Chris Dillen

Citrullination of CXCL8 by peptidylarginine deiminase alters receptor usage, prevents proteolysis, and dampens tissue inflammation

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.

Platelet Factor-4 Variant Chemokine CXCL4L1 Inhibits Melanoma and Lung Carcinoma Growth and Metastasis by Preventing Angiogenesis

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-gamma (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.

Chemokines synergize in the recruitment of circulating neutrophils into inflamed tissue

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 dose‐dependent 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‐1α/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.

In vivo neutrophil recruitment by granulocyte chemotactic protein-2 is assisted by gelatinase B/MMP-9 in the mouse.

Granulocyte chemotactic protein-2 (GCP-2) of the mouse is a potent neutrophil chemotactic and activating factor in vitro and in vivo. Gelatinase B/matrix metalloproteinase-9 is released from neutrophils within 1 h after stimulation with GCP-2. In vitro neutrophil chemotaxis by GCP-2 was not impaired by specific inhibitory monoclonal antibodies (mAb) against gelatinase B, indicating that gelatinase B is not involved in chemotaxis of neutrophils through polycarbonate filters. To investigate if gelatinase B degranulation is involved in in vivo cell migration toward GCP-2, experiments were performed with gelatinase B knockout mice. When mouse GCP-2 was injected intradermally in mice, a dose-dependent neutrophil chemotactic response was observed, and this cell migration was significantly impaired in young mice by genetic gelatinase B knockout. In adult vs. young gelatinase B-deficient mice, such compensatory mechanisms as higher basal neutrophil counts and less impairment of chemotaxis toward local GCP-2 injection were observed. These experiments prove the concept that gelatinase B release under pressure of GCP-2 is a relevant, but not exclusive, effector mechanism of neutrophil chemotaxis in vivo and that known mechanisms, other than the release of gelatinase B, allow for a full-blown chemotactic response and compensate for gelatinase B deficiency in adult life in the mouse.

Interleukin-17 regulates chemokine and gelatinase B expression in fibroblasts to recruit both neutrophils and monocytes

Interleukin 17 (IL-17) is a proinflammatory cytokine, produced only by activated lymphocytes, but with a broad cellular host range. The effects of IL-17 on fibroblasts were investigated by analysis of the induction of chemokine and matrix metalloprotease (MMP) mRNA levels by RT-PCR. IL-17 stimulated CC chemokine (monocyte chemotactic protein-1; MCP-1/JE) and CXC (KC, MIP-2) chemokine and TIMP-1 mRNA expression in fibroblastoid L929 cells. In normal mouse embryonic fibroblasts (MEF) this induction profile by IL-17 was extended with the mRNAs encoding the chemokine granulocyte chemotactic protein-2 (GCP-2) and the proteases MMP-3, MMP-9 and MMP-13. The MMP-9 and GCP-2 induction by IL-17 in MEF, and the absence of induction in L929 cells, were corroborated by gelatin zymography and ELISA, respectively. The induction of MCP-1/CCL2 by IL-17 was confirmed in human diploid fibroblasts. We conclude that IL-17 regulates differentially chemokine and MMP expression by normal and transformed fibroblasts and is indirectly capable of attracting both monocytes and neutrophils to the inflammatory focus.

Bimodal role of endogenous interleukin-6 in concanavalin A-induced hepatitis in mice.

Acute concanavalin A (Con A)‐induced hepatitis in mice is an animal model for hepatic injury induced by activated T cells. The evolution of hepatic involvement can be followed from hour to hour by measuring serum transaminase levels. We investigated the possible role of endogenous interleukin‐6 (IL‐6) in this model. We found serum IL‐6 levels and splenic IL‐6 mRNA during Con A‐induced hepatitis to be significantly lower in interferon‐γ (IFN‐γ)‐deficientmice, which are resistant against the Con A‐induced syndrome, than in wild‐type ones, suggesting that systemic IL‐6 production favors development of hepatic injury. However, IL‐6‐deficient mice proved to be more susceptible to the disease than wild‐type mice, indicating that endogenous IL‐6 plays a predominantly hepatoprotective role. Experiments in which wild‐type mice were treated with anti‐IL‐6 antibodies, before or after Con A challenge, allowed us to reconcile these contrasting observations. The antibody injections resulted in a biphasic alteration of serum IL‐6 levels, initial neutralization being followed by rebound increased levels due to accumulation of IL‐6 in the form of antigen‐antibody complexes. The effect of antibody on disease severity differed depending on the time of injection. Antibody injection at 2.5 h post Con A resulted in delayed disease manifestation, whereas treatment initiated before Con A resulted in accelerated disease. We conclude that endogenous IL‐6 plays a bimodal role. IL‐6 present before Con A challenge as well as that induced in the very early phase after Con A injection triggers hepatoprotective pathways. Continuation of IL‐6 production beyond this early phase, by some other pathway, seems to be harmful to hepato‐cytes. J. Leukoc. Biol. 67: 90–96; 2000.M

Efficient secretion of biologically active mouse tumor necrosis factor alpha by Streptomyces lividans

We have studied the production of mouse tumor necrosis factor alpha (mTNF) with Streptomyces lividans as host. mTNF cDNA was fused to the alpha-amylase-encoding gene (aml) of Streptomyces venezuelae ATCC15068 at 12 amino acids (aa) downstream from the signal-peptidase cleavage site so that the aa surrounding this processing site were conserved. S. lividans containing this construct secreted mTNF at moderately high levels (1-10 micrograms/ml) as a biologically active compound of high specific activity (1 x 10(8) units/mg protein). No unprocessed pre-protein and virtually no processed protein could be detected in the cell lysates. N-terminal aa sequence analysis indicated microheterogeneity (-3 to -6 forms) at the N-terminal site of secreted mTNF. It was demonstrated that this microheterogeneity was due to aminopeptidase activity.

Role of the autocrine chemokines MIP-1α and MIP-1β in the metastatic behavior of murine T cell lymphoma

The ESb‐MP T‐cell line is a highly malignant murine lymphoma, which preferentially metastasizes toward the kidney. This could be a result of the local production of monocyte chemoattractant protein‐1 (MCP‐1) and regulated on activation, normal T expressed and secreted (RANTES), which are chemotactic for ESb‐MP cells. Here, we demonstrate that ESb‐MP cells are already responsive to the chemotactic activity of macrophage inflammatory protein‐1α (MIP‐1α) and MIP‐1β from 1 ng/ml onward. Moreover, upon stimulation with lipopolysaccharide (LPS) or virus, ESb‐MP cells themselves produce significant amounts of MIP‐1 (∼200 ng/ml). Indeed, the major autocrine chemoattractants, isolated from ESb‐MP cells, were intact MIP‐1α and MIP‐1β. Pretreatment with LPS or addition of MIP‐1 inhibited the in vitro migration of ESb‐MP cells toward various chemokines. Moreover, compared with untreated lymphoma cells, LPS‐treated cells produced significantly less metastasis in mice. The results represented here suggest that the role of chemokines in attracting tumor cells at secondary sites depends on a balance between autocrine‐produced and tissue‐derived chemokines. This delicate balance should be considered in the design of antichemokine strategies in different tumor types.

Intradermal air pouch leukocytosis as an in vivo test for nanoparticles

The need for test systems for nanoparticle biocompatibility, toxicity, and inflammatory or adaptive immunological responses is paramount. Nanoparticles should be free of microbiological and chemical contaminants, and devoid of toxicity. Nevertheless, in the absence of contamination, these particles may still induce undesired immunological effects in vivo, such as enhanced autoimmunity, hypersensitivity reactions, and fibrosis. Here we show that artificial particles of specific sizes affect immune cell recruitment as tested in a dermal air pouch model in mice. In addition, we demonstrate that the composition of nanoparticles may influence immune cell recruitment in vivo. Aside from biophysical characterizations in terms of hydrodynamic diameter, zeta potential, concentration, and atomic concentration of metals, we show that – after first-line in vitro assays – characterization of cellular and molecular effects by dermal air pouch analysis is straightforward and should be included in the quality control of nanoparticles. We demonstrate this for innate immunological effects such as neutrophil recruitment and the production of immune-modulating matrix metalloproteases such as MMP-9; we propose the use of air pouch leukocytosis analysis as a future standard assay.

Murine interferon-γ/interleukin-1 fusion proteins used as antigens for the generation of hybridomas producing monoclonal anti-interleukin-1 antibodies

In several biological systems interferon-gamma (IFN-gamma) and interleukin-1 (IL-1) act synergistically. We therefore examined whether it would be possible to construct IFN-gamma/IL-1 hybrid proteins that would be more active than the individual components. Hybrid proteins were examined that consisted of the amino-terminal 118 residues of mouse IFN-gamma and the 156 or 152 carboxyl-terminal residues of mouse IL-1 alpha or IL-1 beta, respectively. They were obtained by ligation of the respective coding sequences and expression of the fused genes under control of the PL promotor in Escherichia coli. Both the IFN-gamma/IL-1 alpha and the IFN-gamma/IL-1 beta fusion proteins were purified by affinity chromatography on an anti-IFN-gamma monoclonal antibody column. Analysis of biological activities showed that these fusion proteins were less active than the individual cytokines. Specific antiviral activity of the IFN-gamma/IL-1 beta hybrids was less than 0.1% that of IFN-gamma and D10.G4.1 T-cell proliferative (IL-1) activity amounted to 0.1% that of mouse IL-1. Affinity-purified preparations of the IFN-gamma/IL-1 alpha hybrid were found to contain variable proportions of a Mr 14,000 degradation product possessing IFN-gamma activity, whereas the undegraded Mr 30,000 fusion protein, while being devoid of detectable IFN-gamma activity, did possess IL-1 activity (1%). Serum from rats immunized with the IFN-gamma/IL-1 alpha hybrid contained high levels of IL-1 alpha-binding and -neutralizing antibodies and IFN-gamma-binding antibodies, but no detectable levels of IFN-gamma-neutralizing antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)