Modulation of post-stroke degenerative and regenerative processes and subacute protection by site-targeted inhibition of the alternative pathway of complement.

BACKGROUND: Complement promotes neuroinflammation and injury in models of stroke. However, complement is also being increasingly implicated in repair and regeneration after central nervous system (CNS) injury, and some complement deficiencies have been shown to provide acute, but not subacute, protection after murine stroke. Here, we investigate the dual role of complement in injury and repair after cerebral ischemia and reperfusion. METHODS: We used complement-deficient mice and different complement inhibitors in a model of transient middle cerebral artery occlusion to investigate complement-dependent cellular and molecular changes that occur through the subacute phase after stroke. RESULTS: C3 deficiency and site-targeted complement inhibition with either CR2-Crry (inhibits all pathways) or CR2-fH (inhibits alternative pathway) significantly reduced infarct size, reduced apoptotic cell death, and improved neurological deficit score in the acute phase after stroke. However, only in CR2-fH-treated mice was there sustained protection with no evolution of injury in the subacute phase. Whereas both inhibitors significantly reduced microglia/macrophage activation and astrogliosis in the subacute phase, only CR2-fH improved neurological deficit and locomotor function, maintained neurogenesis markers, enhanced neuronal migration, and increased VEGF expression. These findings in CR2-fH-treated mice correlated with improved performance in spatial learning and passive avoidance tasks. The complement anaphylatoxins have been implicated in repair and regenerative mechanisms after CNS injury, and in this context CR2-fH significantly reduced, but did not eliminate the generation of C5a within the brain, unlike CR2-Crry that completely blocked C5a generation. Gene expression profiling revealed that CR2-fH treatment downregulated genes associated with apoptosis, TGFß signaling, and neutrophil activation, and decreased neutrophil infiltration was confirmed by immunohistochemistry. CR2-fH upregulated genes for neural growth factor and mediators of neurogenesis and neuronal migration. Live animal imaging demonstrated that following intravenous injection, CR2-fH targeted specifically to the post-ischemic brain, with a tissue half-life of 48.5 h. Finally, unlike C3 deficiency, targeted complement inhibition did not increase susceptibility to lethal post-stroke infection, an important consideration for stroke patients. CONCLUSIONS: Ischemic brain tissue-targeted and selective inhibition of alternative complement pathway provide self-limiting inhibition of complement activation and reduces acute injury while maintaining complement-dependent recovery mechanisms into the subacute phase after stroke.

CD21 and CD19 deficiency: Two defects in the same complex leading to different disease modalities.

PURPOSE: Deficiencies in CD19 and CD81 (forming the CD19-complex with CD21 and CD225) cause a severe clinical phenotype. One CD21 deficient patient has been described. We present a second CD21 deficient patient, with a mild clinical phenotype and compared the immunobiological characteristics of CD21 and CD19 deficiency. METHODS: CD21 deficiency was characterized by flowcytometric immunophenotyping and sequencing. Real-time PCR, in vitro stimulation and next generation sequencing were used to characterize B-cell responses and affinity maturation in CD21(-/-) and CD19(-/-) B cells. RESULTS: A compound heterozygous mutation in CD21 caused CD21 deficiency. CD21(-/-) B cells responded normally to in vitro stimulation and AID was transcribed. Affinity maturation was less affected by CD21 than by CD19 deficiency. CONCLUSIONS: Both CD21 and CD19 deficiencies cause hypogammaglobulinemia and reduced memory B cells. CD19 deficiency causes a more severe clinical phenotype. B-cell characteristics reflect this, both after in vitro stimulation as in affinity maturation.

Deficiency of complement receptors CR2/CR1 in Cr2⁻/⁻ mice reduces the extent of secondary brain damage after closed head injury.

Complement activation at the C3 convertase level has been associated with acute neuroinflammation and secondary brain injury after severe head trauma. The present study was designed to test the hypothesis that Cr2-/- mice, which lack the receptors CR2/CD21 and CR1/CD35 for complement C3-derived activation fragments, are protected from adverse sequelae of experimental closed head injury. Adult wild-type mice and Cr2-/- mice on a C57BL/6 genetic background were subjected to focal closed head injury using a standardized weight-drop device. Head-injured Cr2-/- mice showed significantly improved neurological outcomes for up to 72 hours after trauma and a significantly decreased post-injury mortality when compared to wild-type mice. In addition, the Cr2-/- genotype was associated with a decreased extent of neuronal cell death at seven days post-injury. Western blot analysis revealed that complement C3 levels were reduced in the injured brain hemispheres of Cr2-/- mice, whereas plasma C3 levels remained unchanged, compared to wild-type mice. Finally, head-injured Cr2-/- had an attenuated extent of post-injury C3 tissue deposition, decreased astrocytosis and microglial activation, and attenuated immunoglobulin M deposition in injured brains compared to wild-type mice. Targeting of these receptors for complement C3 fragments (CR2/CR1) may represent a promising future approach for therapeutic immunomodulation after traumatic brain injury.

Genetic depletion of complement receptors CD21/35 prevents terminal prion disease in a mouse model of chronic wasting disease.

The complement system has been shown to facilitate peripheral prion pathogenesis. Mice lacking complement receptors CD21/35 partially resist terminal prion disease when infected i.p. with mouse-adapted scrapie prions. Chronic wasting disease (CWD) is an emerging prion disease of captive and free-ranging cervid populations that, similar to scrapie, has been shown to involve the immune system, which probably contributes to their relatively facile horizontal and environmental transmission. In this study, we show that mice overexpressing the cervid prion protein and susceptible to CWD (Tg(cerPrP)5037 mice) but lack CD21/35 expression completely resist clinical CWD upon peripheral infection. CD21/35-deficient Tg5037 mice exhibit greatly impaired splenic prion accumulation and replication throughout disease, similar to CD21/35-deficient murine prion protein mice infected with mouse scrapie. TgA5037;CD21/35(-/-) mice exhibited little or no neuropathology and deposition of misfolded, protease-resistant prion protein associated with CWD. CD21/35 translocate to lipid rafts and mediates a strong germinal center response to prion infection that we propose provides the optimal environment for prion accumulation and replication. We further propose a potential role for CD21/35 in selecting prion quasi-species present in prion strains that may exhibit differential zoonotic potential compared with the parental strains.

Complement receptors 1 and 2 in murine antibody responses to IgM-complexed and uncomplexed sheep erythrocytes.

Early complement components are important for normal antibody responses. In this process, complement receptors 1 and 2 (CR1/2), expressed on B cells and follicular dendritic cells (FDCs) in mice, play a central role. Complement-activating IgM administered with the antigen it is specific for, enhances the antibody response to this antigen. Here, bone marrow chimeras between Cr2(-/-) and wildtype mice were used to analyze whether FDCs or B cells must express CR1/2 for antibody responses to sheep erythrocytes (SRBC), either administered alone or together with specific IgM. For robust IgG anti-SRBC responses, CR1/2 must be expressed on FDCs. Occasionally, weak antibody responses were seen when only B cells expressed CR1/2, probably reflecting extrafollicular antibody production enabled by co-crosslinking of CR2/CD19/CD81 and the BCR. When SRBC alone was administered to mice with CR1/2(+) FDCs, B cells from wildtype and Cr2(-/-) mice produced equal amounts of antibodies. Most likely antigen is then deposited on FDCs in a way that optimizes engagement of the B cell receptor, making CR2-facilitated signaling to the B cell superfluous. SRBC bound to IgM will have more C3 fragments, the ligands for CR1/2, on their surface than SRBC administered alone. Specific IgM, forming a complex with SRBC, enhances antibody responses in two ways when FDCs express CR1/2. One is dependent on CR1/2(+) B cells and probably acts via increased transport of IgM-SRBC-complement complexes bound to CR1/2 on marginal zone B cells. The other is independent on CR1/2(+) B cells and the likely mechanism is that IgM-SRBC-complement complexes bind better to FDCs than SRBC administered alone. These observations suggest that the immune system uses three different CR1/2-mediated effector functions to generate optimal antibody responses: capture by FDCs (playing a dominant role), transport by marginal zone B cells and enhanced B cell signaling.

CR2+ marginal zone B cell production of pathogenic natural antibodies is C3 independent.

Intestinal ischemia-reperfusion (IR)-induced damage requires complement receptor 2 (CR2) for generation of the appropriate natural Ab repertoire. Pathogenic Abs recognize neoantigens on the ischemic tissue, activate complement, and induce intestinal damage. Because C3 cleavage products act as ligands for CR2, we hypothesized that CR2(hi) marginal zone B cells (MZBs) require C3 for generation of the pathogenic Abs. To explore the ability of splenic CR2(+) B cells to generate the damaging Ab repertoire, we adoptively transferred either MZBs or follicular B cells (FOBs) from C57BL/6 or Cr2(-/-) mice into Rag-1(-/-) mice. Adoptive transfer of wild type CR2(hi) MZBs but not CR2(lo) FOBs induced significant damage, C3 deposition, and inflammation in response to IR. In contrast, similarly treated Rag-1(-/-) mice reconstituted with either Cr2(-/-) MZB/B1 B cells (B1Bs) or FOBs lacked significant intestinal damage and displayed limited complement activation. To determine whether C3 cleavage products are critical in CR2-dependent Ab production, we evaluated the ability of the natural Ab repertoire of C3(-/-) mice to induce damage in response to IR. Infusion of C3(-/-) serum into Cr2(-/-) mice restored IR-induced tissue damage. Furthermore, Rag-1(-/-) mice sustained significant damage after infusion of Abs from C3(-/-) but not Cr2(-/-) mice. Finally, adoptive transfer of MZBs from C3(-/-) mice into Rag-1(-/-) mice resulted in significant tissue damage and inflammation. These data indicate that CR2 expression on MZBs is sufficient to induce the appropriate Abs required for IR-induced tissue damage and that C3 is not critical for generation of the pathogenic Abs.

Down regulation of Fc and complement receptors on B cells in rheumatoid arthritis.

B cell tolerance is regulated by receptors that modulate B cell receptor signaling, such as Fc gamma receptor IIb (FcγRIIb; CD32b) and complement receptors (CR) 1 and 2. Deficiency in these receptors may contribute to autoimmunity. To address this we have investigated the receptor expression in healthy individuals in comparison with rheumatoid arthritis (RA) patients. In healthy subjects we found that women had overall lower FcγRIIb expression on B cells than men that significantly decreased with age. RA patients had fewer FcγRIIb, CR1 and CR2 positive B cells and decreased receptor expressions compared to healthy subjects. Further, the RA B cells displayed a significantly increased proliferative response when cultured with interleukin-2 in vitro. In summary, the dysregulated B cells in RA are associated with lower FcγRIIb, CR1 and CR2 levels. The reduced FcγRIIb expression on B cells in women may influence the increased frequency of autoimmunity in women.

Pathogenic natural antibodies recognizing annexin IV are required to develop intestinal ischemia-reperfusion injury.

Intestinal ischemia-reperfusion (IR) injury is initiated when natural IgM Abs recognize neo-epitopes that are revealed on ischemic cells. The target molecules and mechanisms whereby these neo-epitopes become accessible to recognition are not well understood. Proposing that isolated intestinal epithelial cells (IEC) may carry IR-related neo-epitopes, we used in vitro IEC binding assays to screen hybridomas created from B cells of unmanipulated wild-type C57BL/6 mice. We identified a novel IgM mAb (mAb B4) that reacted with the surface of IEC by flow cytometric analysis and was alone capable of causing complement activation, neutrophil recruitment and intestinal injury in otherwise IR-resistant Rag1(-/-) mice. mAb B4 was found to specifically recognize mouse annexin IV. Preinjection of recombinant annexin IV blocked IR injury in wild-type C57BL/6 mice, demonstrating the requirement for recognition of this protein to develop IR injury in the context of a complex natural Ab repertoire. Humans were also found to exhibit IgM natural Abs that recognize annexin IV. These data in toto identify annexin IV as a key ischemia-related target Ag that is recognized by natural Abs in a pathologic process required in vivo to develop intestinal IR injury.

Enhanced susceptibility to low-dose collagen-induced arthritis in CR1/2-deficient female mice--possible role of estrogen on CR1 expression.

The influence of complement receptor 1 and 2 (CR1/2) was investigated on the susceptibility to low-dose collagen-induced arthritis (CIA) in wild-type (WT) and CR1/2-deficient DBA/1 mice. Significantly enhanced CIA was observed in female CR1/2-deficient mice compared with WT female mice, while male mutant and WT mice showed similar arthritis development. The enhanced CIA was accompanied with higher complement levels and a prolonged IgM anti-collagen type II response. When investigating whether estrogen contributed to the different arthritis susceptibility, we found that ovariectomy rendered WT females more sensitive to low-dose CIA and to the same extent as CR1/2-deficient females, while CR1/2-deficient mice were unaffected by ovariectomy. Notably, the ovariectomized WT mice displayed reduced CR1(+) B220(+) B-cell numbers and CR1 expression compared with sham-operated WT mice, suggesting a stimulatory effect of estrogen on CR1. In accordance, a significant correlation was observed between reduced CR1 expression in B cells and increased age in healthy female blood donors but not in male donors. Our findings demonstrate an important role of CR1/2 in suppressing CIA in female mice under low-antigen conditions. The data suggest that estrogen promote CR1 expression in B cells. These findings provide insight to the increased frequency of rheumatoid arthritis in postmenopausal women.

A unique B2 B cell subset in the intestine.

Over 80% of the body's activated B cells are located in mucosal sites, including the intestine. The intestine contains IgM(+) B cells, but these cells have not been characterized phenotypically or in terms of their developmental origins. We describe a previously unidentified and unique subset of immunoglobulin M(+) B cells that present with an AA4.1(-)CD21(-)CD23(-) major histocompatibility complex class II(bright) surface phenotype and are characterized by a low frequency of somatic hypermutation and the potential ability to produce interleukin-12p70. This B cell subset resides within the normal mucosa of the large intestine and expands in response to inflammation. Some of these intestinal B cells originate from the AA4.1(+) immature B2 cell pool in the steady state and are also recruited from the recirculating naive B cell pool in the context of intestinal inflammation. They develop in an antigen-independent and BAFF-dependent manner in the absence of T cell help. Expansion of these cells can be induced in the absence of the spleen and gut-associated lymphoid tissues. These results describe the existence of an alternative pathway of B cell maturation in the periphery that gives rise to a tissue-specific B cell subset.

Complement receptor CR2/CR1 deficiency protects mice from collagen-induced arthritis and associates with reduced autoantibodies to type II collagen and citrullinated antigens.

Collagen-induced arthritis (CIA), a model of autoimmune inflammatory arthritis, depends upon complement activation and effective B cell responses. To determine the importance of complement receptors CR2/CR1 in CIA, the Cr2-/- genotype was backcrossed onto the DBA/1j strain. CIA was induced by immunization with bovine type II collagen in CFA on days 0 and 21. Cr2-/- mice demonstrated a significantly diminished arthritis severity, decreased antibodies to bovine and murine collagen, and a significant reduction in antibodies to citrullinated antigens. Autoantibodies to citrullinated antigens have been shown to amplify anti-type II collagen passive transfer arthritis. To test the hypothesis that that simple replacement of such antibodies might re-establish severe disease in Cr2-/- mice, monoclonal antibodies to citrullinated antigens were administered to mice during the disease course. Although citrullinated antigens targeted by these antibodies were present within the joints of all mice, addition of these monoclonal antibodies increased disease severity only in Cr2+/+ mice. Taken together, these data suggest that CR2/CR1 are required to develop robust autoimmunity in the CIA model and that amplification of arthritis by antibodies to citrullinated antigens depends on factor(s) absent in arthritic Cr2-/- mice.

Stromal complement receptor CD21/35 facilitates lymphoid prion colonization and pathogenesis.

We have studied the role of CD21/35, which bind derivatives of complement factors C3 and C4, in extraneural prion replication and neuroinvasion. Upon administration of small prion inocula, CD21/35(-/-) mice experienced lower attack rates and delayed disease over both wild-type (WT) mice and mice with combined C3 and C4 deficiencies. Early after inoculation, CD21/35(-/-) spleens were devoid of infectivity. Reciprocal adoptive bone marrow transfers between WT and CD21/35(-/-) mice revealed that protection from prion infection resulted from ablation of stromal, but not hemopoietic, CD21/35. Further adoptive transfer experiments between WT mice and mice devoid of both the cellular prion protein PrP(C) and CD21/35 showed that splenic retention of inoculum depended on stromal CD21/35 expression. Because both PrP(C) and CD21/35 are highly expressed on follicular dendritic cells, CD21/35 appears to be involved in targeting prions to follicular dendritic cells and expediting neuroinvasion following peripheral exposure to prions.

Mice lacking CD21 and CD35 proteins mount effective immune responses against Borrelia burgdorferi infection.

CD21/35(-/-) mice, deficient in CD21 and CD35 (complement receptors 2 and 1, respectively), were infected with Borrelia burgdorferi to assess the role of these receptors in a chronic bacterial infection. Although CD21/35(-/-) mice on both C57BL/6 and BALB/c backgrounds produced less B. burgdorferi-specific antibodies than did wild-type mice, spirochete levels and arthritis severity were similar.

Antibody-independent B cell-intrinsic and -extrinsic roles for CD21/35.

Mice lacking C3, C4 or complement receptor 1/2 (Cr) have defective germinal centers (GC). The requirement for C4 implicates complement fixation by immune complexes (IC) via the classical pathway. Yet, transgenic (Tg) mice that lack circulating antibody but still express membrane IgM (mIgM) have normal GC responses. We showed previously that cross-linking mIgM leads to the deposition of C3 on the B cell surface and that disruption of this pathway diminishes GC responses. Here, we investigate the role of Cr in this process by generating mIgM-Tg mice that lack Cr and serum Ig. These mIgM/Cr-/- mice have smaller, transient GC, with incomplete B cell receptor down-regulation and peanut agglutinin up-regulation, compared to mIgM/Crwt counterparts. BM chimera experiments showed that Cr on B cells is required for normal GC responses. These results establish that Cr ligands generated at the B cell surface are sufficient for normal GC responses and function by signaling Cr on B cells. Unexpectedly, chimera experiments also showed a critical role for Cr on follicular dendritic cells (FDC), even in the absence of IC, indicating novel functions for FDC-expressed Cr beyond the capture of C3-coated IC.

T cell-independent and T cell-dependent immunoglobulin G responses to polyomavirus infection are impaired in complement receptor 2-deficient mice.

Polyomavirus (PyV) infection induces protective T cell-independent (TI) IgM and IgG antibody responses in T cell-deficient mice, but these responses are not generated by immunization with viral proteins or virus like particles. We hypothesized that innate signals contribute to the generation of isotype-switched antiviral antibody responses. We studied the role of complement receptor (CR2) engagement in TI and T cell-dependent (TD) antibody responses to PyV using CR2-deficient mice. Antiviral IgG responses were reduced by 80-40% in CR2-/- mice compared to wild type. Adoptive transfer experiments demonstrated the need for CR2 not only in TD, but also in TI IgG responses to PyV. Transfer of CR2-/- B lymphocytes to SCID mice resulted in TI antiviral IgG responses that corresponded to 10% of that seen in wild-type B cell-reconstituted mice. Thus, our studies revealed a profound dependence of TI and TD antiviral antibody responses on CR2-mediated signals in PyV-infected mice, where the viral antigen is abundant and persistent.

Several genes contribute to the production of autoreactive B and T cells in the murine lupus susceptibility locus Sle1c.

The systemic lupus erythematosus 1 (Sle1) locus mediates the loss of tolerance to nuclear Ags in the NZM2410 mouse model of lupus through intrinsic defects in both B and T cells. Congenic analysis has shown that Sle1 corresponds to at least three genetic loci, Sle1a, Sle1b, and Sle1c. Telomeric Sle1c is associated with abnormal B cell responses to subthreshold stimulation with anti-IgM and C3d and with decreased T-dependent humoral immune responses. We have proposed that these phenotypes resulted from polymorphisms in the C3 complement receptor Cr2 gene. We have also found that Sle1c was associated with the production of histone-specific autoreactive CD4(+) T cells, which correlated with higher activation and proliferative responses, and a reduction in the CD4(+)CD25(+)CD62L(+)forkhead/winged helix transcription factor gene (Foxp3(+)) compartment. In this study we showed, using congenic recombinants, that the decreased humoral immune response and impaired GC formation map to the NZM2410 Cr2 allele. A chronic graft-vs-host disease model also showed that Sle1c produces significantly more autoreactive B cells than B6 controls, and that this phenotype maps to two regions excluding the Cr2 gene. Mixed bone marrow chimera demonstrated that the increased activation, proliferative response, and reduced regulatory T cell compartment were intrinsic to Sle1c-expressing CD4(+) T cells. These phenotypes mapped to the same two loci identified with the chronic graft-vs-host disease model, excluding the Cr2 region. Overall, these results show that Sle1c results in the production of autoreactive B and T cells through the expression of three different genes, one of which is consistent with Cr2, based on the phenotypes of the Cr2-deficient mice, and the other two corresponding to as yet unidentified genes.

B cells from mice prematurely expressing human complement receptor type 2 are unresponsive to T-dependent antigens.

Complement receptor type 2 (CR2/CD21), in association with CD19, plays an important role in enhancing mature B cell responses to opsonized Ags. We have shown that mice expressing a human CR2/CD21 (hCR2/CD21) transgene during the CD43(+)/CD25(-) late pro-B cell stage of B cell development demonstrate marked changes in subsequent B cell ontogeny. In the present study, we show that the humoral immune response to the T cell-dependent Ag, sheep RBC, is muted severely in a manner inversely proportional to B cell expression level of hCR2. Individual Ag-specific IgG isotypes vary in the degree to which they are affected but all are reduced while IgM titers are normal. A substantial reduction in germinal centers, both in size and frequency, in the spleens of immunized hCR2 transgenic mice demonstrates a failure to maintain germinal center reaction. However, both IgM expression levels and LPS-proliferative responses appear fully intact in B cells from hCR2-positive mice, suggesting that this alteration in B cell phenotype is different qualitatively from that of specific Ag-defined anergy models. These data suggest that the unresponsiveness to T-dependent Ags displayed by hCR2-positive B cells is linked to an increase in the level of stimulus required to propel the B cell into a fully activated state and thus a normal humoral immune response to Ags. We conclude that this phenotype and these mice may offer an additional means to dissect mechanisms underlying B cell tolerance and Ag responsiveness both in bone marrow and periphery.

The CD19-CD21 signal transduction complex of B lymphocytes regulates the balance between health and autoimmune disease: systemic sclerosis as a model system.

Cell-surface CD19 functions as a general rheostat for defining intrinsic and antigen receptor-induced signaling thresholds critical for clonal expansion of the B cell pool and humoral immunity. CD19 also governs B cell responses initiated through the CD21 receptor, where complement C3d binding to CD21 links humoral immune responses with the innate immune system. Alterations in this signaling pathway can predispose mice and humans to autoantibody production and systemic autoimmunity. Transgenic mice that overexpress CD19 by 20-170% lose tolerance and generate autoantibodies. Likewise, B cells from CD21-deficient mice overexpress CD19 by approximately 50%, which leads to autoantibody production. Autoimmune patients with systemic sclerosis also overexpress CD19 by approximately 20%, which may contribute to their intrinsic B cell abnormalities and autoantibody production. Thus, chronic B cell activation resulting from augmented CD19 expression or signaling through the CD19 pathway may reveal a prototype autoimmune disease susceptibility pathway in mice and humans.

The virulence function of Streptococcus pneumoniae surface protein A involves inhibition of complement activation and impairment of complement receptor-mediated protection.

Complement is important for elimination of invasive microbes from the host, an action achieved largely through interaction of complement-decorated pathogens with various complement receptors (CR) on phagocytes. Pneumococcal surface protein A (PspA) has been shown to interfere with complement deposition onto pneumococci, but to date the impact of PspA on CR-mediated host defense is unknown. To gauge the contribution of CRs to host defense against pneumococci and to decipher the impact of PspA on CR-dependent host defense, wild-type C57BL/6J mice and mutant mice lacking CR types 1 and 2 (CR1/2(-/-)), CR3 (CR3(-/-)), or CR4 (CR4(-/-)) were challenged with WU2, a PspA(+) capsular serotype 3 pneumococcus, and its PspA(-) mutant JY1119. Pneumococci also were used to challenge factor D-deficient (FD(-/-)), LFA-1-deficient (LFA-1(-/-)), and CD18-deficient (CD18(-/-)) mice. We found that FD(-/-), CR3(-/-), and CR4(-/-) mice had significantly decreased longevity and survival rate upon infection with WU2. In comparison, PspA(-) pneumococci were virulent only in FD(-/-) and CR1/2(-/-) mice. Normal mouse serum supported more C3 deposition on pneumococci than FD(-/-) serum, and more iC3b was deposited onto the PspA(-) than the PspA(+) strain. The combined results confirm earlier conclusions that the alternative pathway of complement activation is indispensable for innate immunity against pneumococcal infection and that PspA interferes with the protective role of the alternative pathway. Our new results suggest that complement receptors CR1/2, CR3, and CR4 all play important roles in host defense against pneumococcal infection.