During infections with Streptococcus pneumoniae (Pn), as well as other extracellular bacteria, the immune system likely encounters a variety of microbial components in soluble form, as well as those associated with the intact bacterium (1, 2). Thus, secreted hydrolases such as hyaluronidases, neuraminidases, and endoglycosidases can mediate bacterial spread and destruction of host tissue through degradation of hyaluronan, mucins, and glycolipids. In addition, during the stationary growth phase, Pn expresses a major autolysin (LytA amidase) which degrades it's own peptidoglycan cell wall, resulting in release of cytoplasmic proteins (3, 4). One such protein is pneumolysin that can induce host cell injury through formation of cell membrane pores (5), and at lower concentrations stimulate release of pro-inflammatory mediators (6) and directly accelerate cell death of neutrophils (7), the major phagocytic cell that mediates innate immunity to extracellular bacteria. In addition, since both capsular polysaccharide (PS) and a number of proteins are covalently attached to the bacterial cell wall peptidoglycan (8, 9), the release of soluble PS-protein conjugates upon bacterial lysis is also likely.
Adaptive immunity to extracellular bacteria is largely mediated by antibody. Although soluble and particulate antigens may exhibit distinct immunologic properties (10-12), their potential cross-regulatory effects on the humoral immune response, following concomitant immunization, as might occur during bacterial infections, is unknown. In particular, the context in which the antigen is expressed may impact on the manner in which it is transported and/or processed within the secondary lymphoid organ. This, in turn may significantly impact on the quality and quantity of the subsequent immune response. The size of the immunogen (13-15), its soluble or particulate nature (16, 17), the valency (18-20) and biochemical nature of the antigenic epitope (21), and the presence of associated innate immune cell activators, such as TLRs (22, 23), and mediators of cellular uptake, such as scavenger receptor ligands (24-26), in turn can influence the outcome of these processes.
We investigated the immunologic consequences of co-immunization with intact Pn and soluble conjugates of Pn-derived proteins and polysaccharides (PS), as a model (27). Co-immunization of mice i.p. with Pn and conjugate resulted in marked inhibition of conjugate-induced PS-specific memory, and primary and memory anti-protein Ig responses. Inhibition occurred with unencapsulated Pn, encapsulated Pn expressing different capsular types of PS than that present in the conjugate, and with conjugate containing protein not expressed by Pn, but not with 1 mm latex beads in adjuvant. Inhibition was long-lasting, occurred only during the early phase of the immune response, but was not associated with tolerance. Pn inhibited the trafficking of conjugate from the splenic marginal zone to the B cell follicle and T cell area, strongly suggesting a potential mechanism for inhibition. These data suggest that during infection, bacterial-associated antigens are the preferential immunogen for anti-bacterial Ig responses.
We will determine whether local s.c. co-immunization with Pn and soluble antigens trafficking to the draining lymph node results in the same type of inhibition as that observed via systemic co-immunization to the spleen. The ability of other intact bacteria, besides Pn to mediate inhibition, and whether inhibition occurs for both small (~< 70kDa) antigens trafficking through the conduit system and large antigens requiring cell transport will be determined. Most importantly, the mechanism of this inhibition will be explored, including the potential ability of Pn to block cell binding, uptake, and/or trafficking of soluble antigens to sites within the lymphoid compartment critical for elicitation of T cell-dependent Ig responses. Use of both flow cytometric analysis and confocal microscopy to accomplish these goals will be employed. Finally, the potential of Pn to inhibit CD4+ T cell activation in response to soluble antigens, will be determined using a transgenic CD4+ T cell approach.
- 1. Jedrzejas, M. J. 2004. Extracellular virulence factors of Streptococcus pneumoniae. Front Biosci 9:891-914.
- 2. Rigden, D. J., M. Y. Galperin, and M. J. Jedrzejas. 2003. Analysis of structure and function of putative surface-exposed proteins encoded in the Streptococcus pneumoniae genome: a bioinformatics-based approach to vaccine and drug design. Crit Rev Biochem Mol Biol 38:143-168.
- 3. Lopez, R., J. L. Garcia, E. Garcia, C. Ronda, and P. Garcia. 1992. Structural analysis and biological significance of the cell wall lytic enzymes of Streptococcus pneumoniae and its bacteriophage. FEMS Microbiol Lett 79:439-447.
- 4. Ronda, C., J. L. Garcia, E. Garcia, J. M. Sanchez-Puelles, and R. Lopez. 1987. Biological role of the pneumococcal amidase. Cloning of the lytA gene in Streptococcus pneumoniae. Eur J Biochem 164:621-624.
- 5. Boulnois, G. J., J. C. Paton, T. J. Mitchell, and P. W. Andrew. 1991. Structure and function of pneumolysin, the multifunctional, thiol-activated toxin of Streptococcus pneumoniae. Mol Microbiol 5:2611-2616.
- 6. Houldsworth, S., P. W. Andrew, and T. J. Mitchell. 1994. Pneumolysin stimulates production of tumor necrosis factor alpha and interleukin-1 beta by human mononuclear phagocytes. Infect Immun 62:1501-1503.
- 7. Martner, A., C. Dahlgren, J. C. Paton, and A. E. Wold. 2008. Pneumolysin released during Streptococcus pneumoniae autolysis is a potent activator of intracellular oxygen radical production in neutrophils. Infect Immun 76:4079-4087.
- 8. Navarre, W. W., and O. Schneewind. 1999. Surface proteins of gram-positive bacteria and mechanisms of their targeting to the cell wall envelope. Microbiol Mol Biol Rev 63:174-229.
- 9. Sorensen, U. B., J. Henrichsen, H. C. Chen, and S. C. Szu. 1990. Covalent linkage between the capsular polysaccharide and the cell wall peptidoglycan of Streptococcus pneumoniae revealed by immunochemical methods. Microb Pathog 8:325-334.
- 10. Kovacsovics-Bankowski, M., K. Clark, B. Benacerraf, and K. L. Rock. 1993. Efficient major histocompatibility complex class I presentation of exogenous antigen upon phagocytosis by macrophages. Proc Natl Acad Sci U S A 90:4942-4946.
- 11. Guermonprez, P., J. Valladeau, L. Zitvogel, C. Thery, and S. Amigorena. 2002. Antigen presentation and T cell stimulation by dendritic cells. Annu Rev Immunol 20:621-667.
- 12. Ziegler, H. K., C. A. Orlin, and C. W. Cluff. 1987. Differential requirements for the processing and presentation of soluble and particulate bacterial antigens by macrophages. Eur J Immunol 17:1287-1296.
- 13. Gretz, J. E., C. C. Norbury, A. O. Anderson, A. E. Proudfoot, and S. Shaw. 2000. Lymph-borne chemokines and other low molecular weight molecules reach high endothelial venules via specialized conduits while a functional barrier limits access to the lymphocyte microenvironments in lymph node cortex. J Exp Med 192:1425-1440.
- 14. Sixt, M., N. Kanazawa, M. Selg, T. Samson, G. Roos, D. P. Reinhardt, R. Pabst, M. B. Lutz, and L. Sorokin. 2005. The conduit system transports soluble antigens from the afferent lymph to resident dendritic cells in the T cell area of the lymph node. Immunity 22:19-29.
- 15. Nolte, M. A., J. A. Belien, I. Schadee-Eestermans, W. Jansen, W. W. Unger, N. van Rooijen, G. Kraal, and R. E. Mebius. 2003. A conduit system distributes chemokines and small blood-borne molecules through the splenic white pulp. J Exp Med 198:505-512.
- 16. Vidard, L., M. Kovacsovics-Bankowski, S. K. Kraeft, L. B. Chen, B. Benacerraf, and K. L. Rock. 1996. Analysis of MHC class II presentation of particulate antigens of B lymphocytes. J Immunol 156:2809-2818.
- 17. Nayak, J. V., D. A. Hokey, A. Larregina, Y. He, R. D. Salter, S. C. Watkins, and L. D. Falo, Jr. 2006. Phagocytosis induces lysosome remodeling and regulated presentation of particulate antigens by activated dendritic cells. J Immunol 177:8493-8503.
- 18. Thyagarajan, R., N. Arunkumar, and W. Song. 2003. Polyvalent antigens stabilize B cell antigen receptor surface signaling microdomains. J Immunol 170:6099-6106.
- 19. Snapper, C. M., M. R. Kehry, B. E. Castle, and J. J. Mond. 1995. Multivalent, but not divalent, antigen receptor cross-linkers synergize with CD40 ligand for induction of Ig synthesis and class switching in normal murine B cells. J. Immunol. 154:1177-1187.
- 20. Snapper, C. M., and J. J. Mond. 1996. A model for induction of T cell-independent humoral immunity in response to polysaccharide antigens. J Immunol 157:2229-2233.
- 21. Mond, J. J., A. Lees, and C. M. Snapper. 1995. T cell-independent antigens type 2. Annu Rev Immunol 13:655-692.
- 22. Blander, J. M., and R. Medzhitov. 2006. Toll-dependent selection of microbial antigens for presentation by dendritic cells. Nature 440:808-812.
- 23. Barton, G. M., and R. Medzhitov. 2002. Control of adaptive immune responses by Toll-like receptors. Curr Opin Immunol 14:380-383.
- 24. Kang, Y. S., J. Y. Kim, S. A. Bruening, M. Pack, A. Charalambous, A. Pritsker, T. M. Moran, J. M. Loeffler, R. M. Steinman, and C. G. Park. 2004. The C-type lectin SIGN-R1 mediates uptake of the capsular polysaccharide of Streptococcus pneumoniae in the marginal zone of mouse spleen. Proc Natl Acad Sci U S A 101:215-220.
- 25. Lanoue, A., M. R. Clatworthy, P. Smith, S. Green, M. J. Townsend, H. E. Jolin, K. G. Smith, P. G. Fallon, and A. N. McKenzie. 2004. SIGN-R1 contributes to protection against lethal pneumococcal infection in mice. J Exp Med 200:1383-1393.
- 26. Arredouani, M., Z. Yang, Y. Ning, G. Qin, R. Soininen, K. Tryggvason, and L. Kobzik. 2004. The scavenger receptor MARCO is required for lung defense against pneumococcal pneumonia and inhaled particles. J Exp Med 200:267-272.
- 27. Chattopadhyay, G., Q. Chen, J. Colino, A. Lees, and C. M. Snapper. 2009. Intact bacteria inhibit the induction of humoral immune responses to bacterial-derived and heterologous soluble T cell-dependent antigens. J Immunol 182:2011-2019.