Coexpression and interaction of CXCL10 and CD26 in mesenchymal cells by synergising inflammatory cytokines: CXCL8 and CXCL10 are discriminative markers for autoimmune arthropathies

Leukocyte infiltration during acute and chronic inflammation is regulated by exogenous and endogenous factors, including cytokines, chemokines and proteases. Stimulation of fibroblasts and human microvascular endothelial cells with the inflammatory cytokines interleukin-1β (IL-1β) or tumour necrosis factor alpha (TNF-α) combined with either interferon-α (IFN-α), IFN-β or IFN-γ resulted in a synergistic induction of the CXC chemokine CXCL10, but not of the neutrophil chemoattractant CXCL8. In contrast, simultaneous stimulation with different IFN types did not result in a synergistic CXCL10 protein induction. Purification of natural CXCL10 from the conditioned medium of fibroblasts led to the isolation of CD26/dipeptidyl peptidase IV-processed CXCL10 missing two NH(2)-terminal residues. In contrast to intact CXCL10, NH(2)-terminally truncated CXCL10(3–77) did not induce extracellular signal-regulated kinase 1/2 or Akt/protein kinase B phosphorylation in CXC chemokine receptor 3-transfected cells. Together with the expression of CXCL10, the expression of membrane-bound CD26/dipeptidyl peptidase IV was also upregulated in fibroblasts by IFN-γ, by IFN-γ plus IL-1β or by IFN-γ plus TNF-α. This provides a negative feedback for CXCL10-dependent chemotaxis of activated T cells and natural killer cells. Since TNF-α and IL-1β are implicated in arthritis, synovial concentrations of CXCL8 and CXCL10 were compared in patients suffering from crystal arthritis, ankylosing spondylitis, psoriatic arthritis and rheumatoid arthritis. All three groups of autoimmune arthritis patients (ankylosing spondylitis, psoriatic arthritis and rheumatoid arthritis) had significantly increased synovial CXCL10 levels compared with crystal arthritis patients. In contrast, compared with crystal arthritis, only rheumatoid arthritis patients, and not ankylosing spondylitis or psoriatic arthritis patients, had significantly higher synovial CXCL8 concentrations. Synovial concentrations of the neutrophil chemoattractant CXCL8 may therefore be useful to discriminate between autoimmune arthritis types

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

Biological Activity of CXCL8 Forms Generated by Alternative Cleavage of the Signal Peptide or by Aminopeptidase-Mediated Truncation

Posttranslational modification of chemokines is one of the mechanisms that regulate leukocyte migration during inflammation. Multiple natural NH(2)-terminally truncated forms of the major human neutrophil attractant interleukin-8 or CXCL8 have been identified. Although differential activity was reported for some CXCL8 forms, no biological data are available for others.status: publishe

Posttranslational modification of the NH2-terminal region of CXCL5 by proteases or peptidylarginine deiminases (PAD) differently affects its biological activity

Posttranslational modifications, e.g. proteolysis, glycosylation and citrullination regulate chemokine function, affecting leukocyte migration during inflammatory responses. Here, modification of CXCL5/epithelial cell-derived neutrophil-activating protein-78 (ENA-78) by proteases or peptidylarginine deiminases (PAD) was evaluated. Slow CXCL5(1-78) processing by the myeloid cell marker aminopeptidase N/CD13 into CXCL5(2-78) hardly affected its in vitro activity, but slowed down the activation of CXCL5 by the neutrophil protease cathepsin G. PAD, an enzyme with a potentially important function in autoimmune diseases, site-specifically deiminated Arg-9 in CXCL5 to citrulline, generating [Cit(9)]CXCL5(1-78). Compared to CXCL5(1-78), [Cit(9)]CXCL5(1-78) less efficiently induced intracellular calcium signaling, phosphorylation of extracellular signal-regulated kinase, internalization of CXCR2 and in vitro neutrophil chemotaxis. In contrast, conversion of CXCL5 into the previously reported natural isoform CXCL5(8 78) provided at least 3-fold enhanced biological activity in these tests. Citrullination, but not NH(2)-terminal truncation, reduced the capacity of CXCL5 to up-regulate the expression of the integrin alpha-chain CD11b on neutrophils. Truncation nor citrullination significantly affected the ability of CXCL5 to upregulate CD11a expression or shedding of CD62L. In line with the in vitro results, CXCL5(8-78) and CXCL5(9-78) induced a more pronounced neutrophil influx in vivo compared to CXCL5(1-78). Administration of 300 pmol of either CXCL5(1-78) or [Cit(9)]CXCL5(1-78) failed to attract neutrophils to the peritoneal cavity. Citrullination of the more potent CXCL5(9-78) lowers its chemotactic potency in vivo and confirms the tempering effect of citrullination in vitro. The highly divergent effects of modifications of CXCL5 on neutrophil influx underline the potential importance of tissue-specific interactions between chemokines and PAD or proteases.status: publishe

Citrullination of TNF-α by peptidylarginine deiminases reduces its capacity to stimulate the production of inflammatory chemokines

Citrullination, a posttranslational modification (PTM) recently discovered on inflammatory chemokines such as interleukin-8 (IL-8/CXCL8) and interferon-γ-inducible protein-10 (IP-10/CXCL10), seriously influences their biological activity. Citrullination or the deimination of arginine to citrulline is dependent on peptidylarginine deiminases (PADs) and has been linked to autoimmune diseases such as multiple sclerosis (MS) and rheumatoid arthritis (RA). Chemokines are to date the first identified PAD substrates with receptor-mediated biological activity. We investigated whether cytokines that play a crucial role in RA, like interleukin-1β (IL-1β) and tumor necrosis factor-alpha (TNF-α), may be citrullinated by PAD and whether such a PTM influences the biological activity of these cytokines. IL-1β and TNF-α were first incubated with PAD in vitro and the occurrence of citrullination was examined by Edman degradation and a recently developed detection method for citrullinated proteins. Both techniques confirmed that human TNF-α, but not IL-1β, was citrullinated by PAD. Citrullination of TNF-α reduced its potency to stimulate chemokine production in vitro on human primary fibroblasts. Concentrations of the inflammatory chemokines CXCL8, CXCL10 and monocyte chemotactic protein-1 (MCP-1/CCL2) were significantly lower in supernatants of fibroblasts induced with citrullinated TNF-α compared to unmodified TNF-α. However, upon citrullination TNF-α retained its capacity to induce apoptosis/necrosis of mononuclear cells, its binding potency to Infliximab and its ability to recruit neutrophils to the peritoneal cavity of mice.status: publishe

Recombinant parvoviruses armed to deliver CXCL4L1 and CXCL10 are impaired in their antiangiogenic and antitumoral effects in a Kaposi sarcoma tumor model due to the chemokines' interference with the virus cycle

Application of oncolytic viruses is a valuable option to broaden the armament of anti-cancer therapies as these combine specific cytotoxic effects and immune-stimulating properties. The self-replicating H-1 parvovirus (H-1PV) is a prototypical oncolytic virus that besides targeting tumor cells also infects endothelial cells, thus combining oncolytic and angiostatic traits. To increase its therapeutic value, H-1PV can be armed with cytokines or chemokines to enhance the immunological response. Some chemokines, more specifically the CXCR3 ligands CXCL4L1 and CXCL10 combine immune-stimulating properties with angiostatic activity. In this study, we explore the therapeutic value of recombinant parvoviruses carrying CXCL4L1 or CXCL10 transgenes (Chi-H1/CXCL4L1 or Chi-H1/CXCL10, respectively) to inhibit the growth of the human Kaposi sarcoma cell line KS-IMM. KS-IMM cells infected by Chi-H1/CXCL4L1 or Chi-H1/CXCL10 released the corresponding chemokine and showed reduced migratory capacity. Therefore, we tested the anti-tumoral capacity of Chi-H1/CXCL4L1 or Chi-H1/CXCL10 in mice. Either in vitro infected KS IMM cells were injected or subcutaneously growing KS IMM xenografts were treated by peri-tumoral injections of the different viruses. Surprisingly, the transgenes did not increase the anti-tumoral effect of natural H-1PV. Further experiments indicated that CXCL4L1 and CXCL10 interfered with expression of the viral NS1 protein in KS-IMM cells. Our results indicate that the outcome of parvovirus-based delivery of CXCR3 ligands might be tumor cell type dependent and hence it's application must be considered carefully.status: publishe

Overview of the naturally occurring CXCL8 isoforms.

<p>Several NH₂-terminally modified isoforms of CXCL8 have been purified from the conditioned medium of leukocytes, fibroblasts and endothelial cells. The NH₂-terminal and COOH-terminal sequences of these isoforms are depicted in the one-letter code (B = citrulline). Based on the criteria formulated by Von Heijne <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023913#pone.0023913-vonHeijne1" target="_blank">[29]</a>, the 99 amino acid precursor molecule may be cleaved by signal peptidases between Cys₂₀ and Glu₂₁ or between Gly₂₂ and Ala₂₃, the latter being more probable. Indeed both resulting CXCL8 proteins have been identified and are indicated in this manuscript as CXCL8(-2-77) and CXCL8(1-77), respectively. CXCL8(1-77) can be cleaved by a number of proteases, resulting in the listed NH₂-terminally truncated forms. Furthermore, CXCL8 can be citrullinated by peptidylarginine deiminase (PAD). These NH₂-terminal modifications differently affect the ability of CXCL8 to recruit neutrophils. The <i>in vivo</i> neutrophil-attracting activity is defined in the boxes at the right. CXCL8(-2-77), CXCL8(1-77), CXCL8(2-77) and CXCL8(3-77) show comparable intermediate activity. The more extensively truncated isoforms CXCL8(6-77), CXCL8(7-77), CXCL(8-77) and CXCL8(9-77), in contrast, display enhanced neutrophil recruiting potency, whereas citrullination significantly lowers the capacity of CXCL8 to guide neutrophils. (MMP, matrix metalloprotease).</p

Neutrophil-attracting activity of the CXCL8 isoforms <i>in vivo</i>.

<p>The neutrophil influx upon i.p. injection of the specified doses of the CXCL8 isoforms in mice was investigated. 2 hours post-injection, the peritoneal cavity was washed and the overall number of leukocytes determined. Differential microscopic counting of Hemacolor-stained cells allowed to define the percentage of neutrophils (panel A) and the total number of neutrophils (panel B) present in the peritoneal cavity. Squares show the median neutrophil influx (formulated as percentages or total counts/ml); the bottom and the top of the rectangle denote the 25th and 75th percentile; whiskers represent the non-outlier range (coefficient 1.5). Outliers are plotted as circles. Control mice (co) were treated with vehicle and illustrate the spontaneous migration of neutrophils to the peritoneal cavity. The Mann-Whitney U test was used for statistical analysis [§, p<0.01; compared with 100 pmol CXCL8(1-77)]. The numbers depicted in (or besides) the rectangular box represent the number of mice treated.</p