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Molecular Immunopathology and Pathophysiology of Inflammatory Injury

L. Feng, Y. Xia, S. Chen, G.E. Garcia, R.C. Blantz,* T. Yamamoto,** C.B. Wilson

* University of California, San Diego, and Veterans Affairs Medical Center, San Diego, CA
** Niigata University School of Medicine, Niigata, Japan

Recruitment of leukocytes is central to the development of inflammation, and numerous chemokines act as chemoattractants for different types of leukocytes through selective receptors. The members of 4 chemokine families, CXC, CC, C, and CX₃C, may interact with more than 1 receptor, and leukocytes may express multiple receptors, creating a potential redundancy that makes therapeutic manipulation a challenge. To address this challenge, we are using in vitro and in vivo studies.

Crescentic and progressive glomerulonephritis induced in WKY rats by antibodies to glomerular basement membrane is a useful in vivo model for the study of lymphocyte and monocyte/macrophage chemoattractants. The development of glomerulonephritis in this model depends on CD8⁺ lymphocytes, and ED-1⁺ macrophages and, to a lesser extent, natural killer cells are major mediators. The lymphocyte and macrophage chemoattractants MCP-1, MIP-1ß, RANTES, lymphotactin, and fractalkine are dramatically induced within 3 days in this model, as are CC chemokine receptors CCR2, CCR5, and CX₃CR1. CX₃CR1 is the receptor for the newly described CX₃C chemokine, fractalkine, which, unlike other chemokines, has a mucinlike stalk with transmembrane and cytoplasmic domains. Leukocytes recovered from nephritic glomeruli in the rat model migrate in response to MCP-1, MIP-1ß, RANTES, and fractalkine in an in vitro chemotaxis assay, indicating the relevance of the model for study.

Fractalkine is expressed predominantly on endothelial cells and mediates both chemotaxis and adhesion of mononuclear cells. IL-1ß, TNF-, lipopolysaccharide, and contact with macrophages induce expression of fractalkine in rat glomerular endothelial cells and rat aortic endothelial cells in a time-dependent manner. The induction correlates with a rapid induction of NF-B--binding activity. An adenovirus vector carrying IM, which is a dominant negative mutant of IB and a potent and specific inhibitor of NF-B, blocked the fractalkine induction, suggesting an essential role of NF-B.

The presence of fractalkine on endothelial cells should be advantageous for attraction and adhesion of CX₃CR1-bearing cells in the rapid glomerular blood flow. Fractalkine, which is largely absent from normal glomerular endothelium, was markedly induced in nephritic glomerular endothelium in the WKY model. Leukocytes recovered from the nephritic glomeruli had higher levels of CX₃CR1 on their surface than did peritoneal macrophages and migrated to fractalkine in an in vitro chemotaxis assay. The in vitro chemotaxis was inhibited by antibodies to CX₃CR1. Treatment with these antibodies blocked glomerular leukocyte infiltration in the WKY model more than did treatment with the chemokine analog vMIP-II, preventing crescent formation and attenuating proteinuria. As controls, normal rabbit serum had no effect, and antibodies to CCR5 were only partially effective in reducing infiltrates. The upregulation of both fractalkine and its receptor, CX₃CR1, appears to be central to cellular infiltration in this model, reflecting the probable role of this membrane-anchored chemokine and its receptor in both chemotaxis and adhesion of leukocytes in the high-flow glomerular circuit.

Certain viruses can encode and synthesize proteins that interfere with the normal host defense response, and vMIP-II, encoded by human herpesvirus 8, has a broad spectrum of in vitro antagonist activity against chemokines. vMIP-II potently inhibited chemotaxis of leukocytes isolated from nephritic WKY glomeruli. In vivo, vMIP-II treatment in nephritic WKY rats significantly inhibited leukocyte infiltration to glomeruli, attenuated crescent formation, and markedly reduced proteinuria. These studies are the first example of using viral antagonists of the host defense system to treat pathologic conditions.

Fractalkine and its receptor present in the brain of rats were characterized to gain insights into use of chemokine-dependent systems in the CNS. Expression of fractalkine was widespread and localized principally to neurons. Recombinant rat CX₃CR1 specifically bound fractalkine and signaled the presence of either membrane-anchored or soluble forms of fractalkine protein. Fractalkine also stimulated chemotaxis and elevated intracellular calcium levels of microglia; these responses were blocked by antibodies to CX₃CR1. After facial motor nerve axotomy, dramatic changes in the levels of CX₃CR1 and fractalkine in the facial nucleus were evident. These data describe unique mechanisms of cellular communication between neurons and microglia involving fractalkine and CX₃CR1 that occur in both normal and pathologic states of the CNS.

To compare inhibition of a chemokine with inhibition of its receptor, we studied MCP-1. Expression of MCP-1 and its receptor, CCR2, correlated with macrophage accumulation in the WKY model. Daily treatment with neutralizing antibodies to MCP-1 caused only a 20% reduction in macrophage infiltration on day 10. In contrast, an antagonist for CCR2 caused a 60% reduction, probably by blocking not only MCP-1 but also MCP-2, MCP-3, and MCP-4. The MCP-1 receptor antagonist was generated by truncating 8 amino acids at the amino-terminus of MCP-1; the truncated molecule binds to CCR2 but does not elicit chemotaxis or calcium mobility of CCR2-bearing cells or macrophages. In contrast, a control MCP-1 analog, which cannot bind to CCR2, had no effect on macrophage accumulation in this model. The study indicates the enhanced usefulness of therapy directed at chemokine receptors rather than at individual chemokines themselves.

The enhanced expression of lymphotactin, the only C chemokine, in the WKY model correlated with lymphocytic infiltration on day 3. Using in situ hybridization immunohistochemistry, we detected lymphotactin in CD8⁺ cells but not in ED-1⁺ macrophages. In the WKY model, we compared daily treatment with antibodies to lymphotactin with treatment with normal rabbit serum or with antibodies to CCR5, a chemokine receptor (for RANTES, MIP-1, and MIP-1ß) also induced in this model. We found, unexpectedly, that antibodies to lymphotactin enhanced glomerular injury, with an increase in infiltration of both CD8⁺ and ED-1⁺ cells. The increased infiltration possibly was due to upregulation of CC and CX3C chemokines, including MCP-1, RANTES, MIP-1, MIP-1ß, and fractalkine, in the rats treated with antibodies to lymphotactin. The detrimental effect was specific; antibodies to CCR5 prepared in the same way had a partially protective effect.

IFN---inducing factor induces proliferation of T cells and enhances the cytolytic activity of natural killer cells. Active recombinant rat IFN---inducing factor expressed and administered in the WKY model increased expression of IFN- in glomeruli by 2.4-fold. Proteinuria and crescentic glomeruli were also dramatically increased.

FTY720, a novel immunosuppressant, has a dramatic effect on lymphocyte homing. To examine whether altered lymphocyte homing could affect immune-mediated local inflammation, we used BN rats with immunologically induced tubulointerstitial nephritis. The rats were given FTY720 or the drug vehicle as a control and were examined on day 11 during the peak infiltration of inflammatory cells. Compared with the drug vehicle, FTY720 reduced monocytic infiltration. We found that treatment with FTY720 decreased the mRNA for chemokines, cytokines, and laminin and fibronectin in the extracellular matrix. Our data suggest that in addition to its direct effect on immune mediation, including lymphocyte homing, FTY720 may decrease production of chemokines, cytokines, and the extracellular matrix.

The NF-B/Rel family plays a central role in the regulation of many proinflammatory and immune responses. In most unstimulated cells, most of the Rel/NK-B factors are present in the cytoplasm as inactive complexes in association with inhibitory proteins termed IBs. Mice deficient in the gene relB have a multiorgan inflammatory syndrome consisting of infiltrations of lymphocytes, neutrophils, and macrophages. Renal fibroblasts obtained from relB-/- mice overexpressed IL-1, IL-1ß, and TNF- when stimulated with lipopolysaccharide. In contrast, peritoneal macrophages had relatively impaired expression of the cytokines after lipopolysaccharide stimulation.

Stimulation with lipopolysaccharide downregulated IB protein because of accelerated degradation of the protein in relB-/- fibroblasts, although NF-B--inducible IB mRNA was upregulated as expected. NF-B--binding activity was increased in lipopolysaccharide-stimulated relB-/- fibroblasts, a condition that led to cytokine overexpression. The overexpression could be blocked by introducing a dominant negative mutant of IB into relB-/- fibroblasts. In relB+/+ fibroblasts, lipopolysaccharide-induced RelA nuclear translocation did not lead to increased B-binding activity. The results suggest that RelB might be involved directly or indirectly in stabilizing IB protein within the nuclei of renal fibroblasts and may inhibit NF-B--dependent gene expression. RelB may function as a limiting factor in the NF-B--IB autoregulatory loop in renal fibroblasts.

PUBLICATIONS

Chen, S., Bacon, K.B., Li, L., Garcia, G.E., Xia, Y., Lo, D., Thompson, D.A., Siani, M.A., Yamamoto, T., Harrison, J.K., Feng, L. In vivo inhibition of CC and CX₃C chemokine-induced leukocyte infiltration and attenuation of glomerulonephritis in Wistar-Kyoto (WKY) rats by vMIP-II. J. Exp. Med. 188:193, 1998.

Feng, L., Jang, B.C., Hwang, D. Inhibitor of protein tyrosine kinase, radicicol, suppresses the expression of cyclooxygenase and pro-inflammatory cytokines in LPS-stimulated rat alveolar macrophages in part by accelerating degradation of mRNA. Adv. Exp. Med. Biol. 407:281, 1997.

Harrison, J.K., Jiang, Y., Chen, S., Xia, Y., Maciejewski, D., McNamara, R.K., Streit, W.J., Salafranca, M.N., Adhikari, S., Thompson, D.A., Botti, P., Bacon, K.B., Feng, L. Role for neuronally derived fractalkine in mediating interactions between neurons and CX₃CR1-expressing microglia. Proc. Natl. Acad. Sci. U.S.A., in press.

Hirose, S., Yamamoto, T., Feng, L., Yaoita, E., Kawasaki, K., Goto, S., Fujinaka, H., Wilson, C.B., Arakawa, M., Kihara, I. Expression and localization of cyclooxygenase isoforms and cytosolic phospholipase A2 in anti-Thy-1 glomerulonephritis. J. Am. Soc. Nephrol. 9:408, 1998.

Jiang, Y., Gram, H., Zhao, M., New, L., Gu, J., Feng, L., De Padova, F., Ulevitch, R.J., Han, J. Characterization of the structure and function of the fourth member of p38 group mitogen-activated protein kinases-p38. J. Biol. Chem. 272:30122, 1997.

Jiang, Y., Salafranca, M.N., Adhikari, S., Xia, Y., Feng, L., Sonntag, M.K., deFiebre, C.M., Pennell, N.A., Streit, W.J., Harrison, J.K. Chemokine receptor expression in cultured glia and rat experimental allergic encephalomyelitis. J. Neuroimmunol. 86:1, 1998.

Matsukawa, A., Miyazaki, S., Maeda, T., Tanase, S., Feng, L., Ohkawara, S., Yoshinaga, M., Yoshimura, T. Production and regulation of MCP-1 in lipopolysaccharide- or monosodium urate crystal-induced arthritis in rabbits: Roles of TNF, IL-1 and IL-8. Lab. Invest., in press.

Okura, Y., Yamamoto, T., Goto, S., Inomata, T., Hirono, S., Hanawa, H., Feng, L., Wilson, C.B., Kihara, I., Izumi, T., Shibata, A., Aizawa, Y., Seki, S., Abo, T. Characterization of cytokine and iNOS mRNA expression in situ during the course of experimental autoimmune myocarditis in rats. J. Mol. Cell. Cardiol. 29:491, 1997.

Schwartz, D., Mendonca, M., Schwartz, I., Xia, Y., Satriano, J., Wilson, C.B., Blantz, R.C. Inhibition of constitutive nitric oxide synthase (NOS) by nitric oxide generated by inducible NOS after lipopolysaccharide administration provokes renal dysfunction in rats. J. Clin. Invest. 100:439, 1997.

Sugisaki, K., Dannenberg, A.M., Jr., Abe, Y., Tsuruta, J., Su, W.-J., Said, W., Feng, L., Yoshimura, T., Converse, P.J., Mounts, P. Nonspecific and immune-specific up-regulation of cytokines in rabbit dermal tuberculous (BCG) lesions. J. Leukoc. Biol. 63:440, 1998.