Depressed activation of the lectin pathway of complement in hereditary angioedema.

The possibility of simultaneous measurement of the classical pathway (CP), mannan-binding lectin (MBL)--lectin pathway (LP) and alternative pathway (AP) of complement activation by the recently developed Wielisa method allowed us to investigate the in vivo significance of the C1-inhibitor (C1INH) in three complement activation pathways. Functional activity of the CP, LP and AP were measured in the sera of 68 adult patients with hereditary angioedema (HAE) and 64 healthy controls. In addition, the level of C1q, MBL, MBL-associated serine protease-2 (MASP-2), C4-, C3- and C1INH was measured by standard laboratory methods. MBL-2 genotypes were determined by polymerase chain reaction. Besides the complement alterations (low CP and C1INH activity, low C4-, C1INH concentrations), which characterize HAE, the level of MASP-2 was also lower (P = 0.0001) in patients compared with controls. Depressed LP activity was found in patients compared with controls (P = 0.0008) in homozygous carriers of the normal MBL genotype (A/A), but not in carriers of variant genotypes (A/O, O/O). Activity of CP correlated with LP in patients (Spearman's r = 0.64; P < 0.0001), but no significant correlation was found in the control group and no correlation with AP was observed. In contrast, the activity of CP and AP correlated (Spearman's r = 0.47; P < 0.0001) in healthy controls, but there was no significant correlation in the HAE patients. We conclude that the activation of LP might also occur in subjects with C1INH deficiency, which is reflected by the low MASP-2 and C4 levels.

Characterization of factor H-related cell membrane molecules expressed by human B lymphocytes and neutrophil granulocytes.

The human factor H protein family comprises six plasma glycoproteins. Earlier we described a membranal factor H-related (mFHR) molecule that is expressed by human B lymphoblastoid cell lines and exerts cofactor activity. In our present study we screened human blood cells for the presence of mFHR proteins and further characterized these molecules. By cytofluorimetry it is shown that the factor H-specific rabbit antiserum reacts strongly with B cells and neutrophil granulocytes, but not with T cells and monocytes. On B lymphocytes mFHR is shown to be down-regulated upon activation of the cells via sIg. In experiments studying which short consensus repeat (SCR) domains are part of the cell membrane proteins we found that antibodies raised against SCRs 1-4, 19-20 and FHR-3 bound to neutrophils but not to B cells. While mFHRs derived both from B cells and granulocytes are shown to bind heparin, their size and structure are different as revealed by Western blotting. A further characteristic of the granulocyte-derived mFHR is its sensitivity to the PI-specific PLCgamma enzyme. These data demonstrate the existence of new members of the FHR protein family, as two distinct, membranal forms are identified. Based on the differences, the B cell derived molecule is termed mFHR-1 and the neutrophil derived protein mFHR-2.

Adjuvant effect of gamma-inulin is mediated by C3 fragments deposited on antigen-presenting cells.

The adjuvant effect of gamma-inulin, a strong activator of the alternative complement pathway, is well-known, but its exact mechanism is not revealed yet. Here, we show that macrophages, isolated from the peritoneal cavity of gamma-inulin-injected mice and used as antigen-presenting cells, enhance the proliferation of antigen-specific T-cells up to 2.5-fold when compared with macrophages of non-treated animals. This effect is abrogated by the presence of anti-C3 F(ab')2 fragments and by prior decomplementation of the donor animals with CVF. It is demonstrated that treatment of mice with the adjuvant results in deposition of C3-fragments onto the surface of peritoneal macrophages, as does in vitro incubation of the cells with gamma-inulin in the presence of fresh autologous serum. Prior incubation of macrophages with gamma-inulin plus serum in vitro enhances subsequent C3 production. Because it has been shown earlier that CR1/2 expressed on activated T-cells and interacting with covalently bound C3-fragments plays an important role in the augmentation of the adaptive response, our present results reveal a mechanism that contributes to the adjuvant effect of gamma-inulin and point to a further link between innate and adaptive immunity.

C5a and C5a(desArg) enhance the susceptibility of monocyte-derived macrophages to HIV infection.

Mononuclear phagocytes, which include circulating blood monocytes and differentiated tissue macrophages, are believed to play a central role in the sexual transmission of HIV infection. The ability of HIV to productively infect these cells may be influenced by action of exogenous or host-derived substances at the site of viral entry. Given the potent capacities of inflammatory mediators to stimulate anaphylatoxic and immunomodulatory functions in mucosa, the effects of complement-derived anaphylatoxins on the susceptibility of monocytes and monocyte-derived macrophages (MDM) to HIV-1 infection were examined. In our in vitro system, the susceptibility to infection was up to 40 times increased in MDM that had been exposed to C5a or C5a(desArg), but not to C3a or C3a(desArg), for 2 days before adding of virus. By contrast, the treatment with complement anaphylatoxins did not affect HIV replication in fresh monocytes. Stimulatory effect of C5a and its desArg derivative on HIV infection correlated with the increase of TNF-alpha and IL-6 secretion from MDM. All these functional effects of C5a and C5a(desArg) were reversible by treatment of cells with the mAb that functionally blocks C5aR. Taken together, these results indicate that C5a and C5a(desArg) may increase the susceptibility of MDM to HIV infection through stimulation of TNF-alpha and IL-6 secretion from these cells.

Mannan-binding lectin and C1q bind to distinct structures and exert differential effects on macrophages.

While the interaction of complement component C1q with cellular proteins is extensively studied, much less is known about the binding of the structurally related molecule, mannan-binding lectin (MBL) to various cells. Here we show by cytofluorimetry that the interaction of MBL with immunocompetent cells is much more restricted than that of C1q. It is shown that under conditions of physiological ionic strength MBL binds to human monocyte-derived macrophages (Mphi) and monocytoid cell lines, but not to T and B lymphocytes, in contrast to C1q, which interacts with all these cells under the same conditions. As opposed to the binding of C1q, low ionic strength does not improve the interaction of MBL with Mphi. No competition for cellular binding sites was found when MBL and C1q were added simultaneously to the cells. Studying the functional consequences of the interaction, we found that the release of TNF-alpha from Mphi is induced by C1q but not by MBL. Production of complement C3 by Mphi is stimulated by C1q strongly, while the effect of MBL is much weaker. C3 produced upon C1q-mediated triggering is shown to opsonize RBC, resulting in enhanced phagocytosis. These results suggest that cell membrane molecules binding MBL and C1 q are not identical; moreover, biological functions exerted by these proteins are also markedly different.

Inhibition of IgE-mediated triggering of mast cells by complement-derived peptides interacting with the Fc epsilon RI.

Mucosal type mast cells, in contrast to the serosal type ones, do not respond to cationic agents, or to the complement-derived peptides C3a and C5a. Earlier we have found that while C3a does not activate the rat mucosal type mast cells (line RBL-2H3), it strongly inhibits the IgE-mediated triggering of these cells, by interfering with the Fc epsilon RI-initiated signaling pathway. In the present study we further investigated the mechanism of this process. It is shown, that C3a interacts with the beta-chain of the Fc epsilon RI complex. Binding of the complement peptide to the cells apparently causes a decrease in the proximity of the IgE-binding Fc epsilon RI. Investigating certain sequences of C3a we found that the inhibition is caused by the C-terminal sequences of the complement-peptide, ranging from positions 56 to 77 and also by a shorter sequence, ranging from positions 56 to 64. The inhibitory effect of these peptides was observed both in the case of RBL-2H3 cells and mouse bone marrow derived mast cells.

HIV-1 induces human monocyte-derived macrophages to produce C3 and to fix C3 on their surface.

Complement components, particularly C3, are known to be involved in the pathogenesis of AIDS and macrophages may serve as a source of C3 at sites of infection. We investigated whether the interaction between HIV-1 and monocytes has any effect on C3 production by the cells. Monocytes isolated from the blood of healthy volunteers were incubated with monocytotropic and T lymphocytotropic HIV-1 strains or with recombinant gp160 and cultured in serum-free medium up to 7 days. Supernatants were tested for secreted C3 by enzyme-linked immunosorbent assay. Our data show that monocytes cultured with either the monocytotropic or the T lymphocytotropic HIV-1 strains produce C3 in large amounts. The effect of both viruses is dose dependent and the amount of C3 induced by HIV was up to 20-fold higher than in the control samples. C3 production was also enhanced by gp160, the envelope protein of the virus. Secretion of IL-6 by the cells was also measured and found to be elevated up to threefold as a consequence of the interaction with the virus. HIV-1-activated monocyte-derived macrophages acquired the capacity to cleave exogenous C3 and to fix generated C3 fragments on their cell membrane.

A further link between innate and adaptive immunity: C3 deposition on antigen-presenting cells enhances the proliferation of antigen-specific T cells.

Murine cells of the B lymphoblastoid line A20 and concanavalin A-elicited peritoneal macrophages are shown to activate and fix C3 fragments covalently when incubated in fresh, autologous serum under conditions allowing the initiation of the alternative complement pathway. For the detection of cell-bound C3, cytofluorimetry was performed using FITC-labeled F(ab')2 fragments of anti-mouse C3. Cell-bound C3 fragments are not internalized or shed by the cells under culture conditions for at least two hours. When the antigen-presenting capacity of serum-treated cells was tested using various antigens and experimental systems, augmentation of the proliferation of antigen-specific T cells was found. This enhancing effect was particularly pronounced at suboptimal antigen doses. The elevation of T cell proliferation induced by C3-opsonized antigen-presenting cells (APC) could be abrogated by F(ab')2 fragments of goat anti-mouse C3, suggesting the involvement of C3 receptors expressed by T cells in the process. Using the 7G6 mAb recognizing murine CR1/CR2, the presence of these complement receptors on activated T cells is demonstrated by cytofluorimetry and immunoprecipitation, as well. These results point to the role of C3 bound to acceptor sites on APC in the facilitation of antigen presentation, providing a further link between innate and adaptive immunity.

Fc gamma R-dependent regulation of the biosynthesis of complement C3 by murine macrophages: the modulatory effect of IL-6.

The effect of murine IgG isotypes on the gene expression and secretion of the third component of complement (C3) has been studied using the monocytoid cell line P388D1 and oil-elicited mouse peritoneal macrophages. It is demonstrated that the binding of IgG2a and IgG2b but not IgG1 and IgG3 augments the biosynthesis of C3 both in the presence and in the absence of the phorbol ester, phorbol myristate acetate in the case of both cell types. The multifunctional cytokine interleukin-6 (IL-6) alone reveals no effect on the gene expression of C3, but increases the effectiveness of mouse IgG2a and IgG2b. Confirming the role of Fc gamma RII, a strong up-regulation of C3 gene expression and C3 secretion was found when macrophages were cultured with the F(ab')2 fragment of the Fc gamma RII-specific monoclonal antibody 2.4G2.

Appearance of acceptor-bound C3b on HLA-DR positive macrophages and on stimulated U937 cells; inhibition of Fc gamma-receptors by the covalently fixed C3 fragments.

The appearance and the functional role of acceptor-bound C3b during differentiation of human monocytes into macrophages were studied. Acceptor-bound C3b could be detected by the immune adherence (IA) test parallel to the expression of antigenic determinants specific to mature cells--i.e. on days 4-5 of culture. Consequently, the capacity of these phagocytes to fix C3b covalently via C3b-acceptors (C3bAs) can be considered as one of the signs of their activation/differentiation. All the mature macrophages positive in the IA test were also found to express HLA-DR antigens on their membrane. Using solubilized extracts of stimulated, 35S-cysteine-labelled cells of the human monocytic cell line, U937, we demonstrate that C3 synthesized by these cells can bind to C3bAs of the same cells. Covalently fixed C3 fragments were found to inhibit Fc gamma-receptor-mediated ingestion of immune complexes and also antibody-dependent cellular cytotoxicity of monocyte-derived macrophages.

Functional cooperation of C3b-acceptors, Fc gamma-receptors and cell-surface proteases on macrophages.

Macrophages are FcR-positive cells, synthetize complement components and express proteolytic enzymes on their surface. In this paper a functional cooperation of C3b acceptor (C3bA) sites, which bind covalently nascent C3b molecules via their metastable binding site, IgG FcRs and cell surface proteases are described and the possible importance of this cooperation in regulation of immune response is discussed. It was found that isolated monocytes did not express C3bA in contrast to cultured macrophages which showed immune adherence positivity. Stimulation of macrophages resulted in enhanced expression of C3bA. C3 synthetized by macrophages was shown to be cleaved by cellular proteases which resulted in the binding of nascent C3b to C3bA. C3bA-nascent C3b interaction inhibited FcR-dependent effector functions, such as immune complex phagocytosis and antibody-dependent cellular cytotoxicity.