Chemokine and cytokine production in susceptible C3H/HeN mice and resistant BALB/c mice during Orientia tsutsugamushi infection

To understand the pathogenesis of scrub typhus, we examined chemokine and cytokine production in susceptible (C3H/HeN) and resistant (BALB/c) mice after infection with O. tsutsugamushi Gilliam. C3H/HeN mice produced high levels of chemokines macrophage inflammatory proteins 1 alpha (MIP-1 alpha ), MIP-2, monocyte chemoattractant protein 1 (MCP-1), and cytokines gamma-interferon (IFN-gamma ), interleukin-12 (IL-12), IL-10, and tumor necrosis factor alpha (TNF-alpha ) in response to O. tsutsugamushi infection, compared to BALB/c mice. Chemokine profiles in infected mice correlated well with the kinetics of inflammatory cell infiltration. Hyperproduction of chemokines and cytokines was observed in another susceptible-infection model (BALB/c-Karp). These results suggest that hyperproduction of chemokines and cytokines are associated with susceptibility during O. tsutsugamushi infection.     

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.     

Inducible expression of inflammatory chemokines in respiratory syncytial virus-infected mice: role of MIP-1alpha in lung pathology

Lower respiratory tract disease caused by respiratory syncytial virus (RSV) is characterized by profound airway mucosa inflammation, both in infants with naturally acquired infection and in experimentally inoculated animal models. Chemokines are central regulatory molecules in inflammatory, immune, and infectious processes of the lung. In this study, we demonstrate that intranasal infection of BALB/c mice with RSV A results in inducible expression of lung chemokines belonging to the CXC (MIP-2 and IP-10), CC (RANTES, eotaxin, MIP-1beta, MIP-1alpha, MCP-1, TCA-3) and C (lymphotactin) families. Chemokine mRNA expression occurred as early as 24 h following inoculation and persisted for at least 5 days in mice inoculated with the highest dose of virus (10(7) PFU). In general, levels of chemokine mRNA and protein were dependent on the dose of RSV inoculum and paralleled the intensity of lung cellular inflammation. Immunohisthochemical studies indicated that RSV-induced expression of MIP-1alpha, one of the most abundantly expressed chemokines, was primarily localized in epithelial cells of the alveoli and bronchioles, as well as in adjoining capillary endothelium. Genetically altered mice with a selective deletion of the MIP-1alpha gene (-/- mice) demonstrated a significant reduction in lung inflammation following RSV infection, compared to control littermates (+/+ mice). Despite the paucity of infiltrating cells, the peak RSV titer in the lung of -/- mice was not significantly different from that observed in +/+ mice. These results provide the first direct evidence that RSV infection may induce lung inflammation via the early production of inflammatory chemokines.     

Polymorphonuclear neutrophils improve replication of Chlamydia pneumoniae in vivo upon MyD88-dependent attraction

Chlamydia pneumoniae, an obligate intracellular bacterium, causes pneumonia in humans and mice. In this study, we show that GR1+/CD45+ polymorphonuclear neutrophils (PMN) surprisingly increase the bacterial load of C. pneumoniae in vivo. Upon intranasal infection of wild-type mice, the lung weight is increased; the cytokines TNF, IL-12p40, and IFN-gamma, as well as the chemokines keratinocyte-derived chemokine, MCP-1, and MIP-2 are secreted; and GR1+/CD45+ PMN are recruited into lungs 3 days postinfection. In contrast, in infected MyD88-deficient mice, which lack a key adaptor molecule in the signaling cascade of TLRs and IL-1R family members, the increase of the lung weight is attenuated, and from the analyzed cyto- and chemokines, only IL-12p40 is detectable. Upon infection, almost no influx of inflammatory cells into lungs of MyD88-deficient mice can be observed. Six days postinfection, however, MyD88-deficient mice were able to produce TNF, IFN-gamma, keratinocyte-derived chemokine, and MCP-1 in amounts similar to wild-type mice, but failed to secrete IL-12p40 and MIP-2. At this time point, the infection increased the lung weight to a level similar to wild-type mice. Curiously, the chlamydial burden in MyD88-deficient mice 3 days postinfection is lower than in wild-type mice, a finding that can be reproduced in wild-type mice by depletion of GR1+ cells. In analyzing how PMN influence the chlamydial burden in vivo, we find that PMN are infected and enhance the replication of C. pneumoniae in epithelial cells. Thus, the lower chlamydial burden in MyD88-deficient mice can be explained by the failure to recruit PMN.     

The effect of MCP-1 depletion on chemokine and chemokine-related gene expression: evidence for a complex network in acute inflammation

The expression of chemokines has been suggested to involve an interdependent network, with the absence of a single chemokine affecting the expression of multiple other chemokines. Monocyte chemoattractant protein (MCP-1), a member of C-C chemokine superfamily, plays a critical role in the recruitment and activation of leukocytes during acute inflammation. To examine the effect of the loss of MCP-1 on expression of the chemokine network, we compared the mRNA expression profiles of MCP-1(-/-) and wild type mice during the acute inflammatory phase of excisional wounds. Utilizing a mouse cDNA array containing 514 chemokine and chemokine related genes, the loss of MCP-1 was observed to cause a significant upregulation of nine genes (Decorin, Persephin, IL-1beta, MIP-2, MSP, IL1ra, CCR5, CCR3, IL-11) and significant downregulation of two genes (CCR4 and CD3Z) in acute wounds. The array data was confirmed by semi-quantitative RT-PCR. The effect of MCP-1 deletion on chemokine expression was further examined in isolated macrophages. Compared to wild type, LPS-stimulated peritoneal macrophages from MCP-1(-/-) mice showed a significant increase in the expression of RANTES, MIP-1beta, MIP-1alpha and MIP-2 mRNA. The data suggest that loss of a single chemokine perturbs the chemokine network not only in the setting of acute inflammation but even in an isolated inflammatory cell, the macrophage.     

The role of macrophage inflammatory protein-1 alpha/CCL3 in regulation of T cell-mediated immunity to Cryptococcus neoformans infection

Macrophage inflammatory protein-1alpha (MIP-1alpha/CCL3) is a CC chemokine required for optimal recruitment of leukocytes in response to cryptococcal Ags. MIP-1alpha is expressed in the lungs by day 6 post Cryptococcus neoformans infection and could play a role in the development of cell-mediated immunity. To address this possibility, wild-type (MIP-1alpha(+/+)) mice and MIP-1alpha knockout (MIP-1alpha(-/-)) mice were infected intratracheally with a highly virulent strain of C. neoformans (145A). MIP-1alpha message was detected in the lungs on days 3, 7, and 14 in MIP-1alpha(+/+) mice, but it was undetectable in MIP-1alpha(-/-) mice. On day 16, MIP-1alpha(-/-) mice had a 7-fold increase in C. neoformans burden in the lungs, but no decrease in pulmonary leukocyte recruitment. MIP-1alpha(+/+) and MIP-1alpha(-/-) mice had similar numbers of recruited lymphocytes and monocytes/macrophages. Notably, MIP-1alpha(-/-) mice had a significantly greater number of eosinophils. MIP-1alpha(-/-) mice had extremely high levels of serum IgE. This switch of immune response to a T(2) phenotype was associated with enhanced IL-4 and IL-13 expression in the lungs of MIP-1alpha(-/-) mice compared with MIP-1alpha (+/+) mice. Progression of pulmonary cryptococcosis in the presence of nonprotective T(2) immunity resulted in profound lung damage in MIP-1alpha(-/-) mice (eosinophilic crystal deposition, destruction of lung parenchyma, and pulmonary hemorrhage). Twelve-week survival was dramatically decreased in MIP-1alpha(-/-) mice. These studies, together with our previous studies, demonstrate that MIP-1alpha plays a role in both the afferent (T(1)/T(2) development) and efferent (T(1)-mediated leukocyte recruitment) phases of cell-mediated immunity to C. neoformans.     

Different chemokines are expressed in human arthritic bone biopsies: IFN-gamma and IL-6 differently modulate IL-8, MCP-1 and rantes production by arthritic osteoblasts

In the present study we analyse chemokine expression in the remodelling of subchondral bone in arthritis patients. Trabecular bone biopsies were tested by immunohistochemistry to identify interleukin (IL)-8, GRO-alpha, MCP-1, RANTES, MIP-1alpha and MIP-1beta expression. Subsequently, we evaluated by immunoassay the effect of interferon (IFN)-gamma and IL-6 on chemokine production by osteoarthritis (OA), rheumatoid arthritis (RA) and post-traumatic (PT) patients' isolated osteoblasts (OB). OB constitutively produced in situ IL-8, GRO-alpha, MCP-1, RANTES and MIP-1alpha. MIP-1beta was positive only in mononuclear cells. In RA many of these chemokines were also produced by mononuclear cells. IFN-gamma significantly down-regulated IL-8 and up-regulated MCP-1 produced by OB from all patients tested, whereas it did not affect the other chemokines analysed. Moreover, IFN-gamma reduced IL-1beta-stimulated IL-8 production but significantly increased both MCP-1 and RANTES. Interestingly, IL-6 significantly downregulated IFN-gamma-induced MCP-1 production, that was significantly lower in OA compared to RA patients. OB expressed chemokines both in vivo and in vitro suggesting that these cells are primary effectors in the bone capable of regulating autocrine/paracrine circuits that affect bone remodelling in these diseases.     

Identification of a gammaherpesvirus selective chemokine binding protein that inhibits chemokine action

Chemokines are involved in recruitment and activation of hematopoietic cells at sites of infection and inflammation. The M3 gene of gammaHV68, a gamma-2 herpesvirus that infects and establishes a lifelong latent infection and chronic vasculitis in mice, encodes an abundant secreted protein during productive infection. The M3 gene is located in a region of the genome that is transcribed during latency. We report here that the M3 protein is a high-affinity broad-spectrum chemokine scavenger. The M3 protein bound the CC chemokines human regulated upon activation of normal T-cell expressed and secreted (RANTES), murine macrophage inflammatory protein 1alpha (MIP-1alpha), and murine monocyte chemoattractant protein 1 (MCP-1), as well as the human CXC chemokine interleukin-8, the murine C chemokine lymphotactin, and the murine CX(3)C chemokine fractalkine with high affinity (K(d) = 1. 6 to 18.7 nM). M3 protein chemokine binding was selective, since the protein did not bind seven other CXC chemokines (K(d) > 1 microM). Furthermore, the M3 protein abolished calcium signaling in response to murine MIP-1alpha and murine MCP-1 and not to murine KC or human stromal cell-derived factor 1 (SDF-1), consistent with the binding data. The M3 protein was also capable of blocking the function of human CC and CXC chemokines, indicating the potential for therapeutic applications. Since the M3 protein lacks homology to known chemokines, chemokine receptors, or chemokine binding proteins, these studies suggest a novel herpesvirus mechanism of immune evasion.     

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.     

Systemic and local CC chemokines production in a murine model of Listeria monocytogenes infection

Repeated intragastric inoculation of Listeria monocytogenes into BALB/c mice resulted in prolonged bacteraemia and severe hepatic infection. Bacteria could also be isolated from the brain tissue of all experimental mice. During the inflammatory process, chemokine concentrations typically increased at the local site in comparison to the systemic level. The liver-to-serum ratio was more pronounced in the case of macrophage inflammatory protein 1 alpha (MIP-1 alpha), suggesting its role in the inflammatory response in the liver. The ratio of brain-to-serum concentration of monocyte chemoattractant protein 1 (MCP-1) remained the same as in the control animals, while it was lower in the infected mice, both in the case MIP-1alpha and in the case of regulated on activation, normal T cell expressed and secreted (RANTES). This is in correlation with slight inflammatory infiltrates found in the brain tissue early in infection.     

Blockade of neurokinin-1 receptor attenuates CC and CXC chemokine production in experimental acute pancreatitis and associated lung injury

Accumulating evidence suggests the neuropeptide substance P (SP) and its receptor neurokinin-1 receptor (NK-1R) play a pivotal role in the pathogenesis of acute pancreatitis (AP). However, the mechanisms remain unclear. The present study investigated whether chemokines as proinflammatory molecules are involved in SP-NK-1R-related pathogenesis of this condition. We observed temporally and spatially selective chemokine responses in secretagogue caerulein-induced AP in mice. CC chemokines monocyte chemotactic protein (MCP)-1 and macrophage inflammatory protein-1alpha (MIP-1alpha) and CXC chemokine MIP-2 were elevated after AP induction. Time-dependent, tissue-specific analysis of their mRNA and protein expression suggested that they are early mediators in the condition and mediate local as well as systemic inflammatory responses. In contrast, another CC chemokine regulated on activation, T cells expressed and secreted (RANTES) was only involved in local pancreatic inflammation at a later stage of the disease. Either prophylactic or therapeutic treatment with a potent selective NK-1R antagonist CP-96,345 significantly suppressed caerulein-induced increase in MCP-1, MIP-1alpha, and MIP-2 expression but had no apparent effect on RANTES expression. The suppression effect of CP-96,345 on MCP-1, MIP-1alpha, and MIP-2 expression was concordantly demonstrated by immunohistochemistry, which, additionally, suggested that chemokine immunoreactivity was localized to acinar cells and the infiltrating leukocytes in the pancreas and alveolar macrophages, epithelial cells, and endothelial cells in the lungs. Our data suggest that SP, probably by acting via NK-1R on various chemokine-secreting cells in the pancreas and lungs, stimulates the release of chemokines that aggravate local AP and the development of its systemic sequelae.     

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.     

IL-13-induced chemokine responses in the lung: role of CCR2 in the pathogenesis of IL-13-induced inflammation and remodeling

IL-13 stimulates inflammatory and remodeling responses and contributes to the pathogenesis of human airways disorders. To further understand the cellular and molecular events that mediate these responses, we characterized the effects of IL-13 on monocyte chemotactic proteins (MCPs) and compared the tissue effects of transgenic IL-13 in mice with wild-type (+/+) and null (-/-) CCR2 loci. Transgenic IL-13 was a potent stimulator of MCP-1, -2, -3, and -5. This stimulation was not specific for MCPs because macrophage-inflammatory protein (MIP)-1alpha, MIP-1beta, MIP-2, MIP-3alpha, thymus- and activation-regulated chemokine, thymus-expressed chemokine, eotaxin, eotaxin 2, macrophage-derived chemokines, and C10 were also induced. The ability of IL-13 to increase lung size, alveolar size, and lung compliance, to stimulate pulmonary inflammation, hyaluronic acid accumulation, and tissue fibrosis, and to cause respiratory failure and death were markedly decreased, whereas mucus metaplasia was not altered in CCR2(-/-) mice. CCR2 deficiency did not decrease the basal or IL-13-stimulated expression of target matrix metalloproteinases or cathepsins but did increase the levels of mRNA encoding alpha1-antitrypsin, tissue inhibitor of metalloproteinase-1, -2, and -4, and secretory leukocyte proteinase inhibitor. In addition, the levels of bioactive and total TGF-beta(1) were decreased in lavage fluids from IL-13 transgenic mice with -/- CCR2 loci. These studies demonstrate that IL-13 is a potent stimulator of MCPs and other CC chemokines and document the importance of MCP-CCR2 signaling in the pathogenesis of the IL-13-induced pulmonary phenotype.     

Role of chemokines and formyl peptides in pneumococcal pneumonia-induced monocyte/macrophage recruitment

Host-derived chemoattractant factors are suggested to play crucial roles in leukocyte recruitment elicited by inflammatory stimuli in vitro and in vivo. However, in the case of acute bacterial infections, pathogen-derived chemoattractant factors are also present, and it has not yet been clarified how cross-talk between chemoattractant receptors orchestrates diapedesis of leukocytes in this context of complex chemoattractant arrays. To investigate the role of chemokine (host-derived) and formyl peptide (pathogen-derived) chemoattractants in leukocyte extravasation in life-threatening infectious diseases, we used a mouse model of pneumococcal pneumonia. We found an increase in mRNA expression of eight chemokines (RANTES, macrophage-inflammatory protein (MIP)-1alpha, MIP-1beta, MIP-2, IP-10, monocyte chemoattractant protein (MCP)-1, T cell activation 3, and KC) within the lungs during the course of infection. KC and MIP-2 protein expression closely preceded pulmonary neutrophil recruitment, whereas MCP-1 protein production coincided more closely than MIP-1alpha with the kinetics of macrophage infiltration. In situ hybridization of MCP-1 mRNA suggested that MCP-1 expression started at peribronchovascular regions and expanded to alveoli-facing epithelial cells and infiltrated macrophages. Interestingly, administration of a neutralizing Ab against MCP-1, RANTES, or MIP-1alpha alone did not prevent macrophage infiltration into infected alveoli, whereas combination of the three Abs significantly reduced macrophage infiltration without affecting neutrophil recruitment. The use of an antagonist to N-formyl peptides, N-t-Boc-Phe-D-Leu-Phe-D-Leu-Phe, reduced both macrophages and neutrophils significantly. These data demonstrate that a complex chemokine network is activated in response to pulmonary pneumococcal infection, and also suggest an important role for fMLP receptor in monocyte/macrophage recruitment in that model.     

Differential regulation of eosinophil chemokine signaling via CCR3 and non-CCR3 pathways

To investigate eosinophil stimulation by chemokines we developed a sensitive assay of leukocyte shape change, the gated autofluorescence/forward scatter assay. Leukocyte shape change responses are mediated through rearrangements of the cellular cytoskeleton in a dynamic process typically resulting in a polarized cell and are essential to the processes of leukocyte migration from the microcirculation into sites of inflammation. We examined the actions of the chemokines eotaxin, eotaxin-2, monocyte chemoattractant protein-1 (MCP-1), MCP-3, MCP-4, RANTES, macrophage inflammatory protein-1alpha (MIP-1alpha), and IL-8 on leukocytes in mixed cell suspensions and focused on the responses of eosinophils to C-C chemokines. Those chemokines acting on CCR3 induced a rapid shape change in eosinophils from all donors; of these, eotaxin and eotaxin-2 were the most potent. Responses to MCP-4 were qualitatively different, showing marked reversal of shape change responses with agonist concentration and duration of treatment. In contrast, MIP-1alpha induced a potent response in eosinophils from a small and previously undescribed subgroup of donors via a non-CCR3 pathway likely to be CCR1 mediated. Incubation of leukocytes at 37 degrees C for 90 min in the absence of extracellular calcium up-regulated responses to MCP-4 and MIP-1alpha in the majority of donors, and there was a small increase in responses to eotaxin. MIP-1alpha responsiveness in vivo may therefore be a function of both CCR1 expression levels and the regulated efficiency of coupling to intracellular signaling pathways. The observed up-regulation of MIP-1alpha signaling via non-CCR3 pathways may play a role in eosinophil recruitment in inflammatory states such as occurs in the asthmatic lung.     

Role of CCR5 in the pathogenesis of IL-13-induced inflammation and remodeling

IL-13 is a major effector at sites of Th2 inflammation and tissue remodeling. In these locations, it frequently coexists with the CCR5 chemokine receptor and its ligands MIP-1alpha/CCL3 and MIP-1beta/CCL4. We hypothesized that CCR5 induction and activation play important roles in the pathogenesis of IL-13-induced tissue responses. To test this hypothesis, we evaluated the effects of IL-13 on the expression of CCR5 in the murine lung. We also compared the effects of lung-targeted transgenic IL-13 in mice treated with anti-CCR5 or an Ab control and mice with wild-type or null CCR5 loci. These studies demonstrate that IL-13 is a potent stimulator of epithelial cell CCR5 expression. They also demonstrate that CCR5 neutralization or a deficiency of CCR5 significantly decreases IL-13-induced inflammation, alveolar remodeling, structural and inflammatory cell apoptosis, and respiratory failure and death. Lastly, these studies provide mechanistic insights by demonstrating that CCR5 is required for optimal IL-13 stimulation of select chemokines (MIP-1alpha/CCL3, MIP-1beta/CCL4, MCP-1/CCL-2), matrix metalloproteinase-9 and cell death regulators (Fas, TNF, TNFR1, TNFR2, Bid), optimal IL-13 inhibition of alpha1-antitrypsin, and IL-13-induction of and activation of caspases-3, -8, and-9. Collectively, these studies demonstrate that CCR5 plays a critical role in the pathogenesis of IL-13-induced inflammation and tissue remodeling.     

Characterization of binding sites for chemokines MCP-1 and MIP-1alpha on human brain microvessels

The presence of binding sites for the beta chemokines monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1alpha (MIP-1alpha) has recently been identified on human brain microvessels. We extend these findings in this report to reveal that such sites exemplify characteristics of the recognized major receptors for MCP-1 and MIP-1alpha: CCR2, and CCR1 and CCR5, respectively. Specifically, labeled MCP-1 binding to isolated brain microvessels was inhibited by unlabeled MCP-1 and MCP-3, the latter another CCR2 ligand, but not by MIP-1alpha. Inhibition of labeled MIP-1alpha binding was achieved with unlabeled MIP-1alpha and RANTES, the latter a beta chemokine that binds to both CCR1 and CCR5, but not by MCP-1. Labeled MIP-1alpha binding was also antagonized by unlabeled MCP-3, which is also recognized by CCR1, and MIP-1beta, which is a ligand for CCR5. Labeled MCP-1 and MIP-1alpha were further observed to be internalized within the endothelial cells of brain microvessels, following their binding to the microvascular surface at 37 degrees C. Additionally, exposure of microvessels to unlabeled MCP-1 or MIP-1alpha was accompanied by the initial loss and subsequent recovery of surface binding sites for these chemokines, which occurred on a time scale consistent with ligand-induced endocytosis and recycling. These collective features bear striking similarity to those that characterize interactions of MCP-1 and MIP-1alpha with their receptors on leukocytes and underscore the concept of cognate chemokine receptors on brain microvascular endothelium.     

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.