The absence of tumor necrosis factor (TNF) causes lethal infection by Leishmania major in normally resistant C57BL/6J (B6.WT) mice. The underlying pathogenic mechanism of this fatal disease has so far remained elusive. We found that B6.WT mice deficient for the tnf gene (B6.TNF−/−) displayed not only a non-healing cutaneous lesion but also a serious infection of the liver upon L. major inoculation. Infected B6.TNF−/− mice developed an enlarged liver that showed increased inflammation. Furthermore, we detected an accumulating monocyte-derived macrophage population (CD45+F4/80+CD11bhiLy6Clow) that displayed a M2 macrophage phenotype with high expression of CD206, arginase-1, and IL-6, supporting the notion that IL-6 could be involved in M2 differentiation. In in vitro experiments, we demonstrated that IL-6 upregulated M-CSF receptor expression and skewed monocyte differentiation from dendritic cells to macrophages. This was countered by the addition of TNF. Furthermore, TNF interfered with the activation of IL-6-induced gp130-signal transducer and activator of transcription (STAT) 3 and IL-4-STAT6 signaling, thereby abrogating IL-6-facilitated M2 macrophage polarization. Therefore, our results support the notion of a general role of TNF in the inflammatory activation of macrophages and define a new role of IL-6 signaling in macrophage polarization downstream of TNF.

Specialized roles for the pro-inflammatory cytokines tumor necrosis factor (TNF) and lymphotoxin (LT) were characterized in TNF/LT alpha -/- and TNF -/- mice established by direct gene targeting of C57BL/6 ES cells. The requirement for LT early in lymphoid tissue organogenesis is shown to be distinct from the more subtle and varied role of TNF in promoting correct microarchitectural organization of leukocytes in LN and spleen. Development of normal Peyer's patch (PP) structure, in contrast, is substantially dependent on TNF. Only mice lacking LT exhibit retarded B cell maturation in vivo and serum immunoglobulin deficiencies. A temporal hierarchy in lymphoid tissue development can now be defined, with LT being an essential participant in general lymphoid tissue organogenesis, developmentally preceeding TNF that has a more varied and subtle role in promotion of correct spatial organization of leukocytes in LN and spleen PP development in TNF -/- mice is unusual, indicating that TNF is a more critical participant for this structure than it is for other lymphoid tissues.

Mice without secreted TNF but with functional, normally regulated and expressed membrane-bound TNF (memTNF(Delta/Delta) mice) were created by knocking-in the uncleavable Delta 1-9,K11E TNF allele. In contrast to TNF-deficient mice (TNF(-/-)), memTNF supported many features of lymphoid organ structure, except generation of primary B cell follicles. Splenic chemokine expression was near normal. MemTNF-induced apoptosis was mediated through both TNF-R1 and TNF-R2. That memTNF is suboptimal for development of inflammation was revealed in experimental autoimmune encephalomyelitis. Disease severity was reduced in memTNF(Delta/Delta) mice relative to wild-type mice, and the nature of spinal cord infiltrates resembled that in TNF(-/-) mice. We conclude that memTNF supports many processes underlying lymphoid tissue structure, but secreted TNF is needed for optimal inflammatory lesion development.

The resolution of infections with the protozoan parasite Leishmania major in mice requires a Th1 response that is closely associated with the expression of IL-12, IFN-γ, and inducible NO synthase. Previous Ab neutralization studies or the use of mice deficient for both TNF receptors suggested that TNF plays only a limited role in the control of parasite replication in vivo. In this study we demonstrate that resistant C57BL/6 (B6.WT) mice locally infected with L. major rapidly succumb to progressive visceral leishmaniasis after deletion of the TNF gene by homologous recombination. A reduction of the parasite inoculum to 3000 promastigotes did not prevent the fatal outcome of the disease. An influence of the altered morphology of secondary lymphoid organs in C57BL/6-TNF−/− (B6.TNF−/−) mice on the course of disease could be excluded by the generation of reciprocal bone marrow chimeras. Although infected B6.TNF−/− mice mounted an L. major-specific IFN-γ response and expressed IL-12, the onset of the immune reaction was delayed. After in vitro stimulation, B6.TNF−/− inflammatory macrophages released 10-fold less NO in response to IFN-γ than B6.WT cells. However, in the presence of a costimulus, e.g., L. major infection or LPS, the production of NO by B6.WT and B6.TNF−/− macrophages was comparable. In vivo, inducible NO synthase protein was readily detectable in skin lesions and draining lymph nodes of B6.TNF−/− mice, but its expression was more disperse and less focal in the absence of TNF. These are the first data to demonstrate that TNF is essential for the in vivo control of L. major.

Although chemokines are sufficient for chemotaxis of various cells, increasing evidence exists for their fine-tuning by selective proteolytic processing. Using a model of immune cell chemotaxis into the CNS (experimental autoimmune encephalomyelitis [EAE]) that permits precise localization of immigrating leukocytes at the blood-brain barrier, we show that, whereas chemokines are required for leukocyte migration into the CNS, additional MMP-2/9 activities specifically at the border of the CNS parenchyma strongly enhance this transmigration process. Cytokines derived from infiltrating leukocytes regulate MMP-2/9 activity at the parenchymal border, which in turn promotes astrocyte secretion of chemokines and differentially modulates the activity of different chemokines at the CNS border, thereby promoting leukocyte migration out of the cuff. Hence, cytokines, chemokines, and cytokine-induced MMP-2/9 activity specifically at the inflammatory border collectively act to accelerate leukocyte chemotaxis across the parenchymal border.