Requirement for complement in antibody responses is not PNAS PLUS explained by the classic pathway activator IgM Christian Rutemarka, Elisabeth Alicotb, Anna Bergmana, Minghe Mab, Andrew Getahunc, Stephan Ellmerichd, Michael C. Carrollb, and Birgitta Heymana,1 aDepartment of Medical Biochemistry and Microbiology, Uppsala University, BMC, SE-751 23 Uppsala, Sweden; bImmune Disease Institute and Program in Cellular and Molecular Medicine, Children’s Hospital Boston, and Department of Pediatrics, Harvard Medical School, Boston, MA 02115; cDepartment of Immunology, University of Colorado School of Medicine and National Jewish Health, K806, Denver, CO 80206; and dCentre for Amyloidosis and Acute Phase Proteins, Division of Medicine, University College London, Royal Free Campus, London NW3 2PF, United Kingdom AUTHOR SUMMARY Complement is primarily known integrin R1 (SIGN-R1), serum as an immune defense system Classical Lectin Alternative amyloid P component (SAP), that leads to the lysis or ruptur- pathway pathway pathway and C-reactive protein (CRP). ing of pathogens, increased Thus, although C1q is required phagocytosis (i.e., the engulf- C1q MBL/ficolins for antibody responses, none of ment of invading organisms or IgMC´-act C1r MASP-1 C3b the known classic pathway foreign material by white blood SIGN-R1 MASP-2 B activators are. fl C1s C4 cells), and in ammation. How- SAP D The classic pathway is pri- ever, complement also plays C4 C2 marily activated by IgM and IgG CRP C2 P a lesser known but crucial role in C3a antibodies, which acquire the the generation of antibody ability to bind C1q after they responses to many antigens (1). C3 C3b iC3b form an immune complex with Complement can be activated by their specific antigen. In naive IMMUNOLOGY three different routes: the classic mice, very little specific antibody pathway, the alternative path- C5 C3d C3d,g is present; therefore, immune way, and the lectin pathway. All C6-C9 complex formation is pre- these pathways lead to the (MAC) sumably rare. Thus, it appears cleavage of a protein, known as paradoxical that the activation C3, into various products, some B of the classic pathway would be of which are the ligands (i.e., CR1/2 FDC crucial for primary antibody free-moving molecules that bind responses. A possible explana- to receptors and thereby activate Fig. P1. Complement system and its role in antibody responses. tion has been suggested by two or suppress different cellular Complement can be activated via three pathways, with each resulting sets of observations. First, spe- pathways) for complement in the formation of a membrane attack complex (MAC). Lack of cific IgM administered with an receptors 1 and 2 (CR1/2) (Fig. complement factors C1q, C2, C4, or C3, or CR1/2 [i.e., receptors for the antigen enhances the antibody P1). Animals lacking comple- C3 products C3b, iC3b, C3dg, and C3d, expressed on B cells and response to that antigen; more- ment factors C1q, C2, C3, or C4, follicular dendritic cells (FDCs)], leads to severe impairment of antibody over, the enhancement is de- or CR1/2, but not mannan- responses (marked red). In contrast, lack of MBL-A and MBL-C of the pendent on the ability of the binding lectin (MBL), factor B, lectin pathway, factor B of the alternative pathway, or factor C5 is IgM molecule to activate com- compatible with normal antibody responses (marked green). Because or C5 have severely impaired the lack of C1q abolishes the classic pathway alone, the observations plement (2, 3). Second, mice primary and secondary antibody show that this pathway is crucial for antibody responses. Here, we lacking secretory IgM have responses; that is, both the ini- show that antibody responses are normal in mice lacking factors lower antibody responses than tial antibody response and the known to initiate the classic pathway: complement-activating IgM WT mice, and the responses can long-term one are impaired. In (IgMC′-act), SIGN-R1, SAP, or CRP (marked green). be restored by the transfer of general, IgG responses (i.e., IgM from normal mouse serum secondary antibody responses) are more affected than IgM ones (4, 5). These findings suggested that natural IgM likely forms (i.e., initial antibody responses). C1q is involved in the classic an immune complex with the antigen in naive mice (albeit with pathway, whereas MBL and factor B are important to the lectin inefficient binding), activates complement, and initiates the pathway and the alternative pathway, respectively. Hence, these classic pathway. Subsequently, early specific IgM formation observations imply that activation of the classic pathway is cru- would further up-regulate the antibody response. We have cial for antibody responses. This is an apparent paradox because sought to test this specific explanation by ascertaining whether (i) the classic pathway is activated mainly by antibodies, which the ability of natural IgM to activate complement is a pre- form a complex with the antigen, and (ii) presumably, extremely low amounts of specific antibodies are present in a naive animal (i.e., when the primary response is initiated). A possible expla- Author contributions: C.R., E.A., A.B., M.M., M.C.C., and B.H. designed research; C.R., E.A., nation is that the classic pathway is initiated by natural IgM, A.B., M.M., A.G., and S.E. performed research; S.E. contributed new reagents/analytic present also in naive mice. To test this, we generated a mouse tools; C.R., E.A., A.B., M.M., M.C.C., and B.H. analyzed data; and C.R., A.B., M.C.C., and strain, Cμ13, in which IgM cannot activate complement owing to B.H. wrote the paper. a small genetic change (a point mutation) in a gene encoding The authors declare no conflict of interest. a particular IgM region. Unexpectedly, the antibody responses in This article is a PNAS Direct Submission. these animals were normal. Moreover, antibody responses were 1To whom correspondence should be addressed. E-mail: [email protected]. normal in mice lacking three other activators of the classic See full research article on page E934 of www.pnas.org. pathway: specific intracellular adhesion molecule-grabbing non- Cite this Author Summary as: PNAS 10.1073/pnas.1109831108. www.pnas.org/cgi/doi/10.1073/pnas.1109831108 PNAS | October 25, 2011 | vol. 108 | no. 43 | 17589–17590 Downloaded by guest on September 26, 2021 requisite for normal antibody responses. For this purpose, we is unlikely that IgG-mediated classic pathway activation explains generated the Cμ13 mice, which carry a point mutation that the positive effect of complement on antibody responses. How- renders IgM unable to activate complement. The mutation is ever, three other endogenous activators of the classic pathway identical to the one in a previous study (2). For confirming have been described: SIGN-R1, SAP, and CRP. We therefore that IgM from Cμ13 mice did not activate complement, the immunized animals lacking any one of these components with number of single B cells producing an antibody to sheep red SRBCs and tested their sera in ELISAs. As before, the IgG anti- blood cells (SRBCs), IgM anti-SRBC, was tested with two sen- SRBC responses were normal. sitive assays: the direct hemolytic plaque-forming cell (PFC) We conclude that classic pathway activation but none of the assay, which only detects B cells producing complement-acti- activators (IgM, SIGN-R1, CRP, and SAP) are required for vating IgM, and the enzyme-linked immunospot (ELISPOT) antibody responses. This is particularly interesting, because assay, which detects all IgM-producing B cells. When Cμ13 mice complement activation by natural IgM has been thought to ex- were immunized with a high dose of SRBCs, they produced plain the requirement for classic pathway activation in primary ∼70,000 ELISPOTs per spleen but only background numbers of antibody responses. These observations imply that spontaneous PFCs (115 per spleen). Thus, Cμ13 B cells were able to produce C1 cleavage is sufficient, an unknown factor is involved, or the IgM anti-SRBC, but this IgM could not activate complement. factors tested here act redundantly. Autoimmune diseases, such Having established this, we asked the crucial question of whether as systemic lupus erythematosus, are associated with deficiencies Cμ13 mice had an impaired antibody response to antigens known in complement components. Therefore, understanding how to depend on a functioning complement system. Cμ13 and WT complement regulates immune responses may have implications controls were immunized with various doses of SRBCs or key- for the understanding of autoimmunity. hole limpet hemocyanin (a large protein antigen). As determined by the sensitive ELISA, primary IgG and IgM and secondary IgG 1. Carroll MC (2004) The complement system in regulation of adaptive immunity. Nat responses were normal in Cμ13 mice. In parallel, antibody Immunol 5:981–986. − − responses in mice lacking C1q (C1qA / ) were tested. As 2. Heyman B, Pilström L, Shulman MJ (1988) Complement activation is required for IgM- mediated enhancement of the antibody response. J Exp Med 167:1999–2004. expected, these responses were severely impaired. Thus, al- 3. Youd ME, Ferguson AR, Corley RB (2002) Synergistic roles of IgM and complement in though secreted IgM (4, 5) and classic pathway activation are antigen trapping and follicular localization. Eur J Immunol 32:2328–2337. both required for the normal primary antibody response, this 4. Ehrenstein MR, O’Keefe TL, Davies SL, Neuberger MS (1998) Targeted gene disruption does not require that IgM activate complement. These obser- reveals a role for natural secretory IgM in the maturation of the primary immune – vations suggested that something other than IgM initiates the response. Proc Natl Acad Sci USA 95:10089 10093. 5. Baumgarth N, et al. (2000) B-1 and B-2 cell-derived immunoglobulin M antibodies are classic pathway.