Biological functions of proteins are influenced by posttranslational modifications such as on/off switching by phosphorylation and modulation by glycosylation. Proteolytic processing regulates cytokine and chemokine activities. In this study, we report that natural posttranslational citrullination or deimination alters the biological activities of the neutrophil chemoattractant and angiogenic cytokine CXCL8/interleukin-8 (IL-8). Citrullination of arginine in position 5 was discovered on 14% of natural leukocyte-derived CXCL8(1–77), generating CXCL8(1–77)Cit5. Peptidylarginine deiminase (PAD) is known to citrullinate structural proteins, and it may initiate autoimmune diseases. PAD efficiently and site-specifically citrullinated CXCL5, CXCL8, CCL17, CCL26, but not IL-1β. In comparison with CXCL8(1–77), CXCL8(1–77)Cit5 had reduced affinity for glycosaminoglycans and induced less CXCR2-dependent calcium signaling and extracellular signal-regulated kinase 1/2 phosphorylation. In contrast to CXCL8(1–77), CXCL8(1–77)Cit5 was resistant to thrombin- or plasmin-dependent potentiation into CXCL8(6–77). Upon intraperitoneal injection, CXCL8(6–77) was a more potent inducer of neutrophil extravasation compared with CXCL8(1–77). Despite its retained chemotactic activity in vitro, CXCL8(1–77)Cit5 was unable to attract neutrophils to the peritoneum. Finally, in the rabbit cornea angiogenesis assay, the equally potent CXCL8(1–77) and CXCL8(1–77)Cit5 were less efficient angiogenic molecules than CXCL8(6–77). This study shows that PAD citrullinates the chemokine CXCL8, and thus may dampen neutrophil extravasation during acute or chronic inflammation.

The involvement of cytokines in the pathogenesis of a generalized, Shwartzman-like lethal inflammatory response to bacterial lipopolysaccharides (LPS) was studied by testing the ability of cytokines or neutralizing anticytokine antibodies to modify the course of the syndrome. The reaction was elicitable in non-SPF NMRI mice by two consecutive injections of S. marcescens LPS: a first injection in the footpad, followed after 24 h by an intravenous dose; the size and route of the preparatory LPS dose were found to be critical. Treatment with mAbs against IFN-gamma was found to completely prevent the reaction. Treatment with IFN-gamma on the other hand, rendered the mice more sensitive to elicitation of the reaction. In contrast, systemic administration of IFN-alpha/beta exerted a desensitizing effect. The role of endogenous cytokines in the pathogenesis of this generalized Shwartzman reaction was also documented by a study of the cytokine levels in the serum of the mice. In comparisons between mice given lethal and nonlethal induction schedules, a good correlation was found between mortality rates and height of IFN or TNF levels, but no correlation was seen with IL-6 levels. Also, in mice that were protected by anti-IFN-gamma antibody, serum IFN and TNF were undetectable, whereas IL-6 levels were as high as in unprotected mice. These data provide evidence that among the cytokines that govern the inflammatory response to LPS, endogenous IFN-gamma occupies a key position. These findings therefore also open perspectives for clinical application of IFN-gamma antagonists.

The platelet factor-4 variant, designated PF-4var/CXCL4L1, is a recently described natural non-allelic gene variant of the CXC chemokine platelet factor-4/CXCL4. PF-4var/CXCL4L1 was cloned, and the purified recombinant protein strongly inhibited angiogenesis. Recombinant PF-4var/CXCL4L1 was angiostatically more active (at nanomolar concentration) than PF-4/CXCL4 in various test systems, including wound-healing and migration assays for microvascular endothelial cells and the rat cornea micropocket assay for angiogenesis. Furthermore, PF-4var/CXCL4L1 more efficiently inhibited tumor growth in animal models of melanoma and lung carcinoma than PF-4/CXCL4 at an equimolar concentration. For B16 melanoma in nude mice, a significant reduction in tumor size and the number of small i.t. blood vessels was obtained with i.t. applied PF-4var/CXCL4L1. For A549 adenocarcinoma in severe combined immunodeficient mice, i.t. PF-4var/CXCL4L1 reduced tumor growth and microvasculature more efficiently than PF-4/CXCL4 and prevented metastasis to various organs better than the angiostatic IFN-inducible protein 10/CXCL10. Finally, in the syngeneic model of Lewis lung carcinoma, PF-4var/CXCL4L1 inhibited tumor growth equally well as monokine induced by IFN-; (Mig)/CXCL9, also known to attract effector T lymphocytes. Taken together, PF-4var/ CXCL4L1 is a highly potent antitumoral chemokine preventing development and metastasis of various tumors by inhibition of angiogenesis. These data confirm the clinical potential of locally released chemokines in cancer therapy. [Cancer Res 2007;67(12):5940-8]

The innate immune response against micro-organisms is mediated by phagocytes, attracted by chemokines and other G protein-coupled receptor (GPCR) ligands. Originally, we observed increased neutrophil migration by the interaction of inflammatory CXC chemokines such as IL-8/CXCL8 and granulocyte chemotactic protein (GCP)-2/CXCL6 with regakine-1, a CC chemokine constitutively present in plasma. We here demonstrate statistically significant synergy between regakine-1 and the neutrophil attractants C5a or IL-8/CXCL8 in inducing neutrophil shape change and migration under agarose. In addition, regakine-1 attracted human bone marrow granulocytes and enhanced their chemotactic response to IL-8/CXCL8 in a dosedependent manner. Thus, plasma chemokines may regulate the number of circulating leukocytes under homeostatic conditions and may facilitate extra recruitment of bone marrow neutrophils during inflammation. Indeed, in vivo, regakine-1 provoked a mild neutrophilia in rabbits upon intravenous injection. We also observed that the CC chemokines regakine-1 and monocyte chemotactic protein-3/CCL7 as well as the CXC chemokine stromal cell-derived factor-1a/CXCL12 co-operated with murine GCP-2 after intraperitoneal co-administration to increase neutrophil influx in mice. These data demonstrate that inducible and constitutive GPCR ligands synergize to enhance inflammation and facilitate a more effective immune response.