of cutaneous, subcutaneous, periodontal, or other submucosal tissues.
Early reports of clinical disorders typified by susceptibility to recurrent soft tissue infections described patients with abnormalities of leukocyte migration in vitro and/or tissue mobilization in vivo.225–229 In contrast to observations in patients with chronic granulomatous disease (see Chap. 189), studies of granulocytes or monocytes in these patients demonstrated neither abnormalities of microbicidal functions nor granulomatous inflammation in infected tissues. Initially, at least for purposes of comparison of individual patients, a distinct subclassification of disorders of leukocyte motility or chemotaxis seemed justified. However, an explosion of literature followed in which defects of chemotaxis in vitro were associated with a wide array of clinical disorders or conditions. Such reports clearly implied but rarely documented that diminished chemotaxis in vitro was associated with diminished availability or delayed infiltration of phagocytes into inflamed tissues. Correlations between abnormalities of cellular motility in vitro and altered exudation in tissues of human subjects have been infrequent because of the imprecisions in the techniques used to evaluate cellular infiltration in vivo in humans (e.g., skin window techniques).
A reliable interpretation of leukocyte functions in vitro must consider the clinical status of the individual patient, because it is important to determine whether abnormal functions result in increased susceptibility to infection or simply reflect other factors surrounding the patient's condition. Certain pharmacologic agents as well as the nutritional status of the patient may transiently influence selected functions tested in vitro.220,230 Blood samples obtained for study during the course of infections may contain an increased percentage of immature myeloid cells that function suboptimally.231 Also, many investigators have reported enhanced, diminished, or otherwise abnormal motility, phagocytosis, oxidative metabolism, and/or other functions of leukocytes in patients with clinical bacterial infections.232–235 In most cases, these abnormalities are found to be transient, probably reflecting cellular influences of inflammatory mediators230,236–238 or products of the infecting organisms. Certain bacterial toxins exert significant inhibitory effects on cellular locomotion as well as on other functions in vitro.230 Some, such as cholera toxin and certain enterotoxins of E. coli, exert primarily intracellular effects (e.g., activate adenyl cyclase and elevate intracellular cyclic AMP levels).239,240 Others preferentially perturb cell membrane properties and include streptolysin O, the clostridial toxins perfringolysin and phospholipase C, a diverse group of staphylococcal toxins (sphingomyelinase C and leukocidin), and proteases (alkaline protease, elastase) elaborated by pathogenic strains of Pseudomonas aeruginosa.241,242 Suggested pathogenic mechanisms related to microbial toxin exposure include the disruption of receptors for chemotactic factors, ligands for complement proteins or reactions from the Fc portion of IgG, alteration of membrane fluidity, and inhibition of cytoskeletal protein assemblage. Finally, certain microbial proteases or other products act directly on humoral mediators of cellular locomotion. For example, elastases elaborated by Pseudomonas aeruginosa cleave C5 (as well as other serum complement proteins) thereby generating complement-derived chemotactic moieties in vitro or in vivo.241
The molecular pathogeneses of a limited number of genetic or secondary disorders characterized by defective migration of leukocytes have been defined. Investigation of these clinical syndromes has provided important insights concerning the physiological and pathologic significance of leukocyte trafficking in vivo, as well as the molecular determinants of these events.
Identification of CD18 Deficiency in Human, Canine, and Bovine—Leukocyte Adhesion Deficiency I (LAD I)
An autosomal recessive trait characterized by recurrent bacterial infections, impaired pus formation and wound healing, and a broad spectrum of functional abnormalities of myeloid and lymphoid cells results from heterogenous mutations of the β subunit of CD18 (β2) integrins. These mutations result in defective biosynthesis of each of heterodimeric glycoproteins of the β2 subfamily including LFA-1 (CD11a/CD18), Mac-1 (CD11b/CD18), and p150,95 (CD11c/CD18).
Before 1980 several reports documented a group of human patients with recurrent bacterial and fungal infections, defective leukocyte motility and phagocytosis, impaired wound healing, and/or delayed umbilical cord severance.243–251 Crowley et al.252 first proposed that defects of neutrophil chemotaxis and associated infectious susceptibility were due to underlying defects of cell adhesion. Moreover, lysates of blood neutrophils in their patient lacked a high-molecular-weight cell-surface glycoprotein termed gp110. Later reports described similar patients lacking leukocyte surface glycoproteins ranging in M r from 130 to 180 kDa.253,254 This M r range for deficient glycoproteins was consistent with that of Mac-1 described by that time in mouse and human as an αβ complex with subunits of M r 165,000 (α) and 95,000 (β) present on macrophages, monocytes, neutrophils, and large granular lymphocytes.255,256 Studies in 1984 by Dana et al.,257 Anderson et al.,258 Beaty et al.,259 and Springer et al.260 revealed that both the α and β subunits of Mac-1 (also designated Mo-1 or CR3) were deficient on patient leukocytes. Anderson et al.,258 Springer et al.,260 and Arnaout et al.261 also showed that the LFA-1 αβ complex, which showed a β subunit identical to that of Mac-1,255 was deficient on patient neutrophils and lymphocytes. A third type of αβ complex, designated p150,95, was also deficient on patient cells.262 These findings led to the proposal that the primary defect in this disorder was related to the β subunit, and that biosynthesis of the β subunit was requisite for surface expression of each of the α subunits.260
Subsequent to these early reports, numerous patients with similar clinical features were shown to lack the Mac-1, LFA-1, and p150,95 glycoproteins on neutrophils, monocytes, lymphocytes, and/or transformed β lymphoblasts or T-cell lines21,250,252–254,257,260–283 as reviewed elsewhere.249 This group of patients, sharing similar clinical features and the same molecular defect, clearly defined a distinct pathologic disorder. Because it was identified in several independent laboratories, it was variably referred to as Mac-1, LFA-1 deficiency disease, Mo-1 deficiency, Leu-cam deficiency, or CR3 deficiency. In the interests of brevity and comprehensiveness, the term leukocyte adhesion deficiency (LAD) was proposed in 1987.249 The term LAD I was adopted in 1992 following discovery of a similar clinical syndrome due to a distinct molecular defect termed LAD II (see below). Very similar clinical disorders recognized in Irish Setter dogs284,285 and Holstein cattle286–290 were subsequently described and shown to be due to defects of the β2 integrin subunit. Thus, the terms canine LAD and bovine LAD were adopted.
The discovery of bovine LAD occurred as part of studies by the United States Department of Agriculture to develop new methods to prevent mastitis in periparturient dairy cows.291 As part of these studies, Kehrli et al.286 identified a Holstein heifer that exhibited marked and progressive blood leukocytosis. Analysis of this animal revealed chronic neutrophilia (exceeding 100,000/FL) associated with the development of fever and chronic diarrhea leading to death at 48 days of age. Prior to death, in vitro studies of blood neutrophils of the calf revealed several functional abnormalities. As the clinical features of this calf were similar to those of human patients with LAD, further postmortem assessments were performed, including immunoblot analyses of neutrophil lysates of the heifer calf, which revealed a total deficiency of Mac-1. Further immunofluorescent analyses indicated diminished amounts of CD18 on blood neutrophils of the calf's dam and sire and diminished amounts of CD18 on neutrophils of 8 of 15 paternal half-sibs, findings that are consistent with an autosomal recessive disorder.286,292 Antigen-specific immune responses were found to be delayed and impaired.293 Genealogic studies revealed that the affected proband calf was related to previously reported Holstein cattle with bovine granulocytopathy syndrome, as reviewed elsewhere,286 and that all shared a common ancestor in their pedigrees. With the availability of cDNA probes to bovine CD18,294 it was later shown that a single mutant CD18 allele was prevalent among Holstein cattle worldwide (see below). Bovine LAD represents one of several genetic disorders identified in dairy cattle that resulted from highly regulated breeding practices as reviewed elsewhere.294
Clinical and Histopathologic Features of LAD I.
Clinical and histopathologic features of LAD I are remarkably similar among affected human, canine, and bovine subjects.249,264,284,287,290 Recurrent necrotic and indolent infections of soft tissues primarily involving skin, mucous membranes, and intestinal tract are the clinical hallmarks of this disease. Superficial infections on body surfaces may invade locally or systemically. Typical small, erythematous, nonpustular skin lesions often progress to large, well-demarcated, ulcerative craters, or “pyoderma gangrenosa,” which heal slowly or with dysplastic eschars.249,264 Staphylococcal or gram-negative enteric bacterial organisms may be cultured from such lesions for up to several weeks despite antimicrobial therapy.
Fulminant progression of gas gangrene of soft tissues of a distal extremity in one patient prompted surgical amputation as a life- saving measure.254 Septicemia progressing from omphalitis associated with delayed umbilical cord severance has been observed in several families.244,245,249,264 Perirectal abscess or cellulitis leading to peritonitis and/or septicemia has been reported in multiple patients, and facial or deep neck cellulitis has been observed to progress from ulcerative mucous membrane lesions of the oral cavity.253,254–264 Recurrent invasive candidal esophagitis, erosive gastritis, acute appendicitis, and necrotizing enterocolitis have been reported in multiple human patients.249 Recurrent or chronic diarrhea is a common and often lethal complication in affected Holstein cattle.291 Recurrent otitis media occurs commonly, and progression to mastoiditis and facial nerve paralysis has been reported. Other common respiratory infections include severe bacterial (pseudomonal) laryngotracheitis, recurrent pneumonitis, and sinusitis.249 Severe gingivitis and/or periodontitis is a major feature among all patients who survive infancy. Acute gingivitis appears in all cases with eruption of the primary dentition. Subsequently, all human and bovine subjects develop characteristic features of a severe, progressive, generalized periodontitis, including gingival proliferation, defective recession, mobility, pathologic migration, and advanced alveolar bone loss associated with periodontal pocket formation and partial or total loss of both the deciduous and permanent dentitions.264,283,289
The recurrent infections observed in affected patients appear to reflect a profound impairment of leukocyte mobilization into extravascular inflammatory sites. Skin windows as well as biopsies of infected tissues demonstrate inflammatory infiltrates totally devoid of neutrophils.254,264,278,288 This histopathologic feature is particularly striking considering that marked peripheral blood leukocytosis (five- to twentyfold normal values) is a constant feature of this disorder.264,284,291 Transfusions of leukocytes result in the appearance of donor neutrophils and monocytes in skin windows and in skin chambers.254 Impaired healing of traumatic or surgical wounds observed in several patients represents a clinical feature not generally observed in patients with neutropenia or dysfunctional neutrophils. Unusual paper-thin or dysplastic cutaneous scars have been found in some patients.264,295 These may reflect the lack of sufficient monocyte infiltration and the lack of inflammatory contributions to healing such as the elaboration of angiogenesis factors.264 The wide spectrum of gram-positive or gram-negative bacterial and fungal infectious microorganisms249,296 is also characteristic of patients with primary neutropenia syndromes. These clinical models also demonstrate insufficient tissue leukocyte infiltration. However, deep-seated granulomatous infections typical of chronic granulomatous disease and other examples of oxidative or nonoxidative intracellular killing deficits have not been observed in LAD I.
Some evidence suggests that human patients with LAD I have an increased susceptibility to viral infection. Most patients have demonstrated normal and self-limiting courses of varicella or other viral respiratory infections, and in one report,264 5 of 10 patients demonstrated no untoward reactions to live viral vaccine administration. However, one patient died of an overwhelming infection with picornavirus involving oral pharynx, glottis, trachea, and lungs, and three patients of the same series had one or more episodes of aseptic (presumably viral) meningitis.264
The severity of clinical infectious complications among human LAD I patients appears to be directly related to the degree of glycoprotein deficiency. Two phenotypes, designated severe and moderate deficiency, are defined.249,264,281 As measured by immunofluorescence flow cytometry and verified by radioimmunoassay and immunoprecipitation techniques, severely deficient patients had essentially undetectable expression of all three complexes264 on their neutrophils. Moderately deficient patients expressed 2.5 to 6 percent of all three αβ complexes. Patients with severe deficiency have either died in infancy or have demonstrated a susceptibility to severe, life-threatening systemic infections (peritonitis, septicemia, pneumonitis, aseptic meningitis). In contrast, among the patients with moderate deficiency, life-threatening infections have been infrequently observed.264 Patients within a kindred demonstrate similar survival periods. For example, in one study, three with moderate disease died at ages 19, 22, and 32 years.297 In other studies of patients with severe disease, five died in their first year and one died at 3 years of age.245,298,299 In some moderately affected patients, skin lesions may disappear after the first few years of life, recurring only with occasional infections. Severe gingivitis is always observed in these patients and may be the presenting symptom.283 Delayed umbilical cord separation occurs more frequently in patients with the severe phenotype, but it is not universally found. Heterozygous carriers of alleles with CD18 mutations among human kindreds or Holstein cattle do not demonstrate any distinctive clinical features.
Overall, more profound functional abnormalities in vitro have been observed among severely deficient patients.249,264 Abnormalities of leukocyte adherence to substrates, and adhesion-dependent functions, including chemotaxis and/or aggregation, were observed among almost all patients studied.223,249,252,264 CD18-deficient neutrophils and monocytes demonstrate profound abnormalities of adhesion to and migration in vitro through endothelial cell monolayers.19,187,300–302 These reflect deficits of the adhesive interactions of Mac-1 and LFA-1 with ICAM-1 and other ligands expressed on vascular endothelial cells.19,187 Deficits of neutrophil transendothelial migration as well as egress into experimental inflammatory sites are directly related to the degree of CD18-deficiency observed among severe, moderate, or heterozygote patient groups. Findings of relatively normal neutrophil emigration into experimental skin windows of heterozygotes are consistent with their normal clinical status.249,264,283
Chemotaxis of neutrophils appears to be affected because it requires adhesion,264,303 and abnormalities of chemotaxis by CD18-deficient cells are most evident when employing assay systems which require adhesion for directed migration.303 Binding and phagocytosis of iC3b-opsonized particles is also deficient, in agreement with the identity of the CR3 with Mac-1.304 In addition, because particles opsonized with iC3b are phagocytosed poorly, they fail to normally trigger the respiratory burst or degranulation.244,247,253,258,264,297,305 Abnormalities of neutrophil or monocyte antibody-dependent cellular cytotoxicity have also been observed in several human patients and bovine subjects.223,264,266,289 In contrast, adherence-independent cellular functions of CD18-deficient cells elicited by chemotactic factors or phorbol esters are generally shown to be normal. These include cell bipolarization,258 complement receptor type-1 (CR1) up-regulation,258 L-selectin down-regulation,60 specific granule release,258,264 superoxide production induced phorbol esters,264 and actin polymerization.250 Intracellular microbicidal activity (e.g., the ability to kill Staphylococcus aureus) in most reported human patients is relatively normal,254,258,264,297 although diminished killing of this test organism was reported in studies of one case of bovine LAD.286 This may reflect the fact that receptors (e.g., FcγR or CR1) other than CR3 (Mac-1) are sufficient to promote a normal level of phagocytosis and intracellular killing in these in vitro assays.258,260,264,306
In vitro assessments of bovine CD18-deficient neutrophils have shown diminished phagocytosis-associated oxidative and secretory functions during ingestion of iC3b opsonized zymosan, findings similar to those with CD18-deficient human neutrophils. Endocytosis of IgG-opsonized Staphylococcus aureus by bovine CD18-deficient neutrophils is significantly reduced, a phenomenon similar to the reported deficits of IgG-Fc receptor mediated endocytosis by human CD18-deficient neutrophils, and indicative of an apparent cooperative interaction of FcγR and Mac-1.267,307,308
The predominance of recurrent bacterial (as opposed to viral or fungal) infections in patients with LAD I implies that the functions of neutrophils or monocytes are more profoundly affected than those of lymphocytes. However, T lymphocyte-mediated killing, proliferative responses, natural killing, and antibody-dependent killing by patients' lymphocytes are deficient compared to adult controls.223,244,266,269,272,278,293,309–310 In primary mixed lymphocyte culture, lymphocytes in several studies have demonstrated profoundly diminished cytotoxic and proliferative responses and diminished interferon production.246,259,269,272 After further stimulation, though, these responses increase to nearly normal levels.269 This may be due to compensatory mechanisms, perhaps alternative accessory adhesion molecules, and may account for the relatively normal functions of B and T lymphocytes observed in most cases. Delayed cutaneous hypersensitivity reactions are normal in most patients tested, and most individuals demonstrate normal specific antibody synthesis.258,275 However, T lymphocyte-dependent antibody responses in vivo (for example, to repeated vaccination with tetanus, diphtheria toxoids, and polio virus) were impaired in some cases, and antibody production in vivo or in vitro in response to influenza virus was found to be abnormal in one patient.271 Thus, responses of lymphocytes in vivo may be deficient in only some of the patients whose β subunit mutation is particularly deleterious to the expression of LFA-1.
Collectively, these findings imply that in vivo functions of granulocytes and monocytes including hyperadherence, aggregation, and adhesion-primed cytotoxic functions are facilitated by an increased surface expression, functional activation, and/or alterations of receptor-density distribution of Mac-1, LFA-1, and p150,95 in response to inflammatory mediators. The profound inability of CD18-deficient cells to localize and function normally in inflamed tissues would appear to reflect their inability to up-regulate and/or activate β2 heterodimers normally required for avid ligation with ICAM-1 and other endothelial ligands or substrates. However, CD18-deficient cells appear capable of normal vascular margination-demargination in vivo,270 findings indicating that CD18-independent adhesive or physical311 determinants interact with vascular endothelium to mediate this physiologic process.
Definition of the Molecular Basis of LAD I.
Prior to the availability of recombinant DNA reagents for phenotypic assessments of LAD I patients and their kindreds, several lines of evidence supported an autosomal recessive pattern of inheritance for this disorder. Both male and female patients were described in initial reports, and applications of monoclonal antibodies to CD18 subunits in family studies showed heterozygous male and female carriers with expression of 50 percent of normal amounts of these proteins on their neutrophils and lymphoid cells.258,264 In three reported families, all the clinically unaffected mothers and fathers, and some of the sibs, were shown to express intermediate levels of CD18 on their leukocytes.264 In one family spanning three generations, an affected son was born to heterozygous parents. The affected son married a heterozygote, and the couple bore an affected son and daughter and two heterozygous daughters. These findings, together with the overall equal numbers of male-female patients recognized worldwide246,249,264,276,297,298,305 and a frequent history of consanguineous matings244,247,264,272,275,299 strongly suggested that LAD I is inherited as an autosomal trait. In one kindred,253,261 X-linked inheritance was suggested, but no definitive evidence for an X-linked form of LAD I presently exists.
Clinical studies showed that patients with LAD I were uniformly deficient in the expression of all three leukocyte integrins,249 suggesting that a primary defect in the common β subunit of Mac-1, LFA-1, and p150,95 accounted for this disease. To date, no patients with LAD I (human, canine, or bovine) have been reported demonstrating a selective deficiency of a single β2 integrin heterodimer. A defect of the β2 (CD18) subunit was also evidenced by initial biosynthesis studies using available monoclonal antibodies directed at both subunits of LFA-1. As shown in studies using EBV-transformed B lymphocytes or mitogen-stimulated T-lymphocyte cell lines, normal individuals synthesize LFA-1α (CD11a) and β (CD18) precursors which associate prior to further carbohydrate processing and transport of αβ complexes to cell surfaces. In contrast, lymphoblasts of patients with LAD I synthesize an apparently normal LFA-1α subunit precursor, but this precursor does not undergo normal carbohydrate processing, does not associate with a β precursor, and is never expressed on the cell surface. Such findings indicated that the α subunit of LFA-1 is apparently degraded in the absence of a normal β subunit.260
Somatic cell complementation studies further implicated a pathologic role of the β subunit in LAD I.265 In hybrids of human and mouse lymphocytes, human LFA-1α and β subunits from healthy individuals associated with mouse LFA-1αβ subunits to form interspecies hybrid αβ complexes. In hybrids of patient and mouse lymphocytes, however, the human α subunit (CD11a) but not β subunit (CD18) was rescued by the formation of interspecies complexes that were expressed on the surface of hybrid cells. Thus, the α subunit of LFA-1 in CD18-deficient cells is competent for biosynthesis and surface expression in the presence of the mouse β2 (CD18) subunit. These same complementation protocols facilitated mapping of the β2 subunit to chromosome 21.
Heterogeneity among patients with LAD I was first described with respect to the extent of β2 integrin deficiency on leukocyte surfaces, as shown by flow cytometric or radiolabeled antibody-binding assays.264 As previously discussed, the severity of clinical features and the magnitude of functional deficits observed in individual patients is directly related to the degree of protein deficiency,264 but the underlying molecular basis for this heterogeneity often remains undefined. The availability of the CD18 subunit cDNA7,8 and a rabbit antiserum reactive with the CD18 precursor312 allowed more precise evaluations of the CD18 subunit of patients' cells. These revealed several distinct phenotypes of CD18 expression and structure among patients previously designated severe or moderate phenotype based on protein expression and clinical features.264 In one study of six unrelated patients and four related patients and members of their kindred, five distinct variations in CD18 were identified (312): (a) the CD18 subunit mRNA and protein precursor were undetectable; (b) the quantities of CD18 mRNA and protein precursor were diminished; (c) an aberrantly large CD18 subunit precursor possibly due to an extra glycosylation site was observed; (d) an aberrantly small CD18 subunit due to a polypeptide chain defect was found; and (e) no gross abnormality in the CD18 subunit mRNA or protein precursor was detectable. Findings similar to the latter phenotype were evident in studies of four other patients reported by Dana et al.313 and one patient described by Dimenshe.248 Despite the presence of apparently normal CD11a and CD18 precursors in this group of patients, neither subunit is normally processed or transported to the cell surface. Among this group of patients, it is unclear why some are of the moderate and some of the severe phenotype.248,312–315 Tables 188-4A and B list, and Fig. 188-2 illustrates, the known specific mutations with the two phenotypes that are discussed below.
Table 188-4: CD18 Mutations |Favorite Table|Download (.pdf) Table 188-4: CD18 Mutations
|A. Identified in Severe Phenotype LAD I Kindreds || || |
|Nucleotide ||Protein ||References |
|605C → T (CCG → CTG) Transcriptional defect: probable splicing deficiency, no detectable mRNA ||P178L ||Back et al.,329 Konno et al.,330 Ohashi et al.428 |
|577G → A (GGG → AGG) Probably homozygous, possible splicing defect (exon 2–3) ||G169R ||Corbi et al.,327 Nelson et al.323 |
|969 + 1G → A; complex splicing defect on exon 7–8; homozygous ||Aberrantly large mRNA due to splicing defect; truncated CD 18 ||Matsuura et al.,326 Kobayashi et al.429 |
|454G → A (GAC → AAC) Homozygous ||D128N ||Matsuura et al.,326 Nunoi et al.331 |
|383G → A (GAC → GGC) Homozygous, cattle ||D128G ||Shuster et al.,291 Kehrli et al. |
|922G → A (GGC → AGC) Homozygous ||G284S ||Wright et al.,320 Taylor et al.325 |
|GGCCCGGCTG191 → 200 deletion and 43 bp insertion at G130-T131; splicing defect of exon 2–3. Homozygous. ||41–43 deletion → frameshift → premature stop codon ||Lopez Rodriquez et al.,328 Lisowski-Grospierre et al.273 |
|1674C → A (TGC → TGA) No detectable mRNA. Second allele not determined. ||C534X ||Lopez Rodriquez et al.,328 Anderson et al.264 |
|605C → T (CCG → CTG) 220 bp deletion (G1730-C1950) Genomic mutation not determined ||P178L; deletion of exon 13 frameshift → premature stop codon ||Back et al.329 Bowen et al.254 |
|577G → A (GGG → AGG) Homozygous or second undefined allele not expressed ||G169R ||Wardlaw et al.,314 Anderson et al.264 |
|2142 deletion T (GAT → GA) Second mutant allele not determined ||D690 frameshift ||Sligh et al.,318 Anderson et al.264 |
|922G → A (CGC → AGC) Homozygous ||G284S ||Law SK (personal communication), Anderson et al.264 |
| B. Identified in Moderate Phenotype LAD I Kindreds || || |
| Nucleotide || Protein || References |
|1155 + 3G → C; deletion of exon 9 (1066–1156) due to defect of 5′ splice acceptor site; homozygous ||Deletion of exon 9 (K332-R361) due to aberrant splicing ||Kishimoto et al.,316 Anderson et al.264 |
|518T → C (CTA → CCA) Low mRNA expression of an undefined second allele ||L149P ||Wardlaw et al.,314 Wright et al.,320 Anderson et al.264 |
|659A → C (AAA → ACA) 1849C → T (CGT → TGT) ||K196T R593C ||Anderson et al.,258 Arnaout et al.315 |
|74T → A (ATG → AAG) 2142 deletion T (GAT → GA) ||M1K D690 frameshift ||Sligh et al.,318 Anderson et al.264 |
|1828C → T (CGG → TGG) and 814-14C → A; aberrant 3′ splice acceptor site (exon 6–7) ||R586W (benign polymorphism) P247 ins (PSSQ) ||Nelson et al.323 Wright et al.320 Ross et al.295 |
|1124A → G (AAT → AGT) ||N351S || |
|1327 deletion of GA; homozygous ||T418 frameshift ||Wright et al.,320 Lau et al.322 |
|922G → A (GGC → AGC) 1849C → T (CGT → TGT) ||G284S R593C ||Wright et al.,320 Davies et al.321 |
|922G → A (GGC → AGC) Second allele not determined ||G284S ||Back et al.324 Bowen et al.254 |
|484T → C (TCC → CCC) 889G → C (GGA → CGA) ||S138P G273R ||Hogg et al.336 |
Mutations in the CD18 gene associated with either severe or moderate phenotypes of leukocyte adhesion deficiency.
In detailed studies of an extended west Texas kindred, including four patients with moderate phenotype, an aberrantly small CD18 precursor of identical size was identified in each case.316 Of 10 relatives of this kindred, 9 were typed as heterozygous based on protein expression.264 Each of these nine relatives showed both normal-sized and abnormally small CD18 precursors. One noncarrier relative with normal neutrophil surface protein values showed only the normal CD18 precursor. Endoglycosidase H digestion of the N-linked carbohydrate from the small CD18 precursor showed that the deficit was in the protein backbone rather than glycosylation.
Detailed assessments of this kindred by Kishimoto et al.316 confirmed that an aberrantly small CD18 precursor is synthesized and degraded in patient cells prior to transport to and processing in the Golgi apparatus. However, studies employing large numbers of 125I-labeled patient lymphoblasts revealed that small amounts of normally sized LFA-1αβ complexes were synthesized and transported to the cell surface, findings consistent with a moderate phenotype.264 S1 nuclease protection studies revealed a 90-nucleotide deletion in the CD18 subunit mRNA. Sequence analysis of PCR amplified cDNA indicated that the deletion resulted in deletion of 30 amino acids in the extracellular domain. Analysis of genomic DNA showed that this 90-bp region is encoded by a single exon (exon 9) in both patient and normal. Sequence analysis of patient genomic DNA revealed a single G to C substitution in the sequence of the 5N splice site, suggesting aberrant RNA splicing. A small amount of apparently normally spliced message was detected (by PCR amplification) in patient cells, which appears to encode a normal CD18 subunit. This apparently accounts for the low but detectable levels (3 to 6 percent of normal) of CD11/CD18 expression in affected patients.
Of significance, the 30-amino-acid deletion recognized in this kindred is located within a 241-amino-acid region of the extracellular domain of CD18 that is highly conserved in evolution. For example, the β subunit of a Drosophila integrin shares 35 percent amino acid identity overall within the entire CD18 subunit, but 50 percent identity in this sequence,317 which, in turn, shares 63 percent amino acid identity with the corresponding region of the fibronectin receptor.316 It is noteworthy that a high percentage of CD18 mutations identified among currently reported kindreds with LAD I are located within this highly conserved segment. Domains within this segment (such as the deleted 30-amino-acid domain described above) are presumably required for αβ precursor association and biosynthesis and may represent critical contact sites between the α and β chain precursors. Notably, the corresponding region of the β3 integrin has been directly implicated in binding to peptides containing Arg-Gly-Asp (RGD) sequences. Moreover, a mutation in this region of the β3 integrin causes Glanzmann thrombasthenia (see Chap. 177). Patients with this mutation have normal expression of IIb-IIIa on platelets but defective ligand binding. Thus, this region of the integrin β subunits appears to be critical for both αβ association and ligand binding.
Detailed studies of another moderate phenotype kindred318 defined distinct mutant alleles of paternal and maternal origin. The paternal allele includes an ATG-to-AAG substitution in the initiation code of the CD18 mRNA (M1K), and the maternal allele (D690 fs, where fs means frameshift) results from a T deletion in codon 690 (GAT → GA), 31 nucleotides 5′ to the beginning of the transmembrane domain. Studies in expression protocols have not been carried out to define which mutant allele accounts for low levels of apparently normal CD18 protein expressed on the patient's cells. However, it is plausible to suggest that the paternal allele (M1K) allows for low levels of mRNA transcription and biosynthesis of (normal) CD18 protein expressed on the patient's cells, based on the observation that an alternative translation initiation codon consistent with the criteria of Kozak319 is created by the paternal allele substitution. Translation via this codon could result in low, albeit detectable, amounts of an essentially normal-sized message and CD18 protein subunit as shown by Kishimoto et al.312 The predicted result of the maternal mutation is a frameshift in the CD18 mRNA predicting a premature termination codon and the biosynthesis of an aberrantly small CD18 subunit lacking a transmembrane domain and cytoplasmic tail. Such an anchorless subunit would possibly be secreted from the cell (unconfirmed), but would not likely target and insert into cell membranes. That this D690 fs mutation (maternal allele) does not allow for any biosynthesis of LFA-1 (and thus can't account for the patient's moderate phenotype) is supported by the observation318 of an identical mutant allele in another (unrelated) compound heterozygote patient with a severe clinical phenotype. Transfection studies employing each of these mutant cDNAs should facilitate a determination of the extent to which one or both are capable of promoting CD11/CD18 expression.
Distinct and diverse CD18 mutant alleles have been defined in other moderate phenotype kindreds, including homozy- gous-deficient or compound heterozygote-deficient patients.314,315,320–323 Studies by Wright320 of a moderate phenotypic 19-year-male described by Lau et al.,322 revealed a GA deletion (at nucleotide 1256) and an abnormally large (4.3 kb) mRNA. The absence of a normal-sized mRNA and the presence of the 1256-mutant allele in all PCR products from both cDNA and genomic DNA clones, suggest that the patient is homozygous deficient and that both abnormalities are encoded by the same allele. This is in agreement with a history of related parents.322 The 1256-GA deletion predicts a frameshift in translation and a complete (severe) deficiency of CD18 expression. However, this does not appear to be the case by other criteria.322 The nature of the 4.3-kb mRNA has not been characterized, and it is not known if it is associated with LAD I.
In another moderate phenotypic patient, Wardlaw et al.314 defined a 446 T-to-C point mutation (CTA → CCA) resulting in a L149P substitution. Cotransfection of this mutant CD18 cDNA with wild-type LFA-1α (CD11a) cDNA in COS cells showed detectable, although markedly diminished, expression of a functionally defective LFA-1 protein. Additional studies of the same patient by Wright et al.320 confirmed the L149 P mutation in one predominant species of cDNA associated with the creation of an MvaI site. Whereas PCR products from this cDNA showed almost complete cleavage with MvcI, only about 50 percent of PCR product from the genomic DNA was cleaved, findings suggesting that this patient has another allele of low mRNA expression yet to be characterized.
Two distinct point mutations including G284S and R593C have been reported by Wright et al.320 in another moderate patient.321 PCR fragments covering both locations showed that they were located on different alleles of maternal (G284S) or paternal (R593C) origin. The basis for detectable CD18 protein expression consistent with a moderate phenotype in this case is unclear. A G284S mutation resulting in an aberrantly small (60-kDa) CD18 subunit protein has been reported in another moderate patient;324 this case presumably represents a compound heterozygote, but the nature of a second mutant allele has not been defined. A G284S mutation has been reported in two additional unrelated severe phenotype patients,325 but the mutant (G284S) alleles are not identical to those described by Wright et al.,320 as indicated by distinct polymorphisms. In contrast to the two former (moderate) cases, both severe cases are homozygous for the G284S mutant allele. Nelson et al.323 defined two distinct mutant CD18 alleles of maternal origin in a male patient with moderate phenotype. These included (a) a 12-bp insertion in the cDNA that added four amino acids (Pro-Ser-Ser-Gln) between Pro247 and Glu248 and arose from a single C-to-A transversion in the 3′ terminus of an intron, which, in turn, generated an aberrant splice receptor site, and (b) an 1828C → T nucleotide transition (CGC → TGG) resulting in an R586W mutation. A third recognized mutation, an 1124A → G nucleotide transition (AAT-AGT) resulting in an N351S substitution, apparently represents a de novo mutation not detected in either parent cell line. COS cell transfection protocols using normal CD11a cDNA and these mutant CD18 cDNAs showed that the double-mutant maternal allele allowed essentially no surface expression of CD11b/CD18 complexes. The R586W mutation alone allowed near-normal expression, findings that suggest that it represents a benign polymorphism. Transfections of N351S mutant cDNA allowed only 22 percent of normal CD11b/CD18 expression recognized by a single anti-CD18 Ma; these findings suggest that this mutation may account for the moderate deficiency phenotype.
Investigations of a series of severe phenotype patients have defined several apparently more deleterious point mutations in the coding region of tile CD18 subunit mRNA.314,320,325–329 As is also true for recognized mutations associated with distinct moderate phenotypes, a disproportionate number of mutant alleles involve the highly conserved domain (exons 5 through 9). These findings provide indirect evidence that this region may be critical to common structural and functional characteristics of β-integrin subunits, including the association of α and β precursors. This cluster includes reported G169R substitutions in two unrelated patients,314,323 P178L substitutions in two additional and (presumably) unrelated patients,329,330 and two mutations involving substitutions of the arginine 128-amino-acid residue by asparagine326,331 or glycine.291 The latter (D128G) mutation represents the molecular basis of bovine LAD currently prevalent worldwide among Holstein cattle as a result of artificial insemination and inbreeding of this allele in the 1970s.291 The deleterious nature of the G169R mutation has been demonstrated by an absence of biosynthesis and expression of LFA-1 αβ complexes following cotransfection of mutant CD18 cDNA of the severe patient described by Wardlaw et al.314 and wild-type CD11a cDNA into COS cells. Of interest, severe mutations within this cluster involve amino acid residues conserved in all other β subunits, while some of the mutant alleles associated with a moderate phenotype involve nonconserved amino acids (e.g., L149P and K196T). Another cluster of severe mutations is also evident within the CD18 segment encoded by exon 13 within the cysteine-rich repeats proposed to impart structural rigidity to β integrin subunits. These include a G553fs allele329 and a C534X nonsense mutation.328
Various splicing defects contribute to these or other described severe phenotype-associated alleles326,328,329,332 and involve both the highly conserved domain and cysteine-rich repeats, as well as other domains. They include: (a) a G-to-A substitution in the exon 7 splice site resulting in aberrant splicing and premature chain termination within the highly conserved domain;326 (b) a probable but undefined splicing defect accounting for a deletion of exon 13329 or impaired transcription332 in two unrelated patients each sharing a mutant P178L allele; and (c) a likely defect of splicing involving the 5′ region (intron 2/3–exon 3/4) of the CD18 gene in a patient described by Corbi et al.327
While this spectrum of CD18 mutations provides an incomplete explanation for the considerable heterogeneity recognized among LAD I patients, some insights concerning the structural and functional requirements for biosynthetics and expression of CD11/CD18 heterodimers are implicit in these descriptions. The deleterious nature of mutations involving the highly conserved domain and the cysteine-rich repeats (among both severe and moderate phenotype patients) supports the likely requirements for these domains in α-β precursor associations and subsequent processing and cell transport of mature CD11/CD18 complexes. In addition to quantitative defects of expression, the loss of functional activity (reflected by impaired binding of anti-CD18 blocking Mab) as a result of site-directed mutagenesis of selected epitopes within the highly conserved domain of CD18 provides additional support for this concept. Moreover, this domain in the β3 integrin (gpIIb) subunit contains motifs required for ligand (fibrinogen, RGD, other) binding by gpIIb/IIIa333 and has been shown to be defective in patients with Glansmann thrombasthenia (see Chap. 177).334
The basis for impaired transcription of CD18 among some recognized LAD I alleles is poorly understood. Wright et al.320 described low levels of mRNA expression associated with undefined314 or P247 (PSSQ) insertion323 alleles. Three additional LAD I patients characterized by low mRNA expression318,326,328 each demonstrate frameshift mutations encoding truncated proteins. Studies of the CD18 promoter elements in a severe phenotypic patient with undetectable CD18 mRNA (by northern blot)328 revealed no abnormalities, but similar studies in other patients may reveal defects in transcriptional regulatory domains contained within a 1-kb domain 5′ to the first exon. Frameshift mutations may affect the stability of mRNA in some patients, but this explanation is not applicable to others. For example, a 10-bp deletion in the patient described by Lopez Rodriquez et al.328 has no apparent effect on mRNA levels. An initiation codon mutation (MIK) described by Sligh et al.318 provides a plausible explanation for a partial deficiency of CD18 expression, but three mutant alleles exclusively associated with partial CD18 expression and a moderate phenotype (L149P, K196T, and R593C) map to regions of the highly conserved or cysteine-rich repeat domains most commonly associated with severe phenotypes.
Kuijpers et al.335 described a LAD I variant. This patient exhibited a moderate LAD I phenotype. There were bacterial infections without apparent pus formation in the presence of a striking granulocytosis, and for which intravenous treatment with high-dose antibiotics was needed. Delayed-type hypersensitivity reaction upon skin testing was absent, and there was delayed wound healing. Specific antibody generation was abnormal, but normal in vitro T cell proliferation responses after immunization were found. Expression levels of CD11/CD18 proteins were completely normal, but leukocyte activation did not result in CD11/CD18 activation and high-avidity ligand binding. No sequence abnormalities in the CD18 integrin proteins were found. In vitro chemotaxis, endothelial transmigration of the neutrophils, and leukocyte aggregation responses were almost absent. During follow-up, a bleeding tendency, which was related to decreased glycoprotein IIb-IIIa (β3 integrin) activation on platelets, became clinically apparent; it was different from previously described cellular adhesion molecule variants. This was the first reported case of a clinical combined immunodeficiency syndrome that apparently resulted from nonfunctional CD11/CD18 molecules.
Hogg et al.336 recently described a patient with clinical features compatible with a moderate phenotype of LAD I, but whose neutrophils expressed LFA-1 and Mac-1 at 40 to 60 percent of normal levels. This level of expression should be adequate for normal integrin function, but both the patient's Mac-1 on neutrophils and LFA-1 on T cells failed to bind ligands such as fibrinogen and ICAM-1, respectively, or to display a β2 integrin activation epitope after adhesion-inducing stimulation of the cells. Sequencing of the patient's two CD18 alleles revealed the mutations S138P and G273R. Both mutations are within the highly conserved domain of CD18, and S138P is a putative divalent cation-coordinating residue in the metal ion-dependent adhesion site (MIDAS) motif.1 After cotransfection of a cell line (K562) with normal α subunits, the mutated S138P β2 subunit was coexpressed but did not support adhesive functions, whereas the G273R mutant was not expressed. This is the second reported example of a LAD I patient exhibiting a failure of β2 integrin function despite adequate levels of cell-surface expression.
Clinical Management of LAD I.
Suggested therapeutic guidelines for LAD I are based on a relatively limited clinical experience. Of major importance is the potential for life-threatening systemic bacterial infections that may extend from commonly infected sites in skin or mucus membranes, especially in severely affected patients. Superficial inflammatory lesions must be managed aggressively with local care and systemic antibiotic regimens. The early use of combination therapy with a staphylocidal agent and an aminoglycoside, or extended-spectrum penicillin agent, is justified in acutely ill or febrile patients in the absence of localizing clinical findings. The use of prophylactic antibiotics for chronic infections (e.g., progressive periodontitis) may be advised, but clinical data documenting the long-term efficacy of this empiric approach is lacking. Trimethoprim B-sulfamethoxazole is widely used in this setting based on its broad antimicrobial spectrum of activity and safety profile.
In patients with progressive and life-threatening infections refractory to antibiotic therapy, granulocyte transfusions are efficacious in attenuating, if not eliminating, infectious episodes. The major limitations of granulocyte transfusions include the short half-life of transfused cells and the need for repeated infusions on a daily basis for the duration of infections, often for several weeks.
Bone marrow transplantation (BMT) with successful engraftment and long-term clinical recovery from disease has been achieved in several patients, although unsuccessful and fatal complications of this approach have been reported.337,338 Because of the inherent risks of myeloid suppression and graft versus host disease (GVHD), some investigators have advised this approach preferentially for severe phenotype patients. Among patients undergoing transplantation, recipients of HLA B identical, as well as HLA mismatched, bone marrow have shown successful engraftment. In the largest reported experience, Thomas et al.338 retrospectively analyzed the outcome of BMT in 14 patients with LAD I. Performed between 1981 and 1993 in two European centers, five patients received bone marrow from HLA B identical donors and nine received T-cell-depleted marrow from two HLA B antigen or haplotype mismatched parents. In five cases, failure of engraftment occurred as a result of either insufficient myeloablation or graft rejection (as seen in two cases of moderate phenotype LAD I). These patients were successfully retransplanted using a conditioning regimen that included total body irradiation and chemotherapy, with the use of anti-LFA-1 and anti-CD2 monoclonal antibodies for moderate patients. Eight patients developed acute GVHD, and chronic GVHD occurred in four cases with fatal complications in three of these patients. At the time of this report,338 10 patients of the series were alive and well 12 months to 12 years after BMT; chimerism was full in 6 cases, and mixed but stable in 4 cases, with variable proportions of normal and CD18-deficient leukocytes in peripheral blood. Mixed chimerism observed in these cases does not appear to reflect slowly progressive graft failure and is compatible with long-term freedom from infectious complications.
Studies demonstrating the retroviral-mediated gene transfer of the human CD18 subunit into EBV-B cells, mouse bone marrow cells, and human bone marrow cells339–341 have set the stage for attempts at somatic cell gene therapy for LAD I. Bauer et al.342 recently reported high levels of transfection of peripheral blood CD34+ cells derived from a patient with severe LAD using a novel retroviral vector. Based on these preliminary observations, it should be possible to engraft autologous cells removed from LAD patients and treated ex vivo with candidate retroviral vectors.
Identification of Leukocyte Adhesion Deficiency II (Lad II, Rambon-Hasharon Syndrome)
Clinical and Histopathologic Features.
Frydman et al.343 described two male Arab patients (ages 5 and 3), both products of unrelated consanguineous matings. Distinctive clinical features of these patients included craniofacial dysmorphism, neurologic deficits (microcephaly, cortical atrophy, central hypotonia, seizures, and developmental delay), and striking peripheral blood neutrophilia. Extreme neutrophilia was noted from the first day of life. Recurrent bacterial infections occurred early in life, and included pneumonia, periodontitis, otitis media, and cellulitis. Mild to moderate skin infections occurred without obvious pus formation. In contrast to most patients with LAD I, these patients' infections were not life threatening.344 After the age of 3 years, the frequency of infections diminished, with the main infectious events being severe periodontitis, a condition also observed in patients with LAD I (Table 188-5).
Table 188-5: Leukocyte Adhesion Deficiencies |Favorite Table|Download (.pdf) Table 188-5: Leukocyte Adhesion Deficiencies
| ||LAD I || |
| || ||LAD II* |
| ||Severe ||Moderate || |
| Clinical Manifestations || || || |
|Severe infections ||++++ ||+ ||+ |
|Neutrophilia || || || |
|Basal ||++ ||+ ||+++ |
|with infection ||++++ ||+++ ||+++ |
|Periodontitis ||++++ ||+++ ||+++ |
|Skin infections ||+++ ||++ ||+ |
|Developmental abnormalities ||– ||– ||+++ |
| Laboratory Findings || || || |
|sLeX expression ||normal ||normal ||absent |
|CD18 integrins ||↓ ↓ ↓, or ||↓ ↓, or ||normal |
|expression ||absent ||dysfunctional || |
|Neutrophil L-selectin ||↓ ↓ ||↓ ||normal |
|Neutrophil motility in vitro ||↓ ↓ ↓ ||↓ ↓ ||↓ ↓ |
|Neutrophil rolling ||normal ||normal ||↓ ↓ ↓ |
|Neutrophil firm adhesion ||↓ ↓ ↓ ||↓ ↓ ||↓ |
|Neutrophil emigration ||↓ ↓ ↓ ↓ ||↓ ↓ ↓ ||↓ ↓ |
|Opsonophagocytosis ||↓ ||↓ ||normal |
|Lymphocyte function ||↓ ||↓ ||normal |
That these patients represent examples of a new, presumably autosomal recessive, syndrome was suggested by the finding that both patients lack the red blood cell H antigen and manifest the Bombay (hh) phenotype. This phenotype is ordinarily due to homozygosity for a rare recessive (h) allele. Individuals with this phenotype generally lack the H antigen, an intermediate in the production of A and B antigens of red blood cells. The H gene product is an α2 fucosyltransferase that catalyzes the addition of a GDP fucose to N-acetylgalactosamine to produce the H antigen.345,346 The Hh system is genetically heterogeneous, and both complete (Bombay) and incomplete (Para-Bombay) deficiencies are recognized. In contrast to the patients described here, individuals with these phenotypes do not have any distinctive clinical features.
The findings of markedly elevated blood neutrophil counts (60 to 150,000 FL) in the patients reported by Frydman prompted studies of leukocyte functions. The prolonged intravascular neutrophil survival seen in the LAD I syndrome,321 which seems to account for the marked leukocytosis, was not seen in LAD II patients.347 In fact, the intravascular half-disappearance time was much shorter than normal, and the neutrophil turnover rate was markedly elevated. The estimated turnover was eight times normal.347 In vitro studies revealed significant defects of random or directed neutrophil migration348 and diminished homotypic aggregation of neutrophils.349 In contrast, opsonophagocytic and bactericidal activities of patient neutrophils were normal, as were lymphocyte proliferation and natural killer cell activities. However, delayed type hypersensitivity reactions were not observed upon intradermal injections of various antigens.347 This may be due to the absence of cutaneous lymphocyte antigen (CLA) involved in E-selectin-dependent lymphocyte homing to the skin.71 Leukocyte surface levels of L-selectin are normal in LAD II, in contrast to observations in LAD I where L-selectin levels on neutrophils are often diminished,350 most likely due to frequent infections leading to some low-level neutrophil activation and subsequent shedding of this adhesion molecule (see selectin regulation above).112
Additional studies were performed to assess the in vivo immune responsiveness and lymphocyte recruitment to the skin in response to keyhole limpet hemocyanin (KLH) in a LAD II patient.351 There was normal priming of KLH-specific T cells, as well as a strong in vivo anti-KLH antibody response, both indicative of a normal T-B cell function and collaboration. Skin biopsies following KLH injection revealed a large number of T cells recruited to the site of challenge, and up-regulation of the endothelial adhesion molecules ICAM-1 (CD54), VCAM-1 (CD106), and E-selectin (CD62E). The recruited T cell showed a normal subset distribution but lacked cutaneous lymphocyte antigen (CLA), the cutaneous homing receptor. However, the clinical symptoms of delayed-type hypersensitivity in the patient (redness and swelling) were severely depressed compared to that in the controls. It appeared that the LAD II patient showed a normal T cell priming and T cell-dependent antibody response, and a significant T cell recruitment to the site of KLH challenge, indicating that fucosylated sugar determinants, such as SLe(x) and CLA, are not strictly required for immune responsiveness and skin homing of some lymphocytes, although some effector functions appear abnormal, possibly as a result of absence of an important effector subset of lymphocytes.
Several features of this syndrome suggest the possibility of a glycosylation defect that might influence the expression of Sialyl-Lewis X (sLeX) (NeuAcα2,3 Gal β1,4 (Fucα1,3) GlcNac), a carbohydrate ligand of the endothelial adhesion molecules E-selectin and P-selectin. LAD II neutrophils exhibited the ability to be activated for the up-regulation of CD18 integrins in vitro, and exhibited the ability to spread on a protein coated glass surface352 (in contrast to LAD I neutrophils264). In addition to this rare Bombay phenotype, both patients were secretor negative and Lewis negative, blood groups associated with Fucα2, Gal and Fucα1, 3/4 GlcNac linkages, respectively.353,354 Because each of these carbohydrate structures contain fucose, Etzioni et al.355 tested the possibility that patient leukocytes were deficient in sLeX, which also contains fucose. Flow cytometric studies using an anti-sLeX monoclonal antibody confirmed a complete absence of this determinant on neutrophils of both patients. Additional studies demonstrated profoundly diminished adherence of patient neutrophils to IL-1 elicited endothelial monolayers bearing high levels of E-selectin. These findings suggest that the clinical features of neutrophilia and recurrent pulmonary infections in this syndrome reflect leukocyte adherence defects due to a lack of sLeX-bearing ligands required for selectin-mediated adhesion to endothelial cells. This concept is supported by intravital microscopic studies of patient neutrophils in rabbit mesenteric venules primed for 4 h with IL-1, a condition in which human neutrophils normally demonstrate rolling margination in vivo.350 Isolated patient neutrophils exhibited markedly reduced rolling adhesion in this model, a finding consistent with the concept that lectin-carbohydrate interactions are necessary for this phase of localization (see Table 188-3).
Characterizations of this novel syndrome provide strong evidence for important adhesive interactions of sLeX-bearing ligands and E-selectin in neutrophil migration and recruitment at sites of inflammation in vivo. This possibility is also supported by in vitro investigations and in vivo animal model studies demonstrating that selectins mediate adhesion of neutrophils (and other leukocyte cell types) to vascular endothelium under flow conditions of postcapillary venules.122,158,356–360 Because P-selectin has also been shown to recognize sLeX-containing ligands,359 it is possible that neutrophils of these patients will be defective in P-selectin-mediated adherence functions. The possibility that sLeX deficiency underlies other neutrophil defects is uncertain, but the observed defects of homotypic aggregation may reflect the requirements for L-selectin interactions with PSGL-1 in this process.361 Future studies to determine the specific biochemical defects and resulting functional and pathologic consequence in this syndrome should allow additional insights concerning the physiological roles of selectin-dependent adhesion reactions.
Molecular Basis of Lad II.
The basic abnormality in LAD II is a general defect in fucose metabolism. Recent observations362 resulting from prenatal diagnosis of LAD II indicated that at 22 weeks of gestation, the fetus was normal in size and without obvious abnormalities. The finding that the previously identified patients exhibited impaired growth only after birth suggested that fucose transported from the mother may be significant. In this regard, B lymphoblasts and fibroblasts from the LAD II patients failed to show binding of fucose-specific lectins unless fed exogenous fucose in vitro. This indicates that a scavenger pathway is intact in LAD II363 and that the defect in fucose metabolism is not limited to hematopoietic cells. These observations support the idea that the growth abnormalities and mental retardation are also related to the fucose abnormality. The exact defect in the de novo pathway of fucose production from GDP-mannose to GDP-fucose is unknown, but Sturla et al.364 found that in cell lysates from a LAD II patient, GDP-D-mannose-4,6-dehydratase (GMD), the first of the two enzymes of the pathway, had a defective activity compared to control subjects. GMD in cell lysates from both parents showed intermediate activity levels. Cloning of GMD from patient and control lymphocytes ruled out a mutation affecting the amino acid sequence of this enzyme, and the purified recombinant proteins from both controls and the patient showed identical specific activities. Because the levels of immunoreactive GMD in cell lysates were comparable in the patient and in controls, the biochemical deficiency of intracellular GMD activity in LAD II seems to be due to mutation(s) affecting some still unidentified GMD-regulating protein.
Abnormalities of Adhesion in Neonates
Developmentally related inflammatory deficits observed in human or animal neonates appear to involve defects of neutrophil localization or emigration at sites of inflammation.365–369 Because specific immunity is limited in the immediate postpartum period, the inflammatory functions of phagocytic cells are an especially important aspect of innate immunity for host defense against microbial invasion.365 Both quantitative and functional deficits of neutrophils/monocytes appear to contribute to infectious susceptibility.302,370–372 However, because these functional deficits are observed commonly among healthy neonates, such abnormalities may play a physiological role in limiting inflammatory responses that would be deleterious in immature hosts.372 For example, injurious pulmonary inflammatory responses to hyperoxia are significantly less in neonatal rats than in adult animals.373
The most consistently observed functional abnormalities of neonatal neutrophils are those involving chemotactic migration in vitro. Migration toward gradients of numerous chemotactic factors, including those released by growing Staphylococcus aureus and E. coli (e.g., f-Met-peptides) and those generated in plasma by antigen-antibody complexes (e.g., C5a),219,374 is significantly reduced. Visual assays demonstrate that the initial event required for directional migration—the ability to orient toward a gradient of chemotactic factors—is significantly impaired.219,375 Depressed migration has been found in healthy neonates 1 to 5 days of age,376,377 although more striking abnormalities have been reported in premature infants. Visual assays using shape change as an index of response to some chemotactic stimuli reveal no deficits219 in dose-response characteristics of neonatal neutrophils, although receptor numbers for C5a appear to be significantly reduced in neonates.378 In addition, there is diminished generation of chemotactic activity (chemotaxigenesis) by virulent Type III Group B Streptococci in neonatal sera, which is directly related to diminished levels of both type-specific anticapsular antibody and serum complement activity.379
The impaired migration of neonatal neutrophils is apparently linked to abnormalities in cell adherence.219,302,371,372,375,380–384 At least three distinct abnormalities of neutrophil adhesion to endothelial cells or protein substrates in vitro have been recognized. Chemotactic factors fail to stimulate adhesion of neonatal neutrophils to artificial or endothelial substrates219,302,382,385 apparently as a result of diminished up-regulation and/or functional activation of the Mac-1 heterodimer.382,384–388 This could be partly due to an increase in the number of immature cells389 or to a reduction in the total cellular content of Mac-1.390 The function and levels of LFA-1 on neonatal neutrophils appear to be equivalent to that on adult neutrophils both in vitro390,391 and in vivo in a neonatal rabbit model of inflammation.182 Expression of LFA-1 on neonatal cells is normal and contributes to both adhesion and transendothelial migration.19,302
Neonatal neutrophils also exhibit diminished adhesion to IL-1 stimulated endothelial cells when studied under conditions of flow (wall shear stress, 2 dynes/cm2) that select for CD18-independent adhesion mechanisms.372,392 This abnormality apparently results from markedly diminished surface levels of L-selectin on circulating blood neutrophils or eosinophils of healthy term neonates both human372,392 and rabbit.393 Monoclonal antibodies recognizing L-selectin reduced the adhesion of adult neutrophils under these experimental conditions to the level of neonatal neutrophils, and they did not further reduce adhesion of neonatal cells. In a rabbit model of peritoneal inflammation, monoclonal antibodies to L-selectin significantly reduce neutrophil influx in adult animals, but not in neonates.393 The basis for the reduced L-selectin on neonatal neutrophils is not yet defined, but may relate to high levels of GM-CSF (and/or other plasma factors), as demonstrated in umbilical cord blood and placental tissues in some studies.394 GM-CSF, similar to chemotactic factors, stimulates the shedding of L-selectin from blood neutrophils in vitro, and significantly diminishes neutrophil emigration into experimental skin windows of adult volunteers.395 Studies of cord blood or neonatal neutrophils by Koenig et al.396 have shown that diminished levels of neutrophil L-selectin are inversely related to total blood neutrophil count, and similar relationships have been reported in neonatal rabbits.393 L-selectin levels are not significantly reduced on neutrophils from healthy premature infants.397–399 Another deficit of selectin-dependent adhesion of neonatal neutrophils is their significantly reduced ability to interact with P-selectin under conditions of flow.391 This appears to be due to a reduced level of P-selectin ligands including PSGL-1.400
Deficits of Mac-1, L-selectin, and PSGL-1 may contribute to diminished leukocyte sequestration in human neonates, but direct evidence for this is currently insufficient. Rebuck skin window studies175,401 reveal levels of neutrophil accumulation much like those in adults, as well as two other abnormalities: (a) a slower and less pronounced shift from the early granulocyte to later mononuclear cell predominance, and (b) a marked eosinophilia in some infants 2 to 21 days of age. Diminished leukocyte mobilization in neonatal rats inoculated intraperitoneally with bacteria or chemotactic agents has been reported,366 and peritoneal influx in a rabbit model was significantly reduced.393
Specific Granule Deficiency
Neutrophils contain subpopulations of granules.402 Azurophil or primary granules appear early in neutrophil development and contain lysosomal enzymes, including lysozyme and myeloperoxidase. Specific or secondary granules develop later, and contain a different set of constituents (e.g., chemotactic receptors, lactoferrin, CD11b/CD18).403,404 The first example of a deficiency of specific granules was recognized in 1972,405 and several laboratories subsequently reported other cases.306,403,406–413 One patient405 appeared to have had an acquired deficiency (associated with a myeloproliferative syndrome), while all others appeared to have genetically determined disease. Each has demonstrated susceptibility to recurrent and severe infections of the skin, mucous membranes, and lung, most commonly due to Staphylococcus aureus, Pseudomonas aeruginosa, other enteric pathogens, and Candida albicans. Detailed descriptions of the histopathology of infected tissues in all patients are not reported, but skin window studies demonstrated diminished leukocyte sequestration in tissues of some individuals who were not neutropenic.403,406 A prognosis for this disorder is not well defined, but most individuals have survived the pediatric age group with supportive and antimicrobial therapy.
Neutrophils from each patient studied have shown distinct morphologic abnormalities, including a severe or total deficiency of specific granules and a variety of nuclear abnormalities including bilobed or multilobed nuclei or nuclear blebs, clefts, or pockets. Eosinophils appear to be affected as well.414 Thus, functional abnormalities in this disorder are not limited to neutrophils, and the secretory proteins absent from patient neutrophils are not limited to specific granules. The membrane-marker alkaline phosphatase is diminished or absent in neutrophils of all but one reported case, and the total cellular content and/or release of the secondary granule markers (lactoferrin, B12 transport protein, cytochrome b, and lysozyme) is diminished in most patients. Neutrophils of these patients also lack defensins localized to the azurophil granules.415 Azurophil granules of patient cells exhibit a decreased density on sucrose gradients when compared with azurophil granules of normal neutrophils.403
Somewhat heterogenous abnormalities of in vitro neutrophil functions have been observed. Defects of chemotaxis and intracellular microbicidal activity are most consistently reported. The basis of impaired neutrophil locomotion in vitro or diminished accumulation in skin windows in vivo is uncertain. However, in studies of two patients,306,403,406 defective neutrophil chemotaxis appeared to be functionally related to abnormalities of adherence. In one patient, there was diminished neutrophil adherence to nylon fibers or endothelial cells, and impaired homotypic aggregation in response to f-Met-Leu-Phe.406 Neutrophils of another patient failed to enhance CR3 (Mac-1) expression in response to f-Met-Leu-Phe, although up-regulation of the CR1 was normal.306 Immunoprecipitation studies employing monoclonal antibodies against CD11b and CR1 showed that Mac-1 (but not CR1) cosediments with specific granule fractions of normal neutrophils. CR1 is apparently associated with unique microvesicular bodies in neutrophils unassociated with specific granules.
The molecular basis for the complex cellular abnormalities of these patients is undefined. Indirect evidence implicates abnormalities of the regulation of gene expression during granulopoiesis resulting in a failure to activate a cassette of genes normally expressed at the myelocyte-metamyelocyte stage of maturation.415,416 Studies of two patients showed no detectable lactoferrin biosynthesis in neutrophils and only trace amounts of lactoferrin transcripts in bone marrow cells.417 However, lactoferrin biosynthesis in nasal secretory glands and other nonmyeloid tissues was normal in these patients, indicating that the abnormality of lactoferrin gene expression is tissue specific and limited to cells of myeloid lineage. Of interest are mice deficient in PU.1, an ETS family transcription factor that is expressed specifically in hematopoietic lineages, which develop neutrophils expressing neutrophil-specific markers but fail to terminally differentiate, as shown by the absence of messages for neutrophil secondary granule components.418 While these cells do not mimic exactly those of patients with specific granule deficiency, they provide an interesting model with some parallels.