Phenotypic perturbation of B cells in the Wiskott-Aldrich syndrome.

Wiskott-Aldrich syndrome (WAS) is an X-linked immunodeficiency/platelet disease due to mutations of WASP, a cytoskeletal regulatory protein of blood cells. Patients exhibit a range of immune defects generally attributed to defective T-cell function, including poor response to immunization, skewed immunoglobulin isotypes, eczema, recurrent infections, autoimmune disease and increased frequency of malignancies. Here we show a deficit of total B-cells in WAS patients of various ages and identify phenotypic perturbations involving complement receptors and CD27. Whereas B-cells of normal healthy donors are overwhelmingly CD21/CD35-positive, B-cells expressing these receptors are significantly reduced in number in WAS patients, and their paucity may cause suboptimal antigen capture and presentation. The frequencies of IgD(-) and IgG(+) patient B-cells were not different from healthy donors (although absolute numbers were decreased), indicating that isotype switching is occurring. In contrast, the frequency of cells positive for CD27, the marker of post germinal centre B-cells, was significantly decreased even among isotype-switched cells, and B-cells resembling germinal centre progenitors (CD10(+)CD27(-)CD38(bright)) were more frequent in adult patients, suggesting impaired germinal centre maturation/differentiation. The documentation of these phenotypic perturbations and deficit of total cells suggest that defects intrinsic to B-cells contribute to the impaired humoral immunity that characterizes this disease.

Early deficit of lymphocytes in Wiskott-Aldrich syndrome: possible role of WASP in human lymphocyte maturation.

Wiskott-Aldrich syndrome (WAS) is an X-linked platelet/immunodeficiency disease. The affected gene encodes WASP, a multidomain protein that regulates cytoskeletal assembly in blood cells. Patients have recurring infections, and their lymphocytes exhibit deficient proliferative responses in vitro. We report an evaluation of peripheral blood lymphocytes of 27 WAS patients, aged one month to 55 years. Whereas NK cells were normal, a significant deficit of T and B lymphocytes was observed. The number of lymphocytes was already decreased in infant patients, suggesting deficient output. Both CD4 and CD8 T lymphocytes were affected; the decrease was most pronounced for naïve T cells. Naïve CD4 lymphocytes of patients showed normal expression of Bcl-2, and Ki-67, and normal survival in vitro, suggesting that their in vivo survival and proliferation are normal. The collective data suggest that the patients' lymphocyte deficit results from deficient output, likely due to abnormal lymphocyte maturation in the thymus and bone marrow. We propose that WASP plays an important role not only in the function of mature T lymphocytes, but also in the maturation of human T and B lymphocytes and that impaired lymphocyte maturation is central to the aetiology of WAS immunodeficiency.

Corneal transplantation in antibody-deficient hosts.

PURPOSE: To examine the role of donor-specific antibodies, with or without complement, in rejection of orthotopic corneal transplants by using mice as recipients in which the genes for the heavy chain of immunoglobulin or the third complement component have been eliminated by homologous recombination. METHODS: BALB/c corneas were transplanted into eyes of B-cell-deficient (n=17) or wild-type control C57BL/6 (n=30) mice and into eyes of complement (C3)-deficient (n=15) or wild-type control 129-C57BL/6 (n=13) mice. After surgery all grafts were evaluated over 8 weeks in a masked manner by biomicroscopy for signs of rejection. RESULTS: The rates of corneal transplant rejection were similar among B-cell-deficient and C3-deficient mice compared with rejection rates in their respective wild-type control subjects. This similarity applied to the time course of rejection and to cumulative survival rates. CONCLUSIONS: Neither donor-specific antibody nor the third component of complement play essential roles in acute rejection of orthotopic corneal allografts in mice.

A critical role for complement in maintenance of self-tolerance.

The role of complement in the maintenance of self-tolerance has been examined in two models: an immunoglobulin transgenic model of peripheral tolerance and a lupus-like murine model of CD95 (Fas) deficiency. We find that self-reactive B lymphocytes deficient in complement receptors CD21/CD35 or transferred into mice deficient in the complement protein C4 are not anergized by soluble self-antigen. In the second model, deficiency in CD21/CD35 or C4 combined with CD95 deficiency results in high titers of anti-nuclear antibodies leading to severe lupus-like disease. These findings suggest a novel role for the complement system in B cell tolerance and provide insight into the genetic association of complement deficiency with susceptibility to systemic lupus erythematosus.

A critical role of natural immunoglobulin M in immediate defense against systemic bacterial infection.

To evaluate the role of natural immunoglobulin (Ig)M in the immediate response against microbial infection, we tested mutant mice that are deficient in secreted (s)IgM in an acute peritonitis model induced by cecal ligation and puncture (CLP). 20% of wild-type mice died within 32 h of CLP, whereas 70% of sIgM-deficient mice died within the same time period. The increased susceptibility was associated with a reduced level of tumor necrosis factor (TNF)-alpha, a decreased neutrophil recruitment and an increased bacterial load in the peritoneum, and elevated levels of endotoxin and proinflammatory cytokines in the circulation. Resistance to CLP by sIgM-deficient mice was restored by reconstitution with polyclonal IgM from normal mouse serum. Reconstitution with a monoclonal IgM specific to phosphatidylcholine, a conserved cell membrane component, has a modest effect but a monoclonal IgM specific to phosphocholine is not protective. These findings demonstrate a critical role of natural IgM in the immediate defense against severe bacterial infection.

A small molecular weight factor in aqueous humor acts on C1q to prevent antibody-dependent complement activation.

PURPOSE: Aqueous humor inhibits activation of the classic complement pathway; however, the mechanism of this inhibition is unknown. We have examined at the molecular level potential factors responsible for the inhibition, and we have tried to determine where in the complement pathway the inhibition takes place. METHODS: Fresh rabbit aqueous humor was size fractionated by centrifuge concentrators and by size exclusion column chromatography, and each fraction was assayed for inhibition of the classic complement pathway in a standard CH50 hemolytic assay. Fractions with inhibitory activity were assayed for protein and the presence of ascorbic acid and were subjected to heat treatment. To identify where in the pathway the inhibitor(s) function, the expression of activated complement components bound to the surface of antibody-coated erythrocytes was analyzed by flow cytometry using fluorescein isothiocyanate-labeled antibodies to specific complement components. In addition, hemolytic assays were performed for the function of individual complement components. RESULTS: The most potent inhibition of the classic pathway was in a fraction of aqueous humor of less than 1.3 kDa. The inhibitory activity in the fraction was unassociated with detectable protein or ascorbic acid, and it remained present after heat treatment. The functional analysis through flow cytometry and hemolytic assays for individual complement components showed that the inhibitor in the less than 1.3-kDa fraction caused a blockade in the complement pathway at the level of C1q. CONCLUSIONS: The aqueous humor contains a unique potent anticomplementary factor that has a molecular weight less than 1.3 kDa. This heat-stable inhibitory factor inhibits the classic pathway at the level of C1q. These results imply that within the eye the complement pathway is inhibited at the earliest steps of its initiation. Such inhibition would prevent production of complement products that mediate inflammation and chemotaxis of inflammatory cells. Therefore, as part of the adaptation of immune privilege, the ocular microenvironment is protected from inflammation induced by antigen-antibody complexes.

Dependence of germinal center B cells on expression of CD21/CD35 for survival.

Affinity-driven selection of B lymphocytes within germinal centers is critical for the development of high-affinity memory cells and host protection. To investigate the role of the CD21/CD35 coreceptor in B cell competition for follicular retention and survival within the germinal center, either Cr2+ or Cr2null lysozyme-specific transgenic B cells were adoptively transferred into normal mice immunized with duck (DEL) or turkey (TEL) lysozyme, which bind with different affinities. In mice injected with high-affinity turkey lysozyme, Cr2null B cells responded by follicular retention; however, they could not survive within germinal centers. This suggests that CD21 provides a signal independent of antigen that is required for survival of B cells in the germinal center.

Linkages of innate and adaptive immunity.

The innate immune system is activated by pathogens or environmental antigens following their binding by recognition molecules such as mannan-binding lectin, C-reactive protein and the mannose receptor. Natural antibody, which represents a collection of germline-encoded antigen recognition molecules, is also important in recognition of pathogens and activation of the innate immune system via the classical pathway of complement activation. The major source of natural antibody is CD5+ B-1 cells which differ from conventional B cells (B-2 cells) firstly because they are thought to require contact with antigen for expansion and maintenance and secondly because in general they do not appear to undergo somatic hypermutation. We review results which support an important role for complement in maintenance of B-1 cells, the effect being mediated by B cell expression of complement receptors CD21 and CD35. We propose that complement and natural antibody are interdependent: clonal selection and expansion of CD5+ B-1 cells is dependent on contact with antigen coated by the complement component C3d, while efficient recognition of pathogens and activation of complement is dependent in a large part on natural antibody. This hypothesis is supported by the finding that mice deficient in CD21 and CD35 have a reduced number of CD5+ B-1 cells and are missing specificities for certain antigens commonly found in wild-type mice, such as lipopolysaccharide, Escherichia coli surface antigens and neoepitopes expressed on hypoxic intestinal endothelium.

Impaired mast cell-dependent natural immunity in complement C3-deficient mice.

The complement system is widely regarded as essential for normal inflammation, not least because of its ability to activate mast cells. However, recent studies have called into question the importance of complement in several examples of mast cell-dependent inflammatory responses. To investigate the role of complement in mast cell-dependent natural immunity, we examined the responses of complement-deficient mice to caecal ligation and puncture, a model of acute septic peritonitis that is dependent on mast cells and tumour necrosis factor-alpha (TNF-alpha). We found that C4- or C3-deficient mice were much more sensitive to caecal ligation and puncture than wild-type (WT) controls (100% versus 20% in 24-h mortality, respectively). C3-deficient mice also exhibited reductions in peritoneal mast cell degranulation, production of TNF-alpha, neutrophil infiltration and clearance of bacteria. Treating the C3-deficient mice with purified C3 protein enhanced activation of peritoneal mast cells, TNF-alpha production, neutrophil recruitment, opsonophagocytosis of bacteria and resistance to caecal ligation and puncture, confirming that the defects were complement-dependent. These results provide formal evidence that complement activation is essential for the full expression of innate immunity in this mast cell-dependent model of bacterial infection.

Endotoxin shock in antibody-deficient mice: unraveling the role of natural antibody and complement in the clearance of lipopolysaccharide.

While the significance of natural Ab is not entirely clear, one proposed role is clearance of bacterial Ags. To determine whether natural Ab was involved in clearance of endotoxin, we have examined novel strains of mice with either a total or selective deficiency in Ig. Recombinase-activating gene-2 (RAG-2(-/-))-deficient mice, which have no serum Ig due to arrested development of B cells at the pro-B stage, demonstrate increased sensitivity to endotoxin that correlates with an impaired clearance. When RAG-2(-/-) mice are reconstituted with pooled sera from normal mice, both survival and clearance of circulating endotoxin are enhanced. To further define the nature of the protective Ab, Bruton's tyrosine kinase (Btk)-deficient mice were characterized in the high dose LPS model. Like RAG-2(-/-) mice, they are highly sensitive to endotoxin and have an impaired clearance of LPS. Reconstitution of Btk(-/-) mice, which have reduced levels of IgG3 and IgM, with purified normal mouse IgM dramatically enhances their ability to clear endotoxin compared with mock (saline)-reconstituted littermates. The cellular source of natural anti-LPS IgM was identified as the peritoneal-residing B-1 cell by enzyme-linked immunospot (ELISPOT) assay. Taken together, these studies demonstrate the important role of natural Ab and complement in the clearance of pathogenic substances from the circulation.

Increased susceptibility to endotoxin shock in complement C3- and C4-deficient mice is corrected by C1 inhibitor replacement.

Endotoxin shock is a life-threatening syndrome associated with a Gram-negative infection and mediated by a systemic inflammatory response. As a major effector of inflammation, the complement system has been implicated in both the pathogenesis and the protection from endotoxin shock. To clarify the role of complement in endotoxin shock, we have used mice totally deficient in either complement component C3 or C4. We found that both the C3- and C4-deficient mice were significantly more sensitive to endotoxin than wild-type controls. The endotoxin-challenged complement-deficient mice failed to clear endotoxin efficiently from the circulation and this led to excess consumption of C1 inhibitor protein (C1 INH), a major regulator of both complement and the contact system of blood coagulation. Replacement of C1 INH rescued the endotoxin-challenged complement-deficient mice from shock and death. These findings suggest a novel therapy for treatment of endotoxemia with C1 INH protein.

CD4+ lymphocyte depletion in HIV-infected patients is associated with gp120-immunoglobulin-complement attachment to CD4+ cells.

The mechanism of CD4+ lymphocyte depletion, which is the main immunological feature in HIV-infected patients, is unclear. We investigated whether gp120-immunoglobulin-complement complexes on the surface of CD4+ cells might be involved in the elimination of CD4+ lymphocytes. The results obtained in 63 HIV-infected patients show that gp120 is attached to a variable degree to CD4+ cells. Importantly, the percentage of CD4+gp120+ lymphocytes is inversely associated with CD4+ lymphocyte counts in the peripheral blood (p = 0.0004). CD4+gp120+ blood lymphocytes bind IgM (p = 0.0027) and IgG antibodies (p = 0.0001) and complement (p = 0.0005). These results suggest that immune complex-mediated cell elimination is an important mechanism of CD4+ cell depletion in patients with AIDS.