Bloodsucking parasites such as ticks have evolved a wide variety of immunomodulatory proteins that are secreted in their saliva, allowing them to feed for long periods of time without being detected by the host immune system. One possible strategy used by ticks to evade the host immune response is to produce proteins that selectively bind and neutralize the chemokines that normally recruit cells of the innate immune system that protect the host from parasites. We have identified distinct cDNAs encoding novel chemokine binding proteins (CHPBs), which we have termed Evasins, using an expression cloning approach. These CHBPs have unusually stringent chemokine selectivity, differentiating them from broader spectrum viral CHBPs. Evasin-1 binds to CCL3, CCL4, and CCL18; Evasin-3 binds to CXCL8 and CXCL1; and Evasin-4 binds to CCL5 and CCL11. We report the characterization of Evasin-1 and -3, which are unrelated in primary sequence and tertiary structure, and reveal novel folds. Administration of recombinant Evasin-1 and -3 in animal models of disease demonstrates that they have potent antiinflammatory properties. These novel CHBPs designed by nature are even smaller than the recently described single-domain antibodies (Hollinger, P., and P.J. Hudson. 2005. Nat. Biotechnol. 23:1126–1136), and may be therapeutically useful as novel antiinflammatory agents in the future.

Results.

Objective. The chemokine receptors CXCR1 and CXCR2 play a role in mediating neutrophil recruitment and neutrophil-dependent injury in several models of inflammation. We undertook this study to investigate the role of these receptors in mediating neutrophil adhesion, subsequent migration, and neutrophildependent hypernociception in a murine model of monarticular antigen-induced arthritis (AIA).Methods. AIA was induced by administration of antigen into the knee joint of previously immunized mice. Intravital microscopy studies were performed to assess leukocyte rolling and adhesion. Mechanical hypernociception was investigated using an electronic pressure meter. Neutrophil accumulation in the tissue was measured by counting neutrophils in the synovial cavity and assaying myeloperoxidase activity. Levels of tumor necrosis factor ␣ (TNF␣) and the chemokines CXCL1 and CXCL2 were quantified by enzyme-linked immunosorbent assay. Histologic analysis was performed to evaluate the severity of arthritis and leukocyte infiltration.Results. Antigen challenge in immunized mice induced production of TNF␣, CXCL1, and CXCL2 and also resulted in neutrophil recruitment, leukocyte rolling and adhesion, and hypernociception. Treatment with reparixin or DF2162 (allosteric inhibitors of CXCR1/CXCR2) decreased neutrophil recruitment, an effect that was associated with marked inhibition of neutrophil adhesion. Drug treatment also inhibited TNF␣ production, hypernociception, and the overall severity of the disease in the tissue.Conclusion. Blockade of CXCR1/CXCR2 receptors inhibits neutrophil recruitment by inhibiting the adhesion of neutrophils to synovial microvessels. As a consequence, there is decreased local cytokine production and reduced hypernociception, as well as ameloriation of overall disease in the tissue. These studies suggest a potential therapeutic role for the modulation of CXCR1/CXCR2 receptor signaling in the treatment of arthritis.

HIV-1 negative factor (Nef) elevates virus replication and contributes to immune evasion in vivo. As one of its established in vitro activities, Nef interferes with T-lymphocyte chemotaxis by reducing host cell actin dynamics. To explore Nef’s influence on in vivo recirculation of T lymphocytes, we assessed lymph-node homing of Nef-expressing primary murine lymphocytes and found a drastic impairment in homing to peripheral lymph nodes. Intravital imaging and 3D immunofluorescence reconstruction of lymph nodes revealed that Nef potently impaired T-lymphocyte extravasation through high endothelial venules and reduced subsequent parenchymal motility. Ex vivo analyses of transendothelial migration revealed that Nef disrupted T-lymphocyte polarization and interfered with diapedesis and migration in the narrow subendothelial space. Consistently, Nef specifically affected T-lymphocyte motility modes used in dense environments that pose high physical barriers to migration. Mechanistically, inhibition of lymph node homing, subendothelial migration and cell polarization, but not diapedesis, depended on Nef’s ability to inhibit host cell actin remodeling. Nef-mediated interference with in vivo recirculation of T lymphocytes may compromise T-cell help and thus represents an important mechanism for its function as a HIV pathogenicity factor.

The pentraxin superfamily is a group of evolutionarily conserved proteins that play important roles in the immune system. The long pentraxin PTX3 protein was originally described as able to be induced by pro-inflammatory stimuli in a variety of cell types. In this study, we evaluated the phenotype of Ptx3 ؊/؊ mice subjected to ischemia followed by reperfusion of the superior mesenteric artery. In reperfused wildtype mice, there was significant local and remote injury as demonstrated by increases in vascular permeability, neutrophil influx, nuclear factor-B activation, and production of CXCL1 and tumor necrosis factor-␣. PTX3 levels were elevated in both serum and intestine after reperfusion. In Ptx3 ؊/؊ mice, local and remote tissue injury was inhibited, and there were decreased nuclear factor-B translocation and cytokine production. Intestinal architecture was preserved, and there were decreased neutrophil influx and significant prevention of lethality in Ptx3 ؊/؊ mice as well. PTX3 given intravenously before reperfusion reversed the protection observed in Ptx3 ؊/؊ mice in a dose-dependent manner, and PTX3 administration significantly worsened tissue injury and lethality in wild-type mice. In conclusion, our studies demonstrate a major role for PTX3 in determining acute reperfusion-associated inflammation, tissue injury, and lethality and suggest the soluble form of this molecule is active in this system. Therapeutic blockade of PTX3 action may be useful in the control of the injuries associated with severe ischemia and reperfusion syndromes.