Selective transport of cytokine-induced neutrophil chemoattractant from the lung to the blood facilitates pulmonary neutrophil recruitment

The CXC chemokines cytokine-induced neutrophil chemoattractant (CINC) and macrophage inflammatory protein-2 (MIP-2) are potent neutrophil chemoattractants in rats. We have previously shown that CINC, unlike MIP-2 and most other proinflammatory cytokines, is elevated in the systemic circulation in response to an intratracheal (IT) challenge. Therefore, we hypothesized that CINC generated within the lung selectively enters the vascular compartment to facilitate pulmonary neutrophil recruitment. Rats were administered IT LPS, and plasma CINC and MIP-2 levels were measured 90 min and 4 h after injection, along with mRNA expression in lung, spleen, liver, and kidney. Ninety minutes and 4 h after IT LPS, CINC and MIP-2 mRNA expression were largely confined to lung homogenate, but of the two chemokines, only CINC was present in plasma. In separate experiments, rats received IT injections of recombinant CINC and/or MIP-2. Here, plasma levels of CINC, but not MIP-2, were significantly increased throughout the 4-h observation period. This finding was verified by individually administering (125)I-labeled forms of each chemokine. Instillation of recombinant MIP-2 or CINC into the lung increased the number of neutrophils recovered in bronchoalveolar lavage fluid at 4 h, and this effect was enhanced when both chemokines were administered together. In addition, intravenous (IV) CINC, but not IV MIP-2, increased pulmonary neutrophil recruitment in response to IT MIP-2. Our results show that CINC, in contrast to MIP-2, is selectively transported from the lung to the systemic circulation, where it promotes neutrophil migration into the lung in response to a chemotactic stimulus.     

Monocyte chemoattractant protein-1 and macrophage inflammatory protein-2 are involved in both excitotoxin-induced neurodegeneration and regeneration

Intrahippocamal injections of kainic acid (KA) significantly increase the expression of monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-2 (MIP-2) in the ipsilateral hippocampus at 2-4 h and 21-45 days post-administration, suggesting the possible involvement of these chemokines in both neurodegenerative and regenerative processes. To examine the possible role of these chemokines on neuronal cell death, hippocampal neurons were incubated with either MCP-1 or MIP-2 in vitro and examined to assess the effects on neuronal cell viability. These treatments resulted in significant neuronal apoptosis that could be abrogated by prior treatment with the caspase-1 inhibitor, Z-VAD-FMK, the caspase-3 inhibitor, Z-DEVD-FMK, the Galphai inhibitor, pertussis toxin, or the MAO-B inhibitor, (-)deprenyl. Furthermore, this chemokine apoptotic effect could also be observed in vivo as intrahippocampal injections of MCP-1 or MIP-2 resulted in the apoptosis of hippocampal neurons, thus supporting a direct role of these chemokines in neuronal death. In contrast, immunohistological analysis of kainic acid lesions on days 21-45 revealed significant expression of MCP-1 and MIP-2 associated with reactive astrocytes and macrophages, respectively, with no apoptotic populations being observed. These results suggested that these chemokines might also mediate distinct biological effects on local microenvironmental cell populations at various stages post truama and during cellular repair. To address this possibility, astrocyte were cultured in the presence or absence of these chemokines and examined by microarray analysis for effects on astrocytes gene expression. A number of genes encoding proteins associated with inflammation, cellular signaling, differentiation, and repair were directly modulated by chemokine treatment. More specifically, the RNA and protein expression of the neurotrophic factor, basic fibroblast growth factor (bFGF), was found to be significantly increased upon culture with MCP-1 and MIP-2. Conditioned media derived from chemokine-stimulated astrocytes also facilitated bFGF-dependent neuronal cell differentiation and promoted survival of H19-7 neurons in vitro, suggesting a possible role for chemokine-activated astrocytes as a source of trophic support. Taken together, these data support possible autocrine and paracrine roles for MCP-1 and MIP-2 in both the "death and life" of hippocampal neurons following CNS injury.     

Mechanical stretch stimulates macrophage inflammatory protein-2 secretion from fetal rat lung cells

Ventilation-induced lung injury has been related to cytokine production. Immaturity and barotrauma are important contributors to the development of bronchopulmonary dysplasia in infants. In the present study, stretch of organotypic cultured fetal rat lung cells was used to simulate ventilation of preterm newborns. Cells were stimulated with lipopolysaccharide (LPS; 100 ng/ml) and/or mechanical stretch. After 4 h, stretch enhanced LPS-induced macrophage inflammatory protein (MIP)-2 production in a force- and frequency-dependent manner. The maximal effect of stretch was seen with 5% elongation at 40 cycles/min. In contrast, after 1 h of stimulation, stretch alone significantly increased MIP-2 production, which was not blocked by cycloheximide, an inhibitor of protein synthesis. At both the 1- and 4-h time points, only LPS increased MIP-2 mRNA levels. Stretch-induced MIP-2 release was associated with cell injury as measured by lactate dehydrogenase release and was not inhibited by gadolinium, a stretch-activated ion channel blocker. Taken together, these results suggest that the major effect of stretch on MIP-2 production from fetal rat lung cells is to stimulate its secretion.     

Production and regulation of cytokine-induced neutrophil chemoattractant in rat ovulation

A cytokine-induced neutrophil chemoattractant (CINC/gro), which belongs to the interleukin (IL)-8 family, acts as a functional chemoattractant for neutrophils in rats. In the present study, we examined whether CINC/gro contributes to the ovulation process in the rat ovulation system. In rat ovaries, CINC/gro was immunohistochemically recognized in the theca layer of the antral follicle but not in the granulosa cells. To clarify the role of CINC/gro in the ovulation process, CINC/gro protein and mRNA were examined during pregnant mare serum gonadotropin (PMSG)-hCG treatment. CINC/gro protein did not increase as a result of PMSG injection. However, it increased rapidly after hCG injection and peaked at 6 h after hCG. CINC/gro mRNA was also strongly expressed after hCG injection. The increase of CINC/gro protein followed increases in IL-1beta and tumor necrosis factor alpha (TNFalpha). In the whole ovarian dispersate culture, FSH, hCG, IL-1beta, and TNFalpha stimulated the production of CINC/gro protein in a dose-dependent manner. In particular, the stimulatory effects of IL-1beta and TNFalpha were stronger than those of gonadotropins. These results suggest that CINC/gro plays an important role in the rat ovulation process by attracting neutrophils. CINC/gro increased just prior to ovulation, and it may be regulated directly by cytokines such as IL-1beta and TNFalpha and indirectly by gonadotropins.     

Role of CC chemokines (macrophage inflammatory protein-1 beta, monocyte chemoattractant protein-1, RANTES) in acute lung injury in rats

The role of the CC chemokines, macrophage inflammatory protein-1 beta (MIP-1 beta), monocyte chemotactic peptide-1 (MCP-1), and RANTES, in acute lung inflammatory injury induced by intrapulmonary deposition of IgG immune complexes injury in rats was determined. Rat MIP-1 beta, MCP-1, and RANTES were cloned, the proteins were expressed, and neutralizing Abs were developed. mRNA and protein expression for MIP-1 beta and MCP-1 were up-regulated during the inflammatory response, while mRNA and protein expression for RANTES were constitutive and unchanged during the inflammatory response. Treatment of rats with anti-MIP-1 beta Ab significantly decreased vascular permeability by 37% (p = 0.012), reduced neutrophil recruitment into lung by 65% (p = 0.047), and suppressed levels of TNF-alpha in bronchoalveolar lavage fluids by 61% (p = 0.008). Treatment of rats with anti-rat MCP-1 or anti-rat RANTES had no effect on the development of lung injury. In animals pretreated intratracheally with blocking Abs to MCP-1, RANTES, or MIP-1 beta, significant reductions in the bronchoalveolar lavage content of these chemokines occurred, suggesting that these Abs had reached their targets. Conversely, exogenously MIP-1 beta, but not RANTES or MCP-1, caused enhancement of the lung vascular leak. These data indicate that MIP-1 beta, but not MCP-1 or RANTES, plays an important role in intrapulmonary recruitment of neutrophils and development of lung injury in the model employed. The findings suggest that in chemokine-dependent inflammatory responses in lung CC chemokines do not necessarily demonstrate redundant function.     

Molecular cloning and characterization of a novel CXC chemokine macrophage inflammatory protein-2 gamma chemoattractant for human neutrophils and dendritic cells

Chemokines play important roles in leukocyte trafficking as well as function regulation. In this study, we described the identification and characterization of a novel CXC chemokine from a human dendritic cell (DC) cDNA library, the full-length cDNA of which contains an open reading frame encoding 111 aa with a putative signal peptide of 34 aa. This CXC chemokine shares greatest homology with macrophage inflammatory protein (MIP)-2alphabeta, hence is designated as MIP-2gamma. Mouse MIP-2gamma was identified by electrocloning and is highly homologous to human MIP-2gamma. Northern blotting revealed that MIP-2gamma was constitutively and widely expressed in most normal tissues with the greatest expression in kidney, but undetectable in most tumor cell lines except THP-1 cells. In situ hybridization analysis demonstrated that MIP-2gamma was mainly expressed by the epithelium of tubules in the kidney and hepatocytes in the liver. Although no detectable expression was observed in freshly isolated or PMA-treated monocytes, RT-PCR analysis revealed MIP-2gamma expression by monocyte-derived DC. Recombinant MIP-2gamma from 293 cells is about 9.5 kDa in size and specifically detectable by its polyclonal Ab developed by the immunization with its 6His-tagged fusion protein. The eukaryotically expressed MIP-2gamma is a potent chemoattractant for neutrophils, and weaker for DC, but inactive to monocytes, NK cells, and T and B lymphocytes. Receptor binding assays showed that MIP-2gamma does not bind to CXCR2. This implies that DC might contribute to the innate immunity through the production of neutrophil-attracting chemokines and extends the knowledge about the regulation of DC migration.     

Role of microtubules in LPS-induced macrophage inflammatory protein-2 production from rat pneumocytes

We have recently demonstrated that primary cultured rat pneumocytes produce macrophage inflammatory protein-2 (MIP-2) in response to lipopolysaccharide (LPS) stimulation. In this study, we found that brefeldin A, by blocking anterograde transport from the endoplasmic reticulum (ER) to the Golgi apparatus, decreased LPS-induced MIP-2 in the culture medium and increased its storage in cells. This suggests that MIP-2 is secreted via a pathway from the ER to the Golgi apparatus, a process commonly regulated by microtubules. We further found that LPS induced depolymerization of microtubules as early as 1 min after LPS stimulation, and it lasted at least for 4 h. Preventing depolymerization of microtubules with paclitaxel (Taxol; 10 nM to 10 microM) partially inhibited LPS-induced MIP-2 production, whereas the microtubule-depolymerizing agents colchicine (1-10 microM) and nocodazole (1-100 microM) increased LPS-induced MIP-2 protein production without affecting MIP-2 mRNA expression. These results suggest that in pneumocytes, LPS-induced microtubule depolymerization is involved in LPS-induced MIP-2 production and that secretion of MIP-2 from pneumocytes is via the ER-Golgi pathway.     

Saliva from Lutzomyia longipalpis induces CC chemokine ligand 2/monocyte chemoattractant protein-1 expression and macrophage recruitment

Saliva of bloodfeeding arthropods has been incriminated in facilitating the establishment of parasite in their host. We report on the leukocyte chemoattractive effect of salivary gland homogenate (SGH) from Lutzomyia longipalpis on saliva-induced inflammation in an air pouch model. SGH (0.5 pair/animal) was inoculated in the air pouch formed in the back of BALB/c or C57BL/6 mice. L. longipalpis SGH induced a significant influx of macrophages in BALB/c but not in C57BL/6 mice. SGH-induced cell recruitment reached a peak at 12 h after inoculation and was higher than that induced by the LPS control. This differential cell recruitment in BALB/c mice was directly correlated to an increase in CCL2/MCP-1 expression in the air pouch lining tissue. In fact, treatment with bindarit, an inhibitor of CCL2/MCP-1 synthesis, and also with a specific anti-MCP-1 mAb resulted in drastic reduction of macrophage recruitment and inhibition of CCL2/MCP-1 expression in the lining tissue. CCL2/MCP-1 production was also seen in vitro when J774 murine macrophages were exposed to L. longipalpis SGH. The SGH effect was abrogated by preincubation with serum containing anti-SGH IgG Abs as well as in mice previously sensitized with L. longipalpis bites. Interestingly, the combination of SGH with Leishmania chagasi induced an increased recruitment of neutrophils and macrophages when compared with L. chagasi alone. Taken together these results suggest that SGH not only induces the recruitment of a greater number of macrophages by enhancing CCL2/MCP-1 production but also synergizes with L. chagasi to recruit more inflammatory cells to the site of inoculation.     

Generation of inflammatory cytokines in zymosan-induced pleurisy in rats: TNF induces IL-6 and cytokine-induced neutrophil chemoattractant (CINC) in vivo

Levels of inflammatory cytokines tumour necrosis factor (TNF), interleukin 1 (IL-1), IL-6, and cytokine-induced neutrophil chemoattractant (CINC), which is a member of the alpha-chemokine family in rats, were measured in the pleural exudates during zymosan-induced pleurisy to examine the relationship between the local production of cytokines and the inflammatory reaction. All four cytokine levels in the pleural exudate began to increase after 1-2 h, preceding the influx of neutrophils, and peaked after 4-5 h. Thereafter, these cytokine levels declined after 24 h, whereas the exudate volume still continued to increase and leukocyte number reached a plateau. Concomitant injection of actinomycin D (10 microg) with zymosan markedly suppressed the neutrophil infiltration, parallel with CINC production in the pleural exudate at 4 h. A transient elevation of IL-6 level, peaking at 5 h, and subsequent rise in the level of an acute-phase protein, T-kininogen, were also observed in the plasma. When recombinant human TNF-alpha (rhTNF-alpha) (20 000 U) was intrapleurally injected a rapid increase in pleural CINC level, followed by neutrophil infiltration, and a sharp rise in IL-6 level in the plasma, followed by an increase in T-kininogen, were demonstrated. These results suggest that CINC produced in the pleural exudate may participate in neutrophil infiltration, that IL-6 induced in the plasma stimulates T-kininogen production, and that endogenous TNF may be partly involved in the induction of CINC and IL-6 in this zymosan inflammation.     

Differential monocyte chemoattractant protein-1 and chemokine receptor 2 expression by murine lung fibroblasts derived from Th1- and Th2-type pulmonary granuloma models.

Recent studies suggest that monocyte chemoattractant protein-1 (MCP-1) is involved in fibrosis through the regulation of profibrotic cytokine generation and matrix deposition. Changes in MCP-1, C-C chemokine receptor 2 (CCR2), procollagen I and III, and TGF beta were examined in fibroblasts cultured from normal lung and from nonfibrotic (i.e., Th1-type) and fibrotic (i.e., Th2-type) pulmonary granulomas. Th2-type fibroblasts generated 2-fold more MCP-1 than similar numbers of Th1-type or normal fibroblasts after 24 h in culture. Unlike normal and Th1-type fibroblasts, Th2-type fibroblasts displayed CCR2 mRNA at 24 h after IL-4 treatment. By flow cytometry, CCR2 was present on 40% of untreated Th2-type fibroblasts, whereas CCR2 was present on <20% of normal and Th1-type fibroblasts after similar treatment. IL-4 increased the number of normal fibroblasts with cell-surface CCR2 but IFN-gamma-treatment of normal and Th2-type fibroblasts significantly decreased the numbers of CCR2-positive cells in both populations. Western blot analysis showed that total CCR2 protein expression was markedly increased in untreated Th2-type fibroblasts compared with normal and Th1-type fibroblasts. IL-4 treatment enhanced CCR2 protein in Th1- and Th2-type fibroblasts whereas IFN-gamma treatment augmented CCR2 protein in normal and Th1-type fibroblasts. All three fibroblast populations exhibited MCP-1-dependent TGF-beta synthesis, but only normal and Th2-type fibroblasts showed a MCP-1 requirement for procollagen mRNA expression. Taken together, these findings suggest that lung fibroblasts are altered in their expression of MCP-1, TGF-beta, CCR2, and procollagen following their participation in pulmonary inflammatory processes, and these changes may be important during fibrosis.     

Differential effects of leukotactin-1 and macrophage inflammatory protein-1 alpha on neutrophils mediated by CCR1

The human CC chemokine leukotactin-1 (Lkn-1) is both a strong chemoattractant for neutrophils, monocytes, and lymphocytes and a potent agonist for CCR1 and CCR3. However, human neutrophils do not migrate when the cells are stimulated with other human CC chemokines, such as human macrophage inflammatory protein-1 alpha (hMIP-1 alpha) and eotaxin, which also use the CCR1 and CCR3 as their receptors. In this report, we demonstrate that while hMIP-1 alpha induced a negligible level of calcium flux and chemotaxis, Lkn-1 produced a high level of calcium flux and chemotaxis in human neutrophils. Lkn-1 cross-desensitized hMIP-1 alpha-induced calcium flux, but hMIP-1 alpha had little effect on the Lkn-1-induced response in human neutrophils. The same pattern was observed in peritoneal neutrophils from wild-type mice, whereas neutrophils from CCR1-/- mice failed to respond to either MIP-1 alpha or Lkn-1. Scatchard analysis revealed a single class of receptor for both hMIP-1 alpha and Lkn-1 on human neutrophils with dissociation constants (Kd) of 3.2 nM and 1.1 nM, respectively. We conclude that CCR1 is a receptor mediating responses to both MIP-1 alpha and Lkn-1 on neutrophils and produces different biological responses depending on the ligand bound.     

Neutralization of the CXC chemokine, macrophage inflammatory protein-2, attenuates bleomycin-induced pulmonary fibrosis

Few studies have addressed the importance of vascular remodeling in the lung during the development of bleomycin-induced pulmonary fibrosis. For fibroplasia and deposition of extracellular matrix to occur, there must be a geometric increase in neovascularization. We hypothesized that net angiogenesis during the pathogenesis of fibroplasia and deposition of extracellular matrix during bleomycin-induced pulmonary fibrosis are dependent in part upon an overexpression of the angiogenic CXC chemokine, macrophage inflammatory protein-2 (MIP-2). To test this hypothesis, we measured MIP-2 by specific ELISA in whole lung homogenates in either bleomycin-treated or control CBA/J mice and correlated these levels with lung hydroxyproline. We found that lung tissue from mice treated with bleomycin, compared with that from saline-treated controls, demonstrated a significant increase in the presence of MIP-2 that was correlated to a greater angiogenic response and total lung hydroxyproline content. Neutralizing anti-MIP-2 Abs inhibited the angiogenic activity of day 16 bleomycin-treated lung specimens using an in vivo angiogenesis bioassay. Furthermore, when MIP-2 was depleted in vivo by passive immunization, bleomycin-induced pulmonary fibrosis was significantly reduced without a change in the presence of pulmonary neutrophils, fibroblast proliferation, or collagen gene expression. This was also paralleled by a reduction in angiogenesis. These results demonstrate that the angiogenic CXC chemokine, MIP-2, is an important factor that regulates angiogenesis/fibrosis in pulmonary fibrosis.     

Neutralization of macrophage inflammatory protein-2 blocks the febrile response induced by lipopolysaccharide in rats

1. This study was aimed to test the hypothesis that macrophage inflammatory protein-2 (MIP-2), a powerful chemotactic cytokine for neutrophils, plays a role in bacterial endotoxin fever.

2. The effect of specific anti-rat MIP-2 antibodies on lipopolysaccharide (LPS)-induced fever was tested. Intraperitoneal injection of LPS resulted in a biphasic fever and a significant increase in serum MIP-2 and prostaglandin (PG) E₂ levels which correlated with the start of fever. Intraperitoneal anti-MIP-2 (500 μg/kg) did not affect the body core temperature of unrestrained rats, but markedly attenuated LPS-induced fever.

3. Treatment with the cyclooxygenase inhibitor ibuprofen (10 mg/kg) resulted in a significant attenuation of LPS-induced fever and a significant decrease of MIP-2 and PGE₂ production.

4. These results indicate that LPS fever in rats is, at least, in part dependent on mechanisms involving neutrophils chemotaxis, and that MIP-2 may be an important mediator in the genesis of fever via prostaglandin-dependent pathways.     

Differential regulation of interleukin-6, macrophage inflammatory protein-1, and JE/MCP-1 cytokine expression in macrophage cell lines.

Activated macrophages produce a number of proinflammatory cytokines including IL-6, JE, MIP-1 alpha and MIP-1 beta. The induction requirements for production of either IL-6 or the MIP-1 related inflammatory proteins (MIP-1 alpha, MIP-1 beta, and JE) have been analyzed independently using fibroblasts, monocytes, or endothelial cells. However, little is known about the regulation of these cytokines in macrophages. Since activated macrophages produce prostaglandins (PGE2) which may participate in the autoregulation of cytokine production by stimulation of adenylate cyclase and the induction of cAMP-dependent signal pathways, we determined the effects of PGE on the production of IL-6 and MIP-1-related proteins. Murine macrophage cell lines were incubated with PGE1, PGE2, cholera toxin, or dibutyryl cAMP in the presence of absence suboptimal doses of LPS. Pharmacologic agents alone did not induce IL-6 production but incubation of macrophages with combinations of adenylate cyclase stimulators and LPS or dcAMP and LPS led to the dose-dependent enhancement of IL-6 secretion and mRNA expression. In contrast, PGE1 inhibits LPS-induced JE, MIP-1 alpha, and MIP-1 beta mRNA expression and this inhibition is partially dependent on a cAMP-mediated pathway of signal transduction. In previous work we demonstrated that IFN-gamma and PMA do not stimulate the production of IL-6 by macrophages. Here we show that incubation of macrophages with either IFN-gamma or PMA induces the expression of JE, MIP-1 alpha and MIP-1 beta mRNA expression. JE mRNA expression is much more responsive to the stimulatory effects of IFN-gamma than are the MIP-1 genes. Finally, PGE inhibits PMA and IFN-gamma-induced JE and MIP-1-related mRNA expression.     

Overexpression of monocyte chemoattractant protein-1 in adipose tissues causes macrophage recruitment and insulin resistance

Adipose tissue expression and circulating concentrations of monocyte chemoattractant protein-1 (MCP-1) correlate positively with adiposity. To ascertain the roles of MCP-1 overexpression in adipose, we generated transgenic mice by utilizing the adipocyte P2 (aP2) promoter (aP2-MCP-1 mice). These mice had higher plasma MCP-1 concentrations and increased macrophage accumulation in adipose tissues, as confirmed by immunochemical, flow cytometric, and gene expression analyses. Tumor necrosis factor-alpha and interleukin-6 mRNA levels in white adipose tissue and plasma non-esterified fatty acid levels were increased in transgenic mice. aP2-MCP-1 mice showed insulin resistance, suggesting that inflammatory changes in adipose tissues may be involved in the development of insulin resistance. Insulin resistance in aP2-MCP-1 mice was confirmed by hyperinsulinemic euglycemic clamp studies showing that transgenic mice had lower rates of glucose disappearance and higher endogenous glucose production than wild-type mice. Consistent with this, insulin-induced phosphorylations of Akt were significantly decreased in both skeletal muscles and livers of aP2-MCP-1 mice. MCP-1 pretreatment of isolated skeletal muscle blunted insulin-stimulated glucose uptake, which was partially restored by treatment with the MEK inhibitor U0126, suggesting that circulating MCP-1 may contribute to insulin resistance in aP2-MCP-1 mice. We concluded that both paracrine and endocrine effects of MCP-1 may contribute to the development of insulin resistance in aP2-MCP-1 mice.     

Macrophage inflammatory protein-2 is a mediator of polymorphonuclear neutrophil influx in ocular bacterial infection

Polymorphonuclear neutrophils (PMN) in Pseudomonas aeruginosa-infected cornea are required to clear bacteria from affected tissue, yet their persistence may contribute to irreversible tissue destruction. This study examined the role of C-X-C chemokines in PMN infiltration into P. aeruginosa-infected cornea and the contribution of these mediators to disease pathology. After P. aeruginosa challenge, corneal PMN number and macrophage inflammatory protein-2 (MIP-2) and KC levels were compared in mice that are susceptible (cornea perforates) or resistant (cornea heals) to P. aeruginosa infection. While corneal PMN myeloperoxidase activity (indicator of PMN number) was similar in both groups of mice at 1 and 3 days postinfection, by 5-7 days postinfection corneas of susceptible mice contained a significantly greater number of inflammatory cells. Corneal MIP-2, but not KC, levels correlated with persistence of PMN in the cornea of susceptible mice. To test the biological relevance of these data, resistant mice were treated systemically with rMIP-2. This treatment resulted in increased corneal PMN number and significantly exacerbated corneal disease. Conversely, administration of neutralizing MIP-2 pAb to susceptible mice reduced both PMN infiltration and corneal destruction. Collectively, these findings support an important role for MIP-2 in recruitment of PMN to P. aeruginosa-infected cornea. These data also strongly suggest that a timely down-regulation of the host inflammatory response is critical for resolution of infection.     

Differential expression of the isoforms for the monocyte chemoattractant protein-1 receptor, CCR2, in monocytes.

Two isoforms of human CCR2, the receptor for monocyte chemoattractant protein-1 (MCP-1), have been identified but their relative expression in monocytes and contribution to inflammatory responses mediated by MCP-1 remain uncertain. All available information on CCR2 expression is based on mRNA data because isoform-specific antibodies were not available until now. To analyze the relative expression of each isoform, we made two antibodies that specifically recognized CCR2A and CCR2B. Examination of receptor protein with these isoform-specific antibodies showed that the total expression of CCR2B in monocytes was about 10-fold higher than that of CCR2A with an equal distribution between the cell surface and intracellular pools. A detailed analysis using purified plasma membranes demonstrated that about 90% of all CCR2 on the cell surface were composed of CCR2B. The relatively abundant expression of CCR2B on the cell surface suggests a principal role of this isoform as a mediator of monocyte responses to MCP-1 in inflammation.     

Involvement of fractalkine and macrophage inflammatory protein-1 alpha in moderate-severe depression

Moderate-severe depression (MSD) is linked to overexpression of proinflammatory cytokines and chemokines. Fractalkine (FKN) and macrophage inflammatory protein-1 alpha (MIP-1alpha) are, respectively, members of CX3C and C-C chemokines, and both are involved in recruiting and activating mononuclear phagocytes in the central nervous system. We analysed the presence of FKN and MIP-1alpha in sera of untreated MSD patients and healthy donors. High FKN levels were observed in all MSD patients as compared with values only detectable in 26% of healthy donors. MIP-1alpha was measurable in 20% of patients, while no healthy donors showed detectable chemokine levels. In conclusion, we describe a previously unknown involvement of FKN in the pathogenesis of MSD, suggesting that FKN may represent a target for a specific immune therapy of this disease.     

Macrophage inflammatory protein-2 is required for neutrophil passage across the epithelial barrier of the infected urinary tract

IL-8 is a major human neutrophil chemoattractant at mucosal infection sites. This study examined the C-X-C chemokine response to mucosal infection, and, specifically, the role of macrophage inflammatory protein (MIP)-2, one of the mouse IL-8 equivalents, for neutrophil-epithelial interactions. Following intravesical Escherichia coli infection, several C-X-C chemokines were secreted into the urine, but only MIP-2 concentrations correlated to neutrophil numbers. Tissue quantitation demonstrated that kidney MIP-2 production was triggered by infection, and immunohistochemistry identified the kidney epithelium as a main source of MIP-2. Treatment with anti-MIP-2 Ab reduced the urine neutrophil numbers, but the mice had normal tissue neutrophil levels. By immunohistochemistry, the neutrophils were found in aggregates under the pelvic epithelium, but in control mice the neutrophils crossed the urothelium into the urine. The results demonstrate that different chemokines direct neutrophil migration from the bloodstream to the lamina propria and across the epithelium and that MIP-2 serves the latter function. These findings suggest that neutrophils cross epithelial cell barriers in a highly regulated manner in response to chemokines elaborated at this site. This is yet another mechanism that defines the mucosal compartment and differentiates the local from the systemic host response.     

Activated protein C inhibits the release of macrophage inflammatory protein-1-alpha from THP-1 cells and from human monocytes

Several lines of evidence have implicated activated protein C (APC) to be an endogenous inhibitor of the inflammatory septic cascade. APC may exhibit direct anti-inflammatory properties, independent of its antithrombotic effects. Chemokines influence the interaction of monocytes at the endothelium during infection and sepsis and are involved in the molecular events leading to an adverse and lethal outcome of sepsis. Defining regulatory mechanisms on the monocytic release profile of the proinflammatory C-C chemokines macrophage inflammatory protein-1-alpha (MIP-1-alpha) and monocyte chemoattractant protein-1 (MCP-1) might have therapeutic implications for the treatment of sepsis. We established a monocytic cell model of inflammation by the addition of lipopolysaccharide (LPS) and examined the effect of human APC on LPS-stimulated chemokine release from the monocytic cell line THP-1. We found that human APC in supra-physiological concentrations of 2.5-10 microg/ml inhibited the LPS-induced release of the chemokines MIP-1-alpha and MCP-1, as measured by enzyme-linked immunosorbent assays (ELISA) at 6 up to 24 h. In addition to experiments on THP-1 cells, recombinant human APC in concentrations of 50 ng/ml was found to have an inhibiting effect on the release of MIP-1-alpha from freshly isolated mononuclear cells of septic patients. The ability of APC to decrease the release of the C-C chemokine MIP-1-alpha from the monocytic cell line THP-1 and from human monocytes may identify a novel immunomodulatory pathway by which APC exerts its anti-inflammatory action and may contribute to control the inflammatory response in sepsis.