L-ficolin binding and lectin pathway activation by acetylated low-density lipoprotein.

L-ficolin, like mannan-binding lectin (MBL), is a lectin pathway activator present in normal human plasma. Upon binding ligand, l-ficolin similarly initiates C4 cleavage via the serine protease MBL-associated serine protease-2 (MASP-2). We sought further insight into l-ficolin binding reactions and MASP-2 activation by passing plasma through GlcNAc-derivatized Sepharose. l-Ficolin bound in 1.0 M NaCl-ethylenediamine tetraacetic acid (EDTA), and remained bound in NaCl-free EDTA, while MASP-2 eluted in proenzyme form ( approximately 20% yield, > 40 000-fold purification). L-Ficolin was eluted with GlcNAc in 1.0 M NaCl ( approximately 10% yield, > 3000-fold purification), with trace amounts of C3, alpha(2)-macroglobulin and both native and activated MASP-2. These preparations were utilized to investigate l-ficolin reactivities with acetylated low-density lipoprotein (A-LDL) as a model ligand in albumin-free systems. L-Ficolin bound strongly to A-LDL in the absence as well as presence of calcium, including saline-EDTA, and was optimal in 1.0 M NaCl-EDTA, but binding failed to occur in EDTA in the absence of NaCl. The addition of l-ficolin to immobilized A-LDL resulted in activation of MASP-2 in unmodified but not ficolin-depleted plasma unless l-ficolin was restored. We conclude that A-LDL is a useful ligand for investigation of l-ficolin function; both binding and activation are optimally examined in systems free of albumin; and ligand binding in 1.0 M NaCl in EDTA can be useful in the isolation of l-ficolin and native MASP-2.

Mechanism of complement-dependent haemolysis via the lectin pathway: role of the complement regulatory proteins.

Mannan-binding lectin (MBL) is an acute phase protein which activates the classical complement pathway at the level of C4 and C2 via two novel serine proteases homologous to C1r and C1s. We recently reported that haemolysis via this lectin pathway requires alternative pathway amplification. The present experiments sought to establish the basis for this requirement, and hence focused on the activity and regulation of the C3 convertases. Complement activation was normalized between the lectin and classical pathways such that identical amounts of bound C4 and of haemolytically active C4,2 sites were present on the indicator cells. Under these conditions, there was markedly less haemolysis, associated with markedly less C3 and C5 deposited, via the lectin pathway than via the classical pathway, particularly when alternative pathway recruitment was blocked by depletion of factor D. Lectin pathway activation was associated with enhanced binding in the presence of MBL of complement control proteins C4bp and factor H to C4b and C3b, respectively, with decreased stability of the C3-converting enzyme C4b,2a attributable to C4bp. Immunodepletion of C4bp and/or factor H increased lectin pathway haemolysis and allowed lysis to occur in absence of the alternative pathway. Thus, the lectin pathway of humans is particularly susceptible to the regulatory effects of C4bp and factor H, due at least in part to MBL enhancement of C4bp binding to C4b and factor H binding to C3b.

Enhancement of lectin pathway haemolysis by immunoglobulins.

We recently reported that indicator sheep erythrocytes (E) coated with mannan and sensitized with mannan-binding lectin (MBL) (E-M-MBL) are lysed by human serum in the absence of calcium via the lectin pathway of complement activation by a process which requires alternative pathway amplification and is associated with increased binding of and control by complement regulatory proteins C4 bp and factor H. In the present study, we investigated the effect of immunoglobulin (Ig) on this haemolysis. Co-sensitization of indicator E with anti-E haemolysin led to threefold enhancement of lectin pathway haemolysis in the absence of calcium, associated with increased binding of C3 and C5. Lysis was enhanced approximately twofold when E-M-MBL were chemically or immunologically coated with IgM or IgA, and fourfold when coated with IgG, prior to lysis in human serum-Mg-ethyleneglycol tetraacetic acid. The presence of haemolysin did not reduce the binding or inhibitory activity of C4 bp, and the enhancing activity of haemolysin was retained in serum depleted of C4 bp. By contrast, binding of factor H was greatly reduced in the presence of haemolysin, which had no enhancing effect in serum depleted of factor H. These experiments demonstrate the ability of IgG, IgM and IgA to enhance lectin pathway cytolysis, and that this enhancement occurs by neutralization of the inhibitory activity of factor H. Immunoglobulin enhancement of lectin pathway cytolysis represents another interaction between the innate and adaptive systems of immunity.

Calcium-independent haemolysis via the lectin pathway of complement activation in the guinea-pig and other species*.

We previously reported that complement-dependent haemolysis of sheep erythrocytes (E) coated with mannan (M) and sensitized with human mannan-binding lectin (MBL) via the lectin pathway in man occurs in Mg-EGTA and requires alternative pathway amplification. Calcium was required for MBL binding to E-M, but once the E-M-MBL intermediate was formed, MBL was retained and haemolysis occurred in the absence of calcium. Comparable or greater lectin pathway haemolysis in the absence of calcium was observed upon incubation of E-M-MBL in guinea-pig, rat, dog and pig sera, and was further investigated in the guinea-pig, in which titres were much higher ( approximately 14-fold) than in man, and in contrast to humans, greater than classical pathway haemolytic activity. As in human serum, no lysis was observed in C4- or C2-deficient guinea-pig serum until purified C4 or C2, respectively, were restored. However, lectin pathway haemolytic activity in the guinea-pig did not require the alternative pathway. Removal (>98%) of factor D activity by three sequential passages through Sephadex G-75, resulting in serum which retained a normal classical pathway but no alternative pathway haemolytic activity, did not reduce the ability of guinea-pig serum to mediate haemolysis via the lectin pathway. Further, the C3-convertase formed via the lectin pathway (E-M-MBL-C4,2) lysed in C2-deficient guinea-pig but not human serum chelated with EDTA, a condition which precludes alternative pathway amplification. Thus, lectin pathway haemolysis occurs efficiently in guinea-pig serum, in the absence of calcium and without requirement for alternative pathway amplification. The guinea-pig provides a model for studying the assembly and haemolytic function of a lectin pathway which contrasts with the lectin pathway of man, and allows for comparisons that may help clarify the role of this pathway in complement biology.

Lysis via the lectin pathway of complement activation: minireview and lectin pathway enhancement of endotoxin-initiated hemolysis.

Lysis via the newly discovered lectin pathway of complement activation is reviewed. Mannan-coated erythrocytes sensitized with MBL are lysed in human serum containing Mg-EGTA via the lectin pathway by a process which requires alternative pathway amplification. The inhibitory activities of C4bp and factor H, which are augmented in the presence of MBL, regulate this hemolysis. Lectin pathway activity is enhanced by IgG, which inhibits H activity, and is inhibited by C-reactive protein, which enhances the activity of H. Lectin pathway hemolysis in Mg-EGTA also is seen in other species, and is particularly intense and does not require alternative pathway amplification in the guinea pig. New investigations using E-RaLPS as the MBL-binding agent allowed comparison with classical pathway activation by rabbit anti-RaLPS using the same indicator cell. E-RaLPS-MBL are lysed in human serum-Mg-EGTA, and alternative pathway amplification is required. The addition of rabbit anti-E to E-RaLPS-MBL leads to significant enhancement of lysis in Mg-EGTA, much greater than Ig enhancement of hemolysis via the alternative pathway. Lectin pathway activation also enhances the antibody-independent C activation of the classical C pathway via C1q by ReLPS, as well as the direct activation of the alternative C pathway by wild type LPS. Thus, potentiation of reactions initiated at sites of IgG deposition and Ig-independent complement activation represents another characteristic of the lectin pathway.

Complement regulation in innate immunity and the acute-phase response: inhibition of mannan-binding lectin-initiated complement cytolysis by C-reactive protein (CRP).

Mannan-binding lectin (MBL) is an acute-phase protein which activates complement at the level of C4 and C2. We recently reported that the alternative pathway also is required for haemolysis via this 'lectin pathway' in human serum. CRP is another acute-phase reactant which activates the classical pathway, but CRP also inhibits the alternative pathway on surfaces to which it binds. Since serum levels of both proteins generally increase with inflammation and tissue necrosis, it was of interest to determine the effect of CRP on cytolysis via the lectin pathway. We report here that although CRP increases binding of C4 to MBL-sensitized erythrocytes, which in turn enhances lectin pathway haemolysis, it inhibits MBL-initiated cytolysis by its ability to inhibit the alternative pathway. This inhibition is characterized by increased binding of complement control protein H and decreased binding of C3 and C5 to the indicator cells, which in turn is attributable to the presence of CRP. Immunodepletion of H leads to greatly enhanced cytolysis via the lectin pathway, and this cytolysis is no longer inhibited by CRP. These results indicate that CRP regulates MBL-initiated cytolysis on surfaces to which both proteins bind by modulating alternative pathway recruitment through H, pointing to CRP as a complement regulatory protein, and suggesting a co-ordinated role for these proteins in complement activation in innate immunity and the acute-phase response.

Requirement for the alternative pathway as well as C4 and C2 in complement-dependent hemolysis via the lectin pathway.

Mannan-binding lectin (MBL) is a C1q-like molecule opsonic for several micro-organisms. MBL can activate C4, C2, and later acting complement components in the presence of serine proteases similar to but distinct from C1r and C1s via the lectin pathway of complement activation. We report here that mannan-coated MBL-sensitized erythrocytes are lysed via the lectin pathway in human serum-Mg-EGTA. The surprising occurrence of MBL-initiated lysis in the absence of calcium contrasts with the calcium requirement for C1q-initiated activation of C4 and C2. C2 is required, and lysis is significantly enhanced when indicator cells presensitized with C4 and then coated with mannan (EAC4-M) are used. The alternative pathway also is required, since lysis is lost when either factor D or factor B is removed and is restored upon reconstitution with the purified protein. Even though MBL is a C-type lectin, it is retained on mannan-coated erythrocytes in the absence of calcium. This contrasts with the absence of calcium-independent retention on mannan immobilized on polystyrene plates or beads, and helps explain the MBL-initiated hemolysis in Mg-EGTA. These investigations show that the alternative pathway as well as C4 and C2 of the classical pathway are required for complement-dependent hemolysis via the lectin pathway and provide a method for assay of lectin pathway-mediated complement activity in human serum that should be useful in unraveling the molecular interactions of this pathway.

Efficacy of HIV-specific and 'antibody-independent' mechanisms for complement activation by HIV-infected cells.

Previous studies in this laboratory have shown that efficient activation of complement (C) on HIV isolates and HIV-infected cells requires the binding of specific anti-HIV antibodies, while other investigators have observed 'antibody-independent' C activation. In an attempt to clarify these disparate findings, we investigated the effect of several variables on C activation by HIV-infected cells using flow cytometric analysis of C3 deposition. Antibody-mediated C activation using pooled sera from infected persons or human MoAbs directed against the V3 region of gp120 was always substantially higher than activation without antibody. Normal human serum (NHS) from a subset of HIV antibody-negative donors did, however, induce low levels of C3 deposition. Differences in C3 activation between the various NHS did not correlate with total haemolytic C levels or mannose-binding protein (MBP) levels. IgM isolated from NHS that induced high levels of C activation was at least partly responsible for the 'antibody-independent' C activation. Although there appeared to be a correlation between NHS that induced C activation and the presence of anti-blood type B IgM, absorption of anti-B did not abrogate the C3 deposition. Additionally, MoAb to the B antigen did not induce C3 deposition. These studies show that IgM in sera from HIV-uninfected donors can induce C3 deposition on HIV-infected cells, but that specific antibody-dependent C activation is substantially more efficient. Therefore, 'antibody-independent' C activation on HIV-infected cells may, in some cases, be more accurately described as HIV-cross-reactive antibody-dependent C activation.

Structure and function of the pentraxins.

Over the past two years, the three-dimensional structure of the serum amyloid P component was defined by X-ray diffraction, the first such visualization of a pentraxin. Binding sites for calcium, ligands and complement were identified. New fusion proteins with amino acid sequence homology to the pentraxins were described, and new insights were gained into pentraxin phylogeny, biosynthesis, ligands, complement activation, leukocyte reactivity and biological functions in vivo.

Activation of the alternative complement pathway by red blood cells from patients with sickle cell disease.

Increased activation of the alternative pathway of complement in serum from sickle cell disease (SCD) patients has been reported. We now show that this selective activation is not an artifact of clotting by measuring increased plasma concentrations of Bb in sickle cell patients compared to controls. Furthermore, we show that red blood cells (RBC) from SCD patients activate the alternative complement pathway more than control RBC in an in vitro system. RBC were incubated in 50% normal human serum chelated with 3.5 mM MgCl2 and 10 mM EGTA to block activation by the classical pathway, but allow alternative pathway activation. SCD RBC yielded significantly more activation, as measured by an EIA for C3, P complexes, than control RBC. Denser SCD RBC produced more activation than control RBC, unfractionated SCD RBC, and less dense SCD RBC. These findings are consistent with the hypothesis that dense irreversibly sickled cells, or membrane spicules or vesicles derived from them, may result in complement activation.

In vivo decrease in the expression of complement receptor 2 on B-cells in HIV infection.

OBJECTIVE: To investigate changes in the expression of complement receptor 2 (CR2) on B-cells from HIV-infected individuals. CR2 is the C3d/Epstein-Barr virus receptor and has been implicated in B-cell activation. Changes in its level of expression may therefore be associated with B-cell dysfunction. DESIGN: Cross-sectional study of HIV-infected adults and age-matched control donors. METHODS: The percentage expression and mean fluorescence intensity of CR2 (and three additional markers: CD19, CD69, and a standard antigen designation: HLA-DR) was measured on CD20+ B-cells using a two-color flow cytometric assay. RESULTS: This study demonstrated a highly significant (P = 0.0001) decrease in the percentage co-expression of CR2 on CD20+ B-cells in HIV-infected individuals, compared with control donors. The mean percentage of CD20+ cells co-expressing CR2 was 71% (s.d., +/- 15%) in the HIV-seropositive patients and 94% (s.d., +/- 4%) in the control group. The pattern of CR2 expression in a number of the patients suggested a decrease in antigen density on the cells. Decreased expression of CR2 did not correlate with disease stage (asymptomatic, AIDS-related complex, or AIDS), nor with CD4+ T-cell percentage or absolute count, in the seropositive group. CONCLUSIONS: The evidence for a role for CR2 in B-cell activation suggests that its decreased expression, which we have demonstrated in HIV-seropositive individuals, may be associated with the B-cell dysfunction observed in HIV infection. Our finding that expression of this marker is decreased even in asymptomatic patients is consistent with reports of early B-cell defects in such individuals. Further investigation of this possible association may shed some light on both the increased incidence of bacterial infections in HIV-infected adults and children and their impaired responses to certain immunizations.

Phenotypic analysis of complement receptor 2+ T lymphocytes: reduced expression on CD4+ cells in HIV-infected persons.

While expression of complement receptor 2 (CR2) (CD21) on some CD4+ cell lines renders them more susceptible to infection by complement-treated human immunodeficiency virus (HIV), coexpression of CR2 and CD4 on peripheral blood lymphocytes has not, until recently, been observed. Several recent studies, however, have found that human T lymphocytes express low levels of CR2. Additionally, complement treatment of HIV before addition to these cells has been reported to increase virus expression in peripheral blood lymphocyte cultures. These findings suggest that complement-mediated enhancement of infection of human T cells could occur in vivo and have prompted us to examine both the phenotypic properties of CD4+CR2+ T cells in healthy persons and the expression of CR2 on CD4+ lymphocytes during HIV infection. As was previously reported, we observed CR2 on a proportion (10-50%) of both CD8+ and CD4+ T cells. Approximately half of CD4+CR2+ cells expressed the memory cell markers CD45RO and CD29, 80% expressed the naive marker CD45RA, while 22% expressed CD25. These values were not substantially different from total CD4+ cells. Stimulation of lymphocytes with phytohaemagglutinin (PHA), OKT3 or calcium ionophore but not with phorbol myristate acetate (PMA) or interleukin-2 (IL-2) decreased expression of CR2 on CD4 cells by half over a 3-day culture period. The per cent of CD4+ cells expressing CR2 was significantly decreased in patients with asymptomatic and symptomatic HIV infection compared to uninfected control donors (P = 0.0001). In contrast, the decrease in CR2 expression was not observed with CD8+ lymphocytes from HIV-infected persons. These results confirm that CR2 is expressed on human T lymphocytes and suggest that a subset of CD4+ lymphocytes is selectively affected in HIV-infected individuals.

Defined chemically cross-linked oligomers of human C-reactive protein: characterization and reactivity with the complement system.

Chemically cross-linked C-reactive protein (CRP) oligomers were prepared and characterized, and C1q binding and C activation were investigated. Purified human CRP was polymerized in the presence of both non-cleavable and cleavable cross-linking agents and further separated by Superose 12 analytical FPLC column chromatography into fractions of 110 KDa (pentameric monomers), 220 KDa (dimers) and 330 KDa (trimers); virtually no larger oligomers were formed under a variety of experimental conditions. CRP subunits were cross-linked both within and between CRP pentamers. CRP trimers retained native CRP antigenicity without expression of neo-CRP epitopes. CRP trimers showed maximal binding and CRP dimers showed partial binding of solid phase C1q while CRP monomers bound virtually no C1q at all; CRP trimers also bound to fluid phase C1q. Binding was Ca++ independent and increased as the ionic strength or pH were lowered, characteristics comparable to binding of aggregated IgG to C1q; it was not inhibited by phosphorylcholine. CRP trimers consumed total C, C1 and C2 haemolytic activities upon incubation in fresh human serum, but much less efficiently than did CRP-protamine complexes or Agg-IgG. CRP trimers failed to deplete alternative C pathway haemolytic activity at all. The stable, chemically defined CRP oligomers described in this report, which bind C1q efficiently but display poor ability to activate the classical C pathway in the absence of an appropriate ligand, should be valuable in further studies of the interactions between CRP and the C system.

Human immunodeficiency virus (HIV)-infected cells and free virus directly activate the classical complement pathway in rabbit, mouse and guinea-pig sera; activation results in virus neutralization by virolysis.

Since animal models of human immunodeficiency virus (HIV) infection are being used increasingly in determining various aspects of virus/host interaction and as models for virus expression, it will be important to assess any significant differences in anti-viral immune responses between animals and humans. Previous studies have shown that incubation of HIV with non-immune sera from several animal species results in virus neutralization, and that rabbit serum can lyse HIV-infected cells. The objectives of the current study were to evaluate the animal complement pathway(s) activated by HIV and HIV-infected cells and determine the mechanism by which complement could mediate viral neutralization. Incubation of HIV-infected cells with mouse, guinea-pig or rabbit sera resulted in cell-surface deposition of C3 fragments. Deposition of C3 fragments did not occur either in the presence of C4-deficient guinea-pig serum or in the absence of Ca2+, indicating that activation by infected cells occurred via the classical pathway. Neutralization of free virus was also mediated by the classical pathway since C4-deficient guinea-pig serum and Ca(2+)-chelated sera lacked activity. Serum treatment of virus resulted in release of HIV reverse transcriptase (RT), suggesting that neutralization occurred by C5b-9-mediated virolysis. RT was also released from simian immunodeficiency virus by animal complement. Antibodies in animal sera were not responsible for the classical pathway activation by free virus or HIV-infected cells. These results define several substantial differences between animal and human complement reactivity with HIV which could significantly affect the ability of HIV to replicate in animals, and which need to be considered in the assessment of animal models of HIV infection.

Direct binding of complement component C1q to human immunodeficiency virus (HIV) and human T lymphotrophic virus-I (HTLV-I) coinfected cells.

Previous studies have shown that coinfection of the human T lymphotrophic virus type I (HTLV-I) chronically infected cell line MT4 with human immunodeficiency virus type 1 (HIV-1) results in cells which spontaneously activate complement via the classical pathway. This complement activation was antibody independent, yet required C2, suggesting either direct C1, C4, or C2 activation. Because some animal retroviruses have been shown to bind human C1q directly, the present study investigated the possible direct binding of C1q by HIV coinfected MT4 cells. Coinfected cells bound both C1q present in serum and highly purified C1q. Binding of C1q resulted in formation of active C1 on the cell surface, which could in turn activate complement as shown by C4 consumption. The C1q binding was not HIV-isolate specific since infection of MT4 cells with any of three diverse isolates all induced C1q binding. Purified collagen-like region (CLR) and globular region (GR) fragments of C1q both bound to coinfected cells, suggesting a mechanism of binding by C1q similar to that of fibronectin-C1q binding. However, culture of coinfected cells in serum-free (fibronectin-free) medium did not reduce C1q binding. A second HTLV-I chronically infected line, SLB-1, also displayed increased binding of C1q after HIV infection. The H9 cell line, which is not HTLV-I infected, did not bind C1q after HIV infection. These results suggest that a retrovirus protein expressed by coinfected cells directly binds C1q resulting in classical complement activation. This type of activation may have profound biological effects in persons coinfected with HIV-1 and HTLV-I.

The potential use of a dopamine neuron antibody and a striatal-derived neurotrophic factor as diagnostic markers in Parkinson's disease.

The cerebral spinal fluid (CSF) of patients with Parkinson's disease (PD) contains an antibody that immunocytochemically reacts with dopamine (DA) neurons in the substantia nigra (SN). This antibody was found in 78% of the CSF samples taken from patients with clinical PD. In contrast, only 3% of the CSF samples taken from control patients or patients with neurologic symptoms other than PD possessed this antibody. The production of this antibody might contribute to disease progression but does not appear to be the etiologic factor responsible for PD. In other experiments, concentrates of the CSF of patients with PD enhanced growth of mesencephalic cultures relative to control CSF. Both the antibody and the growth-promoting activity found in CSF are associated with degeneration of the SN and might therefore be useful as potential diagnostic markers for PD.

Complement and antibody mediate enhancement of HIV infection by increasing virus binding and provirus formation.

Previous studies have shown that infection of complement receptor (CR2)-bearing cells with HIV pretreated with antibody (Ab) plus complement (C) resulted in increased virus expression. The current study was designed to determine whether C-mediated 'enhancement' of HIV-1 production was the result of increased virus infection of cells as assessed by provirus formation and virus binding. Virus was incubated with anti-HIV Ab and/or C and added to CR2-positive MT-2 cells. Increased virus expression by MT-2 cells correlated with increased numbers of HIV-immunofluorescent-positive cells at 24 and 48 h and higher levels of provirus detected 8-28 h after infection. MT-2 cells also bound threefold more Ab-plus-C-treated virus than untreated virus. Serial dilutions of C showed that high levels of C with Ab did not enhance but rather suppressed virus expression. Studies were also performed which showed that terminal C components C5 and C8 were not necessary for the enhancing effect. The increased binding of C-coated HIV to CR-positive cells has important implications for the fate of virus in vivo.

Neutralization of human immunodeficiency virus type 1 by complement occurs by viral lysis.

The ability of complement to inactivate human immunodeficiency virus (HIV) in the presence of specific antibody was evaluated. HIV was treated with complement and/or antibody, and then its titer was determined on the CD4+ H9 cell line. While complement alone had no effect on the HIV titer, complement plus subneutralizing levels of antibody resulted in titer reductions. Complement sources deficient in membrane attack component C5 or C8 did not inactivate antibody-treated HIV, suggesting that neutralization occurred via lysis. This possibility was investigated by assessing release of reverse transcriptase (RT) from the virion. Antibody plus complement, but neither reagent alone, released RT from HIV in a dose-dependent manner. Release of RT did not occur with C5- or C8-deficient sera, also indicating a requirement for membrane attack components. These studies show that complement can neutralize HIV via the classical complement pathway and that this neutralization occurs via C5b-9-mediated viral lysis. Thus, complement may play a major role in resistance to disease by lysing HIV and preventing infection of Fc- and complement receptor-positive cells, as well as CD4+ cells.

Activation of the alternative pathway of complement by twelve different rabbit-mouse chimeric transfectoma IgA isotypes.

Previous experiments have resulted in the identification and cloning of 13 nonallelic genes encoding the constant region of rabbit IgA H chains. The genes, C alpha 1 to C alpha 13, were each cloned into an expression vector containing the VDJ gene of a dansyl (DNS)-binding murine hybridoma and the constructs were then transfected into SP2/0 cells that were producing murine kappa-L chains from the DNS-binding hybridoma. Of the 13 resulting transfectomas, 12 were shown, by ELISA, to secrete DNS-binding chimeric rabbit-mouse IgA molecules. These transfectoma antibodies, representing 12 different isotypes, are of high affinity and provide a unique source of Ag-specific IgA for comparison of the functions of the multiple IgA isotypes. One such function for antibodies is activation of C by either the classical or alternative pathway. We have used the DNS-binding IgA transfectoma antibodies in C assays based on binding of rabbit C3 to IgA-Ag complexes in an ELISA. The results demonstrated that all 12 IgA isotypes are capable of activating C by the alternative pathway but that none can activate C by the classical pathway. Control experiments demonstrated that activation was hapten dependent and was not caused by endotoxin contamination. These data demonstrate that Ag-specific IgA molecules, unmodified by heat or chemical aggregation, activate C by the alternative pathway but not by the classical pathway.