Regulation of macrophage chemokine expression by lipopolysaccharide in vitro and in vivo.

The host response to Gram-negative LPS is characterized by an influx of inflammatory cells into host tissues, which is mediated, in part, by localized production of chemokines. The expression and function of chemokines in vivo appears to be highly selective, though the molecular mechanisms responsible are not well understood. All CXC (IFN-gamma-inducible protein (IP-10), macrophage inflammatory protein (MIP)-2, and KC) and CC (JE/monocyte chemoattractant protein (MCP)-1, MCP-5, MIP-1alpha, MIP-1beta, and RANTES) chemokine genes evaluated were sensitive to stimulation by LPS in vitro and in vivo. While IL-10 suppressed the expression of all LPS-induced chemokine genes evaluated in vitro, treatment with IFN-gamma selectively induced IP-10 and MCP-5 mRNAs, but inhibited LPS-induced MIP-2, KC, JE/MCP-1, MIP-1alpha, and MIP-1beta mRNA and/or protein. Like the response to IFN-gamma, LPS-mediated induction of IP-10 and MCP-5 was Stat1 dependent. Interestingly, only the IFN-gamma-mediated suppression of LPS-induced KC gene expression was IFN regulatory factor-2 dependent. Treatment of mice with LPS in vivo also induced high levels of chemokine mRNA in the liver and lung, with a concomitant increase in circulating protein. Hepatic expression of MIP-1alpha, MIP-1beta, RANTES, and MCP-5 mRNAs were dramatically reduced in Kupffer cell-depleted mice, while IP-10, KC, MIP-2, and MCP-1 were unaffected or enhanced. These findings indicate that selective regulation of chemokine expression in vivo may result from differential response of macrophages to pro- and antiinflammatory stimuli and to cell type-specific patterns of stimulus sensitivity. Moreover, the data suggest that individual chemokine genes are differentially regulated in response to LPS, suggesting unique roles during the sepsis cascade.     

Differential regulation of epithelial-derived C-C chemokine expression by IL-4 and the glucocorticoid budesonide.

Airway epithelial cells are a rich source of eosinophil-selective C-C chemokines. We investigated whether cytokines and the topical glucocorticoid budesonide differentially regulate RANTES, monocyte chemoattractant protein-4 (MCP-4), and eotaxin mRNA and protein expression in the human bronchial epithelial cell line BEAS-2B and in primary human bronchial epithelial cells by Northern blot analysis and ELISAs. Eotaxin and MCP-4 mRNA expression induced by TNF-alpha alone or in combination with IFN-gamma was near-maximal after 1 h, peaked at 4 and 8 h, respectively, remained unchanged up to 24 h, and was protein synthesis independent. In contrast, RANTES mRNA was detectable only after 2 h and slowly increased to a peak at 24 h, and was protein synthesis dependent. Induction of eotaxin and MCP-4 mRNA showed a 10- to 100-fold greater sensitivity to TNF-alpha compared with RANTES mRNA. IL-4 and IFN-gamma had selective effects on chemokine expression; IL-4 selectively up-regulated the expression of eotaxin and MCP-4 and potentiated TNF-alpha-induced eotaxin, while IFN-gamma markedly potentiated only the TNF-alpha-induced expression of RANTES. Although budesonide inhibited the expression of chemokine mRNA to a variable extent, it effectively inhibited production of eotaxin and RANTES protein. Budesonide inhibited both RANTES- and eotaxin promoter-driven reporter gene activity. Budesonide also selectively accelerated the decay of eotaxin and MCP-4 mRNA. These results point to IL-4 as a possible mediator by which Th2 cells may induce selective production of C-C chemokines from epithelium and indicate that glucocorticoid inhibit chemokine expression through multiple mechanisms of action.     

Effects of dietary omega3 and omega6 lipids and vitamin E on chemokine levels in autoimmune-prone MRL/MpJ-lpr/lpr mice

Elevated levels of chemokines, such as Regulated upon Activation, Normal T cell Expressed and Secreted (RANTES), Monocyte Chemotactic Protein-1 (MCP-1), Macrophage Inflammatory Protein-1alpha (MIP-1alpha), and Macrophage Inflammatory Protein-1beta (MIP-1beta) have been found in rheumatoid arthritis (RA) and juvenile arthritis (JA), and they may be associated with the pathogenesis of these diseases. These chemokines are implicated in the migration of specific leukocytes into the joints. Omega-3 (omega3) fatty acid rich-fish oil (FO) and vitamin E may delay the progress of certain autoimmune diseases. The present study was designed to understand the effects of dietary lipids (omega-6 and omega-3 fatty acids) and vitamin E on the production of chemokines in autoimmune-prone MRL/lpr (a mouse model for RA) and congenic control MRL/++ mice. The MRL mice were fed for 4.5 months omega-6 and omega-3 diets that varied in lipid sources (corn oil; CO and fish oil; FO) and vitamin E levels (269 I.U./kg and 694 I.U./kg diet). Spleen cells were isolated and cultured aseptically in the presence of PHA for 48 h at 37 degrees C and the levels of chemokines (RANTES, JE/MCP-1 and MIP-1alpha) were determined in the cell-free supernatants. The levels of RANTES and JE/MCP-1 were significantly higher in MRL/lpr mice compared to MRL/++ mice. The FO had differential effect on RANTES and MCP-1 production by spleen cells. The production of RANTES and JE/MCP-1 by spleen cells in mice fed the FO diets was significantly lower than in mice fed the CO diets (p < 0.0001). The levels of vitamin E did not affect the production of RANTES and JE/MCP-1. The levels of vitamin E had a significant effect on MIP-1alpha as the spleen cells of mice fed diets containing 694 IU/kg diet of vitamin E produced significantly higher levels of MIP-1alpha compared to the group of mice fed the diets containing 269 IU of vitamin E (p < 0.0001). The data obtained from this study in MRL/lpr and MRL/++ mice suggest that FO diets containing omega-3 fatty acids are beneficial in decreasing the levels of certain pro-inflammatory chemokines (RANTES and MCP-1) thereby delaying the onset of and severity of autoimmune symptoms in MRL/lpr mouse model.     

The expression of the cytomegalovirus chemokine receptor homolog US28 sequesters biologically active CC chemokines and alters IL-8 production

We hypothesized that US28, a cytomegalovirus (CMV) CC chemokine receptor homolog, plays a role in modulating the host antiviral defense. Monocyte chemotaxis was induced by supernatants from fibroblasts infected with a US28 deletion mutant of CMV (CMV Delta US28) due to endogenously produced CC chemokines MCP-1 and RANTES. However, these chemokines were sequestered from the supernatants of CMV-infected cells that did express US28. US28 was also capable of sequestering exogenously added RANTES. Surprisingly, cells infected with CMV Delta US28 transcribed and secreted increased levels IL-8, a CXC chemokine, when compared to CMV-infected cells. Finally, because chemokines are potent mediators of immune cell migration through the endothelium, we characterized the CC chemokine binding potential of CMV-infected endothelial cells. We propose that US28 functions as a 'chemokine sink' by sequestering endogenously and exogenously produced chemokines and alters the production of the CXC chemokine IL-8, suggesting that CMV could significantly alter the inflammatory milieu surrounding infected cells.     

Toll-like receptor (TLR) 2-9 agonists-induced cytokines and chemokines: I. Comparison with T cell receptor-induced responses

The cells of innate and adaptive immunity, although activated by different ligands, engage in cross talk to ensure a successful immune outcome. To better understand this interaction, we examined the demographic picture of individual TLR (TLRs 2-9) -driven profiles of eleven cytokines (IFN-alpha/beta, IFN-gamma, IL-12p40/IL-12p70, IL-4, 1L-13, TNF-alpha, IL-1beta, IL-2, IL-10) and four chemokines (MCP-1, MIP1beta, IL-8, and RANTES), and compared them with direct T-cell receptor triggered responses in an assay platform using human PBMCs. We find that T-cell activation by a combination of anti-CD3/anti-CD28/PHA induced a dominant IL-2, IL-13, and Type-II interferon (IFN-gamma) response without major IL-12 and little Type-I interferon (IFN-alphabeta) release. In contrast, TLR7 and TLR9 agonists induced high levels of Type-I interferons. The highest IFN-gamma levels were displayed by TLR8 and TLR7/8 agonists, which also induced the highest levels of pro-inflammatory cytokines IL-12, TNF-alpha, and IL-1beta. Amongst endosomal TLRs, TLR7 displayed a unique profile producing weak IL-12, IFN-gamma, TNF-alpha, IL-1beta, and IL-8. TLR7 and TLR9 resembled each other in their cytokine profile but differed in MIP-1beta and MCP1 chemokine profiles. Gram positive (TLR2, TLR2/6) and gram negative (TLR4) pathogen-derived TLR agonists displayed significant similarities in profile, but not in potency. TLR5 and TLR2/6 agonists paralleled TLR2 and TLR4 in generating pro-inflammatory chemokines MCP-1, MIP-1beta, RANTES, and IL-8 but yielded weak TNF-alpha and IL-1 responses. Taken together, the data show that diverse TLR agonists, despite their operation through common pathways induce distinct cytokine/chemokine profiles that in turn have little or no overlap with TCR-mediated response.     

Effects of dietary ω3 and ω6 lipids and vitamin E on chemokine levels in autoimmune-prone MRL/MpJ-lpr/lpr mice

Elevated levels of chemokines, such as Regulated upon Activation, Normal T cell Expressed and Secreted (RANTES), Monocyte Chemotactic Protein-1 (MCP-1), Macrophage Inflammatory Protein-1α (MIP-1α), and Macrophage Inflammatory Protein-1β (MIP-1β) have been found in rheumatoid arthritis (RA) and juvenile arthritis (JA), and they may be associated with the pathogenesis of these diseases. These chemokines are implicated in the migration of specific leukocytes into the joints. Omega-3 (ω3) fatty acid rich-fish oil (FO) and vitamin E may delay the progress of certain autoimmune diseases. The present study was designed to understand the effects of dietary lipids (ω-6 and ω-3 fatty acids) and vitamin E on the production of chemokines in autoimmune-prone MRL/lpr (a mouse model for RA) and congenic control MRL/++ mice. The MRL mice were fed for 4.5 months ω-6 and ω-3 diets that varied in lipid sources (corn oil; CO and fish oil; FO) and vitamin E levels (269 I.U./kg and 694 I.U./kg diet). Spleen cells were isolated and cultured aseptically in the presence of PHA for 48 h at 37°C and the levels of chemokines (RANTES, JE/MCP-1 and MIP-1α) were determined in the cell-free supernatants. The levels of RANTES and JE/MCP-1 were significantly higher in MRL/lpr mice compared to MRL/++ mice. The FO had differential effect on RANTES and MCP-1 production by spleen cells. The production of RANTES and JE/MCP-1 by spleen cells in mice fed the FO diets was significantly lower than in mice fed the CO diets (p < 0.0001). The levels of vitamin E did not affect the production of RANTES and JE/MCP-1. The levels of vitamin E had a significant effect on MIP-1α as the spleen cells of mice fed diets containing 694 IU/kg diet of vitamin E produced significantly higher levels of MIP-1α compared to the group of mice fed the diets containing 269 IU of vitamin E (p < 0.0001). The data obtained from this study in MRL/lpr and MRL/++ mice suggest that FO diets containing ω-3 fatty acids are beneficial in decreasing the levels of certain pro-inflammatory chemokines (RANTES and MCP-1) thereby delaying the onset of and severity of autoimmune symptoms in MRL/lpr mouse model.     

Chemokine production and pattern recognition receptor (PRR) expression in whole blood stimulated with pathogen-associated molecular patterns (PAMPs)

Recognition of conserved bacterial structures called pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs), may lead to induction of a variety of "early immediate genes" such as chemokines. In the current study, we have in an ex vivo whole blood model studied the induction of the chemokines MIP-1alpha, MCP-1 and IL-8 by various PAMPs. The rate of appearance of Escherichia coli-Lipopolysaccharide (LPS) induced chemokines differed. The production of MIP-1alpha and IL-8 was after 1 h of stimulation significantly higher when compared to unstimulated whole blood, whereas MCP-1 was not significantly elevated until after 3 h. At peak levels the MIP-1alpha concentration induced by E. coli-LPS was 3-5-fold higher than MCP-1 and IL-8. By specific cell depletion, we demonstrated that all three chemokines were mainly produced by monocytes. However, the mRNA results showed that IL-8 was induced in both monocytes and granulocytes. The production of all three chemokines, induced by the E. coli-LPS and Neisseria meningitidis-LPS, was significantly inhibited by antibodies against CD14 and TLR4, implying these receptors to be of importance for the effects of LPS in whole blood. The chemokine production induced by lipoteichoic acid (LTA) and non-mannose-capped lipoarabinomannan (AraLAM) was, however, less efficiently blocked by antibodies against CD14 and TLR2. E. coli-LPS and LTA induced a dose-dependent increase of CD14, TLR2 and TLR4 expression on monocytes in whole blood. These data show that PAMPs may induce chemokine production in whole blood and that antibodies against PRRs inhibit the production to different extent.     

Chemokine responses and accumulation of inflammatory cells in the lungs of mice infected with highly virulent Cryptococcus neoformans: effects of interleukin-12

We examined the mechanisms involved in the development of lung lesions after infection with Cryptococcus neoformans by comparing the histopathological findings and chemokine responses in the lungs of mice infected with C. neoformans and assessed the effect of interleukin (IL) 12 which protects mice from lethal infection. In mice infected intratracheally with a highly virulent strain of C. neoformans, the yeast cells multiplied quickly in the alveolar spaces but only a poor cellular inflammatory response was observed throughout the course of infection. Very little or no production of chemokines, including MCP-1, RANTES, MIP-1alpha, MIP-1beta and IP-10, was detected at the mRNA level using RT-PCR as well as at a protein level in MCP-1, RANTES and MIP-1alpha. In contrast, intraperitoneal administration of IL-12 induced the synthesis of these chemokines and a marked cellular inflammatory response involving histiocytes and lymphocytes in infected mice. Our findings were confirmed by flow cytometry of intraparenchymal leukocytes obtained from lung homogenates which showed IL-12-induced accumulation of inflammatory cells consisting mostly of macrophages and CD4+ alphabeta T cells. On the other hand, C-X-C chemokines including MIP-2 and KC, which attract neutrophils, were produced in infected and PBS-treated mice but treatment with IL-12 showed a marginal effect on their level, and neutrophil accumulation was similar in PBS- and IL-12-treated mice infected with C. neoforman. Our results demonstrate a close correlation between chemokine levels and development of lung lesions, and suggest that the induction of chemokine synthesis may be one of the mechanisms of IL-12-induced protection against cryptococcal infection.     

CKbeta-8 [CCL23], a novel CC chemokine, is chemotactic for human osteoclast precursors and is expressed in bone tissues

We have previously demonstrated that a tartrate-resistant acid phosphatase (TRAP)-positive subpopulation of mononuclear cells isolated from collagenase digests of human osteoclastoma tissue exhibits an osteoclast phenotype and can be induced to resorb bone. Using these osteoclast precursors as a model system, we have assessed the chemotactic potential of 16 chemokines. Three CC chemokines, the recently described CKbeta-8, RANTES, and MIP-1alpha elicited significant chemotactic responses. In contrast, 10 other CC chemokines (MIP-1beta, MCP-1, MCP-2, MCP-3, MCP-4, HCC-1, eotaxin-2, PARC, SLC, ELC) and 3 CXC chemokines (IL-8, GROalpha, SDF-1) were inactive. None of these chemokines showed any chemotactic activity for either primary osteoblasts derived from human bone explants or the osteoblastic MG-63 cell line. The identity of the osteoclast receptor that mediates the chemotactic response remains to be established. However, all three active chemokines have been reported to bind to CCR1 and cross-desensitization studies demonstrate that RANTES and MIP-1alpha can partially inhibit the chemotactic response elicited by CKbeta-8. CKbeta-8, the most potent of the active CC chemokines (EC(max) 0.1-0.3 nM), was further characterized with regard to expression in human bone and cartilage. Although expression is not restricted to these tissues, CKbeta-8 mRNA was shown to be highly expressed in osteoblasts and chondrocytes in human fetal bone by in situ hybridization. In addition, CKbeta-8 protein was shown to be present in human osteophytic tissue by immunolocalization. These observations suggest that CKbeta-8, and perhaps other chemokines, may play a role in the recruitment of osteoclast precursors to sites of bone resorption.     

Functional IL-2 receptor beta (CD122) and gamma (CD132) chains are expressed by fibroblast-like synoviocytes: activation by IL-2 stimulates monocyte chemoattractant protein-1 production

The expression of the IL-2R alpha-, beta-, and gamma-chains, CD25, CD122, and CD132, respectively, was investigated on fibroblast-like synoviocytes (FLS) and dermal fibroblasts (DF). Both protein and mRNA for CD122 and CD132 were observed but there was no evidence of CD25 expression. Quantification of the Ag binding sites for CD122 showed that FLS expressed 4 times more receptor molecules than DF. The functional capability of these receptors was confirmed by the production of monocyte chemoattractant protein-1 (MCP-1) in direct response to stimulation by IL-2, which could be inhibited by neutralizing anti-CD122 mAb. Both rheumatoid arthritis (RA) and osteoarthritis (OA) FLS and DF spontaneously produced MCP-1 in culture over a similar range of concentrations. However, RA and OA FLS produced significantly greater levels of MCP-1 following stimulation by IL-2 and IL-1 beta; RA FLS produced significantly more MCP-1 than OA FLS. Addition of exogenous IL-2 caused a slight, but significant, decrease in MCP-1 production by DF. The addition of neutralizing anti-CD122 mAb to FLS cultures partially, but significantly, reduced the IL-2-induced MCP-1 secretion, but did not effect either the spontaneous or IL-1 beta-induced secretion of MCP-1. Increased tyrosine phosphorylation was observed in FLS lysates following 30-min incubation with IL-2. In conclusion, in the inflamed synovium, as activated T cells migrate through the sublining and lining layer, T cell-derived IL-2 may activate FLS to secrete MCP-1, thus recruiting macrophages into the rheumatoid synovium and perpetuating inflammation.     

Acute ethanol administration downregulates human immunodeficiency virus-1 glycoprotein 120-induced KC and RANTES production by murine Kupffer cells and splenocytes

Glycoprotein 120 from HIV-1, HIV-2 and SIV is known to stimulate secretion of chemokines by mononuclear cells. Thus, this work tests the hypothesis that acute ethanol intoxication suppresses HIV-1 gp120-induced chemokine production by murine Kupffer cells and splenocytes. Male Balb/c mice were given ethanol (1.70 g/Kg) by intragastric gavage in 0.1 ml volume of saline. Five minutes after ethanol administration, mice received an intravenous injection of HIV-1 gp120 (5 microg/Kg). After 24 hr, serum samples, splenocytes and Kupffer cells were obtained. Isolated cells were cultured in DMEM for 24 hr to determine production of chemokines and cytokines in vitro. Chemokines (MIP-2, KC, RANTES, MIP-1 alpha and MCP-1) and cytokines (IL-1 beta, TNF alpha, IL-10, gamma-IFN) were measured by ELISA. M-RNA abundance of these mediators was determined by RT-PCR. Results show that HIV-1 gp120 treatment was associated with significant elevations in serum KC and RANTES. No changes were observed with regard to other chemokines and cytokines. Oral administration of ethanol significantly suppressed HIV-1gp120-induced KC and RANTES release. KC and RANTES-mRNA expression and protein release by splenocytes and Kupffer cells were up-regulated by HIV-1 gp120. Such up-regulation was attenuated by ethanol treatment. These data show that acute ethanol administration attenuates HIV-1 gp120-induced chemokine release in vivo by isolated splenocytes and Kupffer cells. Through this mechanism, previous in vivo ethanol use may compromise the ability of HIV-1 gp120 to induce chemokine-mediated inhibition of HIV-1 entry into target cells.     

Host response to malaria during pregnancy: placental monocyte recruitment is associated with elevated beta chemokine expression

Malaria during pregnancy is associated with poor birth outcomes, particularly low birth weight. Recently, monocyte infiltration into the placental intervillous space has been identified as a key risk factor for low birth weight. However, the malaria-induced chemokines involved in recruiting and activating placental monocytes have not been identified. In this study, we determined which chemokines are elevated during placental malaria infection and the association between chemokine expression and placental monocyte infiltration. Placental malaria infection was associated with elevations in mRNA expression of three beta chemokines, macrophage-inflammatory protein 1 (MIP-1) alpha (CCL3), monocyte chemoattractant protein 1 (MCP-1; CCL2), and I-309 (CCL1), and one alpha chemokine, IL-8 (CXCL8); all correlated with monocyte density in the placental intervillous space. Placental plasma concentrations of MIP-1 alpha and IL-8 were increased in women with placental malaria and were associated with placental monocyte infiltration. By immunohistochemistry, we localized placental chemokine production in malaria-infected placentas: some but not all hemozoin-laden maternal macrophages produced MIP-1 beta and MCP-1, and fetal stromal cells produced MCP-1. In sum, local placental production of chemokines is increased in malaria, and may be an important trigger for monocyte accumulation in the placenta.     

Enhancing effect of IL-1, IL-17, and TNF-alpha on macrophage inflammatory protein-3alpha production in rheumatoid arthritis: regulation by soluble receptors and Th2 cytokines.

Macrophage inflammatory protein (MIP)-3alpha is a chemokine involved in the migration of T cells and immature dendritic cells. To study the contribution of proinflammatory cytokines and chemokines to the recruitment of these cells in rheumatoid arthritis (RA) synovium, we looked at the effects of the monocyte-derived cytokines IL-1beta and TNF-alpha and the T cell-derived cytokine IL-17 on MIP-3alpha production by RA synoviocytes. Addition of IL-1beta, IL-17, and TNF-alpha induced MIP-3alpha production in a dose-dependent manner. At optimal concentrations, IL-1beta (100 pg/ml) was much more potent than IL-17 (100 ng/ml) and TNF-alpha (100 ng/ml). When combined at lower concentrations, a synergistic effect was observed. Conversely, the anti-inflammatory cytokines IL-4 and IL-13 inhibited MIP-3alpha production by activated synoviocytes, but IL-10 had no effect. Synovium explants produced higher levels of MIP-3alpha in RA than osteoarthritis synovium. MIP-3alpha-producing cells were located in the lining layer and perivascular infiltrates in close association with CD1a immature dendritic cells. Addition of exogenous IL-17 or IL-1beta to synovium explants increased MIP-3alpha production. Conversely, specific soluble receptors for IL-1beta, IL-17, and TNF-alpha inhibited MIP-3alpha production to various degrees, but 95% inhibition was obtained only when the three receptors were combined. Similar optimal inhibition was also obtained with IL-4, but IL-13 and IL-10 were less active. These findings indicate that interactions between monocyte and Th1 cell-derived cytokines contribute to the recruitment of T cells and dendritic cells by enhancing the production of MIP-3alpha by synoviocytes. The inhibitory effect observed with cytokine-specific inhibitors and Th2 cytokines may have therapeutic applications.     

Proinflammatory response of alveolar epithelial cells is enhanced by alveolar macrophage-produced TNF-alpha during pulmonary ischemia-reperfusion injury

Pulmonary ischemia-reperfusion (IR) injury entails acute activation of alveolar macrophages followed by neutrophil sequestration. Although proinflammatory cytokines and chemokines such as TNF-alpha and monocyte chemoattractant protein-1 (MCP-1) from macrophages are known to modulate acute IR injury, the contribution of alveolar epithelial cells to IR injury and their intercellular interactions with other cell types such as alveolar macrophages and neutrophils remain unclear. In this study, we tested the hypothesis that following IR, alveolar macrophage-produced TNF-alpha further induces alveolar epithelial cells to produce key chemokines that could then contribute to subsequent lung injury through the recruitment of neutrophils. Cultured RAW264.7 macrophages and MLE-12 alveolar epithelial cells were subjected to acute hypoxia-reoxygenation (H/R) as an in vitro model of pulmonary IR. H/R (3 h/1 h) significantly induced KC, MCP-1, macrophage inflammatory protein-2 (MIP-2), RANTES, and IL-6 (but not TNF-alpha) by MLE-12 cells, whereas H/R induced TNF-alpha, MCP-1, RANTES, MIP-1alpha, and MIP-2 (but not KC) by RAW264.7 cells. These results were confirmed using primary murine alveolar macrophages and primary alveolar type II cells. Importantly, using macrophage and epithelial coculture methods, the specific production of TNF-alpha by H/R-exposed RAW264.7 cells significantly induced proinflammatory cytokine/chemokine expression (KC, MCP-1, MIP-2, RANTES, and IL-6) by MLE-12 cells. Collectively, these results demonstrate that alveolar type II cells, in conjunction with alveolar macrophage-produced TNF-alpha, contribute to the initiation of acute pulmonary IR injury via a proinflammatory cascade. The release of key chemokines, such as KC and MIP-2, by activated type II cells may thus significantly contribute to neutrophil sequestration during IR injury.     

Inhibition of endotoxin-induced macrophage chemokine production by VIP and PACAP in vitro and in vivo

Inflammatory chemokines recruit immune cells which initiate and maintain the inflammatory response. Although such a response is necessary for the elimination of the antigen, the inflammation has to be eventually resolved. Peptides such as vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP), released following antigenic stimulation, contribute to the termination of an inflammatory response primarily by inhibiting the production of proinflammatory cytokines. Here we investigated the effects of VIP and PACAP on chemokine production. We report that VIP and PACAP inhibit the expression of the macrophage-derived CXC chemokines MIP-2 and KC (IL-8), and of the CC chemokines MIP-1a, MIP-1b, MCP-1 and RANTES in vivo and in vitro. The decrease of chemokine gene expression correlates with an inhibitory effect of VIP/PACAP on NFkB binding. In an in vivo model of acute peritonitis, the inhibition of chemokine production by VIP/PACAP leads to a significant reduction in the recruitment of PMNs, macrophages and lymphocytes into the peritoneal cavity. These findings support the proposed role of VIP and PACAP as key endogenous anti-inflammatory agents, and describe a novel mechanism, i.e., the inhibition of the production of macrophage-derived chemokines.     

IL-17RA signaling amplifies antibody-induced arthritis

Objective

To investigate the role of IL-17RA signaling in the effector phase of inflammatory arthritis using the K/BxN serum-transfer model.

Methods

Wild-type and Il17ra^−/− mice were injected with serum isolated from arthritic K/BxN mice and their clinical score was recorded daily. Mice were also harvested on days 12 and 21 and ankles were analyzed for cytokine and chemokine mRNA expression by qPCR on day 12 and for bone and cartilage erosions by histology on day 21, respectively. The induction of cytokine and chemokine expression levels by IL-17A in synovial-like fibroblasts was also analyzed using qPCR.

Results

Il17ra^−/− mice were partially protected from clinical signs of arthritis and had markedly fewer cartilage and bone erosions. The expression of several pro-inflammatory mediators, including the chemokines KC/CXCL1, MIP-2/CXCL2, LIX/CXCL5 MIP-1γ/CCL9, MCP-3/CCL7, MIP-3α/CCL20, the cytokines IL-1β, IL-6, RANKL and the matrix metalloproteinases MMP2, MMP3, and MMP13 were decreased in the ankles of Il17ra^−/− mice compared to wild-type mice. Many of these proinflammatory genes attenuated in the ankles of Il17ra^−/− mice were shown to be directly induced by IL-17A in synovial fibroblasts in vitro.

Conclusions

IL-17RA signaling plays a role as an amplifier of the effector phase of inflammatory arthritis. This effect is likely mediated by direct activation of synovial fibroblasts by IL-17RA to produce multiple inflammatory mediators, including chemokines active on neutrophils. Therefore, interrupting IL-17RA signaling maybe a promising pharmacological target for the treatment of inflammatory arthritis.     

Role of the pregnancy-specific glycoprotein in regulation of the cytokine and chemokine profiles of intact mononuclear cells

The effect of human pregnancy-specific glycoprotein (PSG) on the cytokine and chemokine production in vitro by intact mononuclear cells was studied by the method of flow fluorimetry. PSG inhibited production of the proinflammatory cytokines IL-6, IL-8, IL-17, IFN-γ, and TNF-α and chemokines CCL3/MIP-1α, CCL4/MIP-1β, CCL2/MCP-1; at the same time, PSG stimulated IL-12(p70) production. Simultaneously with increasing the VEGF level, PSG inhibited production of IL-9, IL-13, G-CSF, and GM-CSF. The PSG effect discovered can be interpreted as a contribution into the immune tolerance formation during pregnancy.     

A small synthetic molecule capable of preferentially inhibiting the production of the CC chemokine monocyte chemotactic protein-1.

Blocking chemokine production or action is a major target for pharmacological intervention in different human diseases. Bindarit (2-methyl-2-[[1-(phenylmethyl)-1H-indazol-3yl]methoxy]propan oic acid) dose-dependently inhibited MCP-1 and TNF-alpha production induced in vitro in monocytes by LPS and Candida albicans. It did not affect the production of the cytokines IL-1, IL-6, or the chemokines IL-8, MIP-1alpha and RANTES. In the air pouch model in mice, oral treatment reduced monocyte recruitment and local MCP-1 production, induced by carrageenan or IL-1 injection. In NZB/W mice, a model of lupus nephritis, oral treatment prolonged survival and delayed the onset of proteinuria. The results presented here show that bindarit is a preferential inhibitor of the production of MCP-1 in vitro and in vivo and suggest that its beneficial effects in models of joint and kidney inflammation are related to its anti-MCP-1 action. It is therefore possible to selectively and differentially regulate chemokines by targeting their production with small synthetic molecules.