Stephen T. Smiley

Stat6 Is Required for Mediating Responses to IL-4 and for the Development of Th2 Cells

Interleukin-4 (IL-4) stimulation of cells leads to the activation of multiple signaling pathways, one of which involves Stat6. We have generated Stat6-deficient mice by gene targeting in embryonic stem cells to determine the role of this transcription factor in mediating the biologic functions of IL-4. IL-4-induced increases in the cell surface expression of both MHC class II antigens and IL-4 receptor are completely abrogated, and lymphocytes from Stat6-deficient animals fail to proliferate in response to IL-4. Stat6-deficient B cells do not produce IgE following in vivo immunization with anti-IgD. In addition, Stat6-deficient T lymphocytes fail to differentiate into Th2 cells in response to either IL-4 or Il-13. These results demonstrate that, despite the existence of multiple signaling pathways activated by IL-4, Stat6 is essential for mediating responses to IL-4 lymphocytes.

Fibrinogen Stimulates Macrophage Chemokine Secretion Through Toll-Like Receptor 4

Extravascular fibrin deposition is an early and persistent hallmark of inflammatory responses. Fibrin is generated from plasma-derived fibrinogen, which escapes the vasculature in response to endothelial cell retraction at sites of inflammation. Our ongoing efforts to define the physiologic functions of extravasated fibrin(ogen) have led to the discovery, reported here, that fibrinogen stimulates macrophage chemokine secretion. Differential mRNA expression analysis and RNase protection assays revealed that macrophage inflammatory protein-1α (MIP-1α), MIP-1β, MIP-2, and monocyte chemoattractant protein-1 are fibrinogen inducible in the RAW264.7 mouse macrophage-like cell line, and ELISA confirmed that both RAW264.7 cells and primary murine thioglycolate-elicited peritoneal macrophages up-regulate the secretion of monocyte chemoattractant protein-1 >100-fold upon exposure to fibrinogen. Human U937 and THP-1 precursor-1 (THP-1) monocytic cell lines also secreted chemokines in response to fibrinogen, upon activation with IFN-γ and differentiation with vitamin D3, respectively. LPS contamination could not account for our observations, as fibrinogen-induced chemokine secretion was sensitive to heat denaturation and was unaffected by the pharmacologic LPS antagonist polymyxin B. Nevertheless, fibrinogen- and LPS-induced chemokine secretion both apparently required expression of functional Toll-like receptor 4, as each was diminished in macrophages derived from C3H/HeJ mice. Thus, innate responses to fibrinogen and bacterial endotoxin may converge at the evolutionarily conserved Toll-like recognition molecules. Our data suggest that extravascular fibrin(ogen) induces macrophage chemokine expression, thereby promoting immune surveillance at sites of inflammation.

Targeting of the chemokine receptor CCR1 suppresses development of acute and chronic cardiac allograft rejection

Although mononuclear cell infiltration is a hallmark of cellular rejection of a vascularized allograft, efforts to inhibit rejection by blocking leukocyte-endothelial cell adhesion have proved largely unsuccessful, perhaps in part because of persistent generation of chemokines within rejecting grafts. We now provide, to our knowledge, the first evidence that in vivo blockade of specific chemokine receptors is of therapeutic significance in organ transplantation. Inbred mice with a targeted deletion of the chemokine receptor CCR1 showed significant prolongation of allograft survival in 4 models. First, cardiac allografts across a class II mismatch were rejected by CCR1(+/+) recipients but were accepted permanently by CCR1(-/-) recipients. Second, CCR1(-/-) mice rejected completely class I- and class II-mismatched BALB/c cardiac allografts more slowly than control mice. Third, levels of cyclosporin A that had marginal effects in CCR1(+/+) mice resulted in permanent allograft acceptance in CCR1(-/-) recipients. These latter allografts showed no sign of chronic rejection 50-200 days after transplantation, and transfer of CD4(+) splenic T cells from these mice to naive allograft recipients significantly prolonged allograft survival, whereas cells from CCR1(+/+) mice conferred no such benefit. Finally, both CCR1(+/+) and CCR1(-/-) allograft recipients, when treated with a mAb to CD4, showed permanent engraftment, but these allografts showed florid chronic rejection in the former strain and were normal in CCR1(-/-) mice. We conclude that therapies to block CCR1/ligand interactions may prove useful in preventing acute and chronic rejection clinically.

Beneficial effects of targeting CCR5 in allograft recipients.

BACKGROUND The chemokine receptor, CCR5, and its three high-affinity ligands, macrophage inflammatory protein- (MIP) 1alpha, MIP-1beta, and regulated on activation normal T cell expressed and secreted (RANTES), are expressed by infiltrating mononuclear cells during the rejection of clinical and experimental organ allografts, although the significance of these molecules in the pathogenesis of rejection has not been established. METHODS We studied intragraft events in four allograft models. First, we studied cardiac transplants in fully MHC-mismatched mice that were deficient in CCR5 or two of its ligands, MIP-1alpha or RANTES. Second we tested the effects of a neutralizing rat anti-mCCR5 monoclonal antibody on allograft survival. Third we assessed whether a subtherapeutic course of cyclosporine would potentiate enhance survival in CCR5-deficient recipients. Finally, we tested the effect of targeting CCR5 in a class II-mismatched model. RESULTS Whereas mice deficient in expression of MIP-1alpha or RANTES reject fully MHC-mismatched cardiac allografts normally, CCR5-/- mice, or CCR5+/+ mice treated with a neutralizing mAb to mCCR5, show enhanced allograft survival. MHC class II-disparate mismatched are permanently accepted in CCR5-/- but not CCR5+/+ recipients. Finally, the beneficial effects of targeting of CCR5 are markedly synergistic with the effects of cyclosporine, resulting in permanent engraftment without development of chronic rejection. CONCLUSIONS We conclude that CCR5 plays a key role in the mechanisms of host T cell and macrophage recruitment and allograft rejection, such that targeting of CCR5 clinically may be of therapeutic significance.

Differential effects of cyclosporine A, methylprednisolone, mycophenolate, and rapamycin on CD154 induction and requirement for NFkappaB: implications for tolerance induction.

BACKGROUND Recent experimental data indicate that the targeting of the costimulatory molecule CD40-ligand (CD154) may well offer an opportunity for tolerance induction in transplant recipients and patients with autoimmune diseases, although the optimal therapeutic strategy for clinical application of CD154 monoclonal antibody (mAb) is unclear. METHODS We undertook vascularized heterotopic cardiac allograft transplantation in completely MHC-mismatched mice, treated recipients with CD154 mAb plus various immunosuppressive agents, and performed flow cytometric analysis of CD154 expression by T cells activated in vitro in the presence of corresponding immunosuppressive agents. We also tested the extent to which CD154 induction was NFkappaB-dependent by using NFkappaB/p50-deficient mice as allograft recipients and as source of cells for in vitro studies of CD154 induction, and through use of proteasome inhibitors to block IkappaBalpha degradation and NFKB activation in wild-type mice. RESULTS Concomitant use of cyclosporin A or methylprednisolone, but not rapamycin or mycophenolate, inhibited CD154 mAb-induced allograft survival. The differential effects of these agents on CD154 mAb-induced tolerance correlated with their capacity to inhibit activation-induced CD154 expression on CD4+ T cells. Full expression of CD154 expression was found to require NF-kappaB activation, and CD154 mAb was ineffective in NF-kappaB/p50 deficient allograft recipients or control mice in which NF-kappaB activation was blocked by proteasome inhibition. CONCLUSIONS Strategies to use CD154 mAb clinically must take into account the effects of immunosuppressive agents on CD154 induction, which seems to be at least partially NF-kappaB dependent. Our data suggest that ligation of surface-expressed CD154 provides an important signal that modulates T cell activation and thereby contributes to the effects of CD154 mAb, in addition to previously recognized actions involving blockade of CD40/CD154-dependent cell activation and activation-induced cell death.

Current challenges in the development of vaccines for pneumonic plague

Inhalation of Yersinia pestis bacilli causes pneumonic plague, a rapidly progressing and exceptionally virulent disease. Extensively antibiotic-resistant Y. pestis strains exist and we currently lack a safe and effective pneumonic plague vaccine. These facts raise concern that Y. pestis may be exploited as a bioweapon. Here, I review the history and status of plague vaccine research and advocate that pneumonic plague vaccines should strive to prime both humoral and cellular immunity.

Systemic but Not Local Infections Elicit Immunosuppressive IL-10 Production by Natural Killer Cells

Surviving infection represents a balance between the proinflammatory responses needed to eliminate the pathogen, and anti-inflammatory signals limiting damage to the host. IL-10 is a potent immunosuppressive cytokine whose impact is determined by the timing and localization of release. We show that NK cells rapidly express IL-10 during acute infection with diverse rapidly disseminating pathogens. The proinflammatory cytokine IL-12 was necessary and sufficient for NK cell induction of IL-10. NK cells from mice with systemic parasitic infection inhibited dendritic cell release of IL-12 in an IL-10-dependent manner, and NK cell depletion resulted in elevated serum IL-12. These data suggest an innate, negative feedback loop in which IL-12 limits its own production by eliciting IL-10 from NK cells. In contrast to disseminating pathogens, locally restricted infections did not elicit NK cell IL-10. Thus systemic infections uniquely engage NK cells in an IL-10-mediated immunoregulatory circuit that functions to alleviate inflammation.

Cell-Mediated Protection against Pulmonary Yersinia pestis Infection

ABSTRACT Pulmonary infection with the bacterium Yersinia pestis causes pneumonic plague, an often-fatal disease for which no vaccine is presently available. Antibody-mediated humoral immunity can protect mice against pulmonary Y. pestis infection, an experimental model of pneumonic plague. Little is known about the protective efficacy of cellular immunity. We investigated the cellular immune response to Y. pestis in B-cell-deficient μMT mice, which lack the capacity to generate antibody responses. To effectively prime pulmonary cellular immunity, we intranasally vaccinated μMT mice with live replicating Y. pestis. Vaccination dramatically increased survival of μMT mice challenged intranasally with a lethal Y. pestis dose and significantly reduced bacterial growth in pulmonary, splenic, and hepatic tissues. Vaccination also increased numbers of pulmonary T cells, and administration of T-cell-depleting monoclonal antibodies at the time of challenge abrogated vaccine-induced survival. Moreover, the transfer of Y. pestis-primed T cells to naive μMT mice protected against lethal intranasal challenge. These findings establish that vaccine-primed cellular immunity can protect against pulmonary Y. pestis infection and suggest that vaccines promoting both humoral and cellular immunity will most effectively combat pneumonic plague.

Immune defense against pneumonic plague

Summary: Yersinia pestis is one of the worlds most virulent human pathogens. Inhalation of this Gram‐negative bacterium causes pneumonic plague, a rapidly progressing and usually fatal disease. Extensively antibiotic‐resistant strains of Y. pestis exist and have significant potential for exploitation as agents of terrorism and biowarfare. Subunit vaccines comprised of the Y. pestis F1 and LcrV proteins are well‐tolerated and immunogenic in humans but cannot be tested for efficacy, because pneumonic plague outbreaks are uncommon and intentional infection of humans is unethical. In animal models, F1/LcrV‐based vaccines protect mice and cynomolgus macaques but have failed, thus far, to adequately protect African green monkeys. We lack an explanation for this inconsistent efficacy. We also lack reliable correlate assays for protective immunity. These deficiencies are hampering efforts to improve vaccine efficacy. Here, I review the immunology of pneumonic plague, focusing on evidence that humoral and cellular defense mechanisms collaborate to defend against pulmonary Y. pestis infection.

Fibrin-mediated Protection Against Infection-stimulated Immunopathology

Fibrin, a product of the blood coagulation cascade, accompanies many type 1 immune responses, including delayed-type hypersensitivity, autoimmunity, and graft rejection. In those settings, fibrin is thought to exacerbate inflammation and disease. Here, we evaluate roles for coagulation during infection with Toxoplasma gondii, a pathogen whose control requires robust type 1 immunity. We establish that fibrin prevents infection-stimulated blood loss, thereby performing a protective function that is essential for survival. Remarkably, fibrin does not simply protect against vascular damage caused directly by the infectious agent, but rather, protects against hemorrhage evoked by interferon-γ, a critical mediator of type 1 immunity. This finding, to our knowledge, is the first to document a beneficial role for coagulation during type 1 immunity, and suggests that fibrin deposition protects host tissue from collateral damage caused by the immune system as it combats infection.