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Hemofiltrate C-C chemokine (HCC)-1 is a recently cloned C-C chemokine that is structurally similar to macrophage inflammatory protein (MIP)-1alpha. Unlike most chemokines, it is constitutively secreted by tissues and is present at high concentrations in normal human plasma. Also atypical for chemokines, HCC-1 is reported not to be chemotactic for leukocytes. In this paper, we have investigated the chemokine receptor usage and downstream signaling pathways of HCC-1. Cross-desensitization experiments using THP-1 cells suggested that HCC-1 and MIP-1alpha activated the same receptor. Experiments using a panel of cloned chemokine receptors revealed that HCC-1 specifically activated C-C chemokine receptor (CCR)1, but not closely related receptors, including CCR5. HCC-1 competed with MIP-1alpha for binding to CCR1-transfected cells, but with a markedly reduced affinity (IC50 = 93 nM versus 1.3 nM for MIP-1alpha). Similarly, HCC-1 was less potent than MIP-1alpha in inducing inhibition of adenylyl cyclase in CCR1-transfected cells. HCC-1 induced chemotaxis of freshly isolated human monocytes, THP-1 cells, and CCR1-transfected cells, and the optimal concentration for cell migration (100 nM) was approximately 100-fold lower than that of MIP-1alpha (1 nM). These data demonstrate that HCC-1 is a chemoattractant and identify CCR1 as a functional HCC-1 receptor on human monocytes.

CD40 is a member of the TNF family of receptors that has been shown to play a crucial role in enhancing dendritic cell activity and fostering anti-tumor immune responses. In this study, we demonstrate the in vitro properties and in vivo efficacious activity of the CD40 agonist antibody, CP-870,893. CP-870,893 is a fully human, IgG2 antibody that selectively interacts with CD40 at a site distinct from its ligand-binding region with a KD of 0.4 nM. It enhances the expression of MHC class II, CD54, CD86, and CD23 on human B cells in vitro. CP-870,893 also enhances dendritic cell activity as evidenced by cytokine secretion (IL-12, IL-23, IL-8), the upregulation of CD86 and CD83, and the ability to prime T cells to secrete IFNγ. In SCID-beige mice, a single parenteral injection of CP-870,893 was therapeutically effective against several CD40(pos) human tumors (B-cell lymphoma, breast, colon, and prostate) indicating direct effects on tumor cell survival and/or growth. When mice were co-implanted with human T cells and dendritic cells, the activity of CP-870,893 against CD40(pos) tumors increased, and efficacy was also observed against CD40(neg) and CD40(low) tumors demonstrating the ability of CP-870,893 to enhance anti-tumor immune function in vivo. These studies suggest that CP-870,893 has the potential to be efficacious against a wide range of tumor types through both direct and immune-mediated effects.

Objective: The CCR2 receptor plays a crucial role in monocyte recruitment and has been implicated as a contributing factor to atherosclerosis. CCR2 receptor deletion leads to significant inhibition of lesion development. Our objective was to determine if CCR2 receptor blockade with a small molecule would have a beneficial effect of decreasing established lesions., Methods and Results: We demonstrated that CCR2 blockade had no significant effect on advanced lesions or the progression of fatty streaks. CCR2 blockade in mice resulted in elevations in plasma CCL2 levels and a significant reduction in the plasma Ly-6C(hi) subpopulations of monocytes expressing the CCR2 receptor. Neither CCL2 elevation nor margination of the Ly-6C(hi) population was observed in CCR2(-/-) mice., Conclusions: CCR2 receptor blockade with a small molecule antagonist at dose levels showing efficacy in several inflammatory models did not show a beneficial effect in murine models of atherosclerosis. Elevations in CCL2 and margination of Ly-6C(hi) cells demonstrate that the role of CCR2 in controlling monocyte levels goes beyond the control of monocyte emigration., (Copyright 2009. Published by Elsevier Ireland Ltd.)

The identification of chemokines and their receptors as potent mediators of leukocyte infiltration raised interest in the potential role of these proteins on disease pathogenesis. This is exemplified by the chemokine receptor, CCR1, which has been shown to be up-regulated in a number of human diseases, the implications of which have been suggested by animal models where inhibition of CCR1 or its ligands have shown beneficial effects. These data support the possibility that a CCR1 antagonist will provide therapeutic benefit to patients with inflammatory diseases. Over the last several years, several of these antagonists entered clinical trials, including CP-481,715 (Pfizer) and MLN3897 (Millennium) for rheumatoid arthritis, BX471 (Berlex / Scherring AG) for multiple sclerosis, and AZD-4818 (Astra-Zeneca) for COPD. This review will describe the evidence that supported the role of CCR1 in these diseases, the results from clinical trials, and provide perspectives on what has been learned from these trials for potential application / consideration to other studies with chemokine receptor antagonists.

CCR6 is expressed in a number of dermatological inflammatory diseases. Here, we report that mice sensitized with the hapten oxazolone had increased numbers of CCR6+ T cells in the draining lymph nodes. Using CCR6-/- mice, we assessed the role of CCR6 on the development of contact hypersensitivity. After hapten sensitization and re-challenge, ear swelling in CCR6-/- animals was reduced 80% as compared with wild-type (WT) control mice. This decreased level of inflammation was not related to an inhibition in T-cell activation, because CCR6-/- lymph node cells from sensitized mice produced threefold higher levels of IFN-gamma in culture than cells from sensitized WT mice and, when these cells were directly injected into the site of hapten challenge, induced a robust inflammatory response. However, intravenous injection of CCR6-/- lymph node cells from sensitized mice were unable to prime naive mice to re-challenge whereas cells from primed WT mice were able to sensitize animals. These results suggest that CCR6 plays an important role in directing the trafficking of activated T cells into the skin and suggests that a CCR6 antagonist could be useful to treat skin-mediated inflammatory reactions.

The synthesis, biological activity, and pharmacokinetic profile of CCR1 antagonists are described.

CD40-mediated interactions play an important role in the response to a variety of diseases, including cancer. Engagement of CD40 on antigen-presenting cells, namely dendritic cells (DC), by CD40L leads to maturation and up-regulation of co-stimulatory molecules B7.1 and B7.2 (CD80 and CD86). These molecules are requisite to subsequent antigen-specific activation of T cells. T-cell activation is a critical aspect of specific anti-tumour immune responses that have become the focus of a variety of cancer immunotherapy approaches. Clinical trials involving immunologic interventions have shown clinical responses confirming that the immune system can be harnessed for the treatment of cancer. However, the clinical response rate has been low, signifying the need for new immunotherapeutic strategies. To this end, an agonist antibody specific for CD40, CP-870,893, has been developed. A fully autologous mixed tumour cell/lymph node cell model was utilized to demonstrate that CP-870,893 promotes the responsiveness of lymph node-derived T cells to autologous tumour. Specifically, T cells from the tumour-draining lymph nodes are not responsive to autologous tumour cells; however, in the presence of CP-870,893, this unresponsiveness is reversed, as indicated by lymph node cell proliferation and cytokine secretion. Monocyte-derived DC treated with CP-870,893 consistently display a mature phenotype: up-regulation of CD80, CD83, CD86 and HLA-DR expression, increased Mip1alpha and IL-12 secretion, and the loss of exogenous antigen-presenting capability subsequent to treatment with the antibody. These data indicate that CP-870,893 binds to and activates DC, ultimately driving a specific anti-tumour T-cell response.

Innate inflammatory events promoting antiviral defense in the liver against murine cytomegalovirus (MCMV) infection have been characterized. However, the mechanisms that regulate the selective recruitment of inflammatory T lymphocytes to the liver during MCMV infection have not been defined. The studies presented here demonstrate the expression of monokine induced by gamma interferon (IFN-gamma; Mig/CXCL9) and IFN-gamma-inducible protein 10 (IP-10/CXCL10) in liver leukocytes and correlate their production with the infiltration of MCMV-specific CD8 T cells into the liver. Antibody-mediated neutralization of CXCL9 and CXCL10 and studies using mice deficient in CXCR3, the primary known receptor for these chemokines, revealed that CXCR3-dependent mechanisms promote the infiltration of virus-specific CD8 T cells into the liver during acute infection with MCMV. Furthermore, CXCR3 functions augmented the hepatic accumulation of CD8 T-cell IFN-gamma responses to MCMV. Evaluation of protective functions demonstrated enhanced pathology that overlapped with transient increases in virus titers in CXCR3-deficient mice. However, ultimate viral clearance and survival were not compromised. Thus, CXCR3-mediated signals support the accumulation of MCMV-specific CD8 T cells that contribute to, but are not exclusively required for, protective responses in a virus-infected tissue site.

Background and Objectives: The chemokine receptor CCR1 is believed to play a role in several inflammatory diseases, primarily by promoting the migration of leukocytes through the endothelial barrier. Thus, a possible strategy for treating inflammatory diseases is inhibition of leukocyte infiltration by antagonising CCR1. Recently, CP-481,715 has been described as a potent and specific antagonist of CCR1. The aims of this study were to assess the safety, pharmacokinetics and pharmacodynamics of CP-481,715 along with drug interactions with ciclosporin., Subjects and Methods: This was a phase I randomised, double-blind, placebo-controlled study with CP-481,715 in 78 healthy male volunteers. Subjects were administered escalating CP-481,715 doses of up to 3000 mg with food and after fasting in the single-dose study. In the drug interaction study, which was a single-dose, two-way crossover study, 12 subjects received a 300 mg dose of CP-481,715 as a suspension of polymorph A under fasted conditions, both with and without prior administration of ciclosporin., Results and Conclusions: All doses of CP-481,715 were well tolerated, with linear pharmacokinetics up to the 300 mg dose. The pharmacodynamic activity of CP-481,715 was detected ex vivo by demonstrating a dose-related and linear increase in the amount of macrophage inflammatory protein-1alpha, CCL3, required to induce CD11b upregulation. Analysis of vital signs indicated no consistent clinical effects, and statistical analysis of ECG characteristics demonstrated no significant prolongation of the corrected QT interval. A drug-drug interaction study with ciclosporin demonstrated that CP-481,715 clearance was decreased by ciclosporin, consistent with its ability to compete with P-glycoprotein. Phase II studies may be warranted to see if CP-481,715 exhibits efficacy in treating inflammatory diseases such as rheumatoid arthritis, multiple sclerosis or transplant rejection.

We previously described the in vitro characteristics of the potent and selective CCR1 antagonist, CP-481,715. In addition to being selective for CCR1 vs other chemokine receptors, CP-481,715 is also specific for human CCR1 (hCCR1), preventing its evaluation in classical animal models. To address this, we generated mice whereby murine CCR1 was replaced by hCCR1 (knockin) and used these animals to assess the anti-inflammatory properties of CP-481,715. Cells isolated from hCCR1 knockin mice were shown to express hCCR1 and migrate in response to both murine CCR1 and hCCR1 ligands. Furthermore, this migration is inhibited by CP-481,715 at dose levels comparable to those obtained with human cells. In animal models of cell infiltration, CP-481,715 inhibited CCL3-induced neutrophil infiltration into skin or into an air pouch with an ED50 of 0.2 mg/kg. CP-481,715 did not inhibit cell infiltration in wild-type animals expressing murine CCR1. In a more generalized model of inflammation, delayed-type hypersensitivity, CP-481,715 significantly inhibited footpad swelling and decreased the amount of IFN-gamma and IL-2 produced by isolated spleen cells from sensitized animals. It did not, however, induce tolerance to a subsequent challenge. These studies illustrate the utility of hCCR1 knockin animals to assess the activity of human specific CCR1 antagonists; demonstrate the ability of the CCR1 antagonist CP-481,715 to inhibit cell infiltration, inflammation, and Th1 cytokine responses in these animals; and suggest that CP-481,715 may be useful to modulate inflammatory responses in human disease.

T cells infiltrating the inflamed liver express high levels of CXCR 3 and show enhanced migration to CXCR 3 ligands in chemotactic assays. Moreover, CXCR 3 ligands are up-regulated on hepatic endothelium at sites of T-cell infiltration in chronic hepatitis, and their presence correlates with outcome of inflammatory liver disease. We used a flow-based adhesion assay with human hepatic endothelium to investigate the function of CXCR 3 on lymphocyte adhesion to and transmigration through hepatic endothelium under physiological conditions of blood flow. To more accurately model the function of in vivo activated CXCR 3(high) lymphocytes, we isolated T cells from human liver tissue and studied their behavior in flow-based adhesion assays. We demonstrate that CXCR 3 not only promoted the adhesion of effector T cells to endothelium from flow but also drove transendothelial migration. Moreover, these responses could be stimulated either by endogenous CXCR 3 ligands secreted by the endothelium or by exogenous CXCR 3 ligands derived from other cell types and presented by the endothelium. This study thus demonstrates that activation of CXCR 3 promotes lymphocyte adhesion and transendothelial migration under flow and that human hepatic endothelium can present functionally active chemokines secreted by other cell types within the liver.

The chemokines are a large gene superfamily with critical roles in development and immunity. The chemokine receptor CXCR3 appears to play a major role in the trafficking of activated Th1 lymphocytes. There are at least three major ligands for CXCR3: mig/CXCL9, IP-10/CXCL10 and I-TAC/CXCL11, and of these three ligands, CXCL11 is the least well-characterized. In this study, we have cloned a rat ortholog of CXCL11, evaluated its function, and examined its expression in the Th-1-mediated disease, experimental autoimmune encephalomyelitis (EAE) in the rat. Based on its predicted primary amino-acid sequence, rat I-TAC/CXCL11 was synthesized and shown to induce chemotaxis of activated rat T lymphocytes in vitro and the in vivo migration of T lymphocytes when injected into the skin. I-TAC/CXCL11 expression, as determined by RT-PCR, increased in lymph node and spinal cord tissue collected from rats in which EAE had been actively induced, and in spinal cord tissue from rats in which EAE had been passively induced. The kinetics of expression were similar to that of CXCR3 and IP-10/CXCL10, although expression of both CXCR3 and IP-10/CXCL10 was more intense than that of I-TAC/CXCL11 and increased more rapidly in both lymph nodes and the spinal cord. Only minor levels of expression of the related chemokine mig/CXCL9 were observed. Immunohistochemistry revealed that the major cellular source of I-TAC/CXCL11 in the central nervous system (CNS) during EAE is likely to be the astrocyte. Together, these data indicate that I-TAC/CXCL11 is expressed in the CNS during the clinical phase of EAE. However, the observation that I-TAC/CXCL11 is expressed after receptor expression is detected suggests that it is not essential for the initial migration of CXCR3-bearing cells into the CNS.

The present manuscript details the discovery and early fundamental structure-activity relationship studies involving compound 3, a novel hydroxyethylene peptide isostere derived molecule that provides micromolar inhibition of CCL3 binding to its receptor CCR1. Initial studies established this screening hit as a legitimate lead for further medicinal chemistry optimization.

The synthesis, biological activity, and pharmacokinetic profile of novel CCR1 antagonists are described.

The present manuscript details structure-activity relationship studies of lead structure 1, which led to the discovery of CCR1 antagonists >100-fold more potent than 1.

Chemokines are 8- to 10-kDa proteins that regulate leukocyte infiltration into inflammatory sites. The therapeutic potential of inhibiting these proteins is supported by their increased expression in human diseases, numerous studies in animal models of disease and, in some cases, by human genetic association studies. These findings, combined with the ability of chemokines to interact with 7-transmembrane G protein-coupled receptors, render them attractive drug discovery targets. This article reviews the evidence that supports a role for the chemokine receptor CCR1 in the pathogenesis of autoimmune diseases, progress made in identifying low molecular-weight antagonists and the current status of agents undergoing clinical evaluation.

The chemokines CCL3 and CCL5, as well as their shared receptor CCR1, are believed to play a role in the pathogenesis of several inflammatory diseases including rheumatoid arthritis, multiple sclerosis, and transplant rejection. In this study we describe the pharmacological properties of a novel small molecular weight CCR1 antagonist, CP-481,715 (quinoxaline-2-carboxylic acid [4(R)-carbamoyl-1(S)-(3-fluorobenzyl)-2(S),7-dihydroxy-7-methyloctyl]amide). Radiolabeled binding studies indicate that CP-481,715 binds to human CCR1 with a Kd of 9.2 nm and displaces 125I-labeled CCL3 from CCR1-transfected cells with an IC50 of 74 nm. CP-481,715 lacks intrinsic agonist activity but fully blocks the ability of CCL3 and CCL5 to stimulate receptor signaling (guanosine 5'-O-(thiotriphosphate) incorporation; IC50 = 210 nm), calcium mobilization (IC50 = 71 nm), monocyte chemotaxis (IC50 = 55 nm), and matrix metalloproteinase 9 release (IC50 = 54 nm). CP-481,715 retains activity in human whole blood, inhibiting CCL3-induced CD11b up-regulation and actin polymerization (IC50 = 165 and 57 nm, respectively) on monocytes. Furthermore, it behaves as a competitive and reversible antagonist. CP-481,715 is >100-fold selective for CCR1 as compared with a panel of G-protein-coupled receptors including related chemokine receptors. Evidence for its potential use in human disease is suggested by its ability to inhibit 90% of the monocyte chemotactic activity present in 11/15 rheumatoid arthritis synovial fluid samples. These data illustrate that CP-481,715 is a potent and selective antagonist for CCR1 with therapeutic potential for rheumatoid arthritis and other inflammatory diseases.

CCR2, and its principle ligand MCP-1/CCL2, have been well documented for their ability to induce monocyte infiltration and promote the pathogenesis of rheumatoid arthritis and atherosclerosis. In order to assess additional roles for CCR2, we inserted allogeneic implants into CCR2-/- and MCP-1-/- mice and characterized T cell responses and the regulatory role of CCR2 on MCP-1 expression. The results demonstrate a marked decrease in lymphocyte infiltration in both CCR2-/- and MCP-1-/- animals. In contrast, IL-12 and CTL function were only suppressed in CCR2-/- animals. Further, whereas MCP-1 was only transiently elevated in the inflammatory fluid of WT animals, levels were sustained within the implants (5000pg/ml; >8 days) and serum (243pg/ml) of CCR2-/- mice. Higher levels of MCP-1 were also observed in the culture supernatants of CCR2-/- macrophages as compared to WT cells despite no difference in mRNA levels. Evidence that MCP-1 levels are regulated by receptor binding and internalization was suggested by its rapid decline when added to WT macrophages at 37 degrees C but not 4 degrees C. These studies indicate that CCR2 plays an important role in regulating T cell responses and controlling the level of MCP-1 at inflammatory sites.

Eosinophils are recruited to sites of inflammation via the action of a number of chemical mediators, including PAF, leukotrienes, eotaxins, ECF-A and histamine. Although many of the cell-surface receptors for these mediators have been identified, histamine-driven chemotaxis has not been conclusively attributed to any of the three known histamine receptor subtypes, suggesting the possibility of a 4th histamine-responsive receptor on eosinophils. We have identified and cloned a novel G protein-coupled receptor (GPCR), termed Pfi-013, from an IL-5 stimulated eosinophil cDNA library which is homologous to the human histamine H3 receptor, both at the sequence and gene structure level. Expression data indicates that Pfi-013 is predominantly expressed in peripheral blood leukocytes, with lower expression levels in spleen, testis and colon. Ligand-binding studies using Pfi-013 expressed in HEK-293Galpha15 cells, demonstrates specific binding to histamine with a Kd of 3.28 +/- 0.76 nM and possesses a unique rank order of potency against known histaminergic compounds in a competitive ligand-binding assay (histamine > clobenpropit > iodophenpropit > thioperamide > R-alpha-methylhistamine > cimetidine > pyrilamine). We have therefore termed this receptor human histamine H4. Chemotaxis studies on isolated human eosinophils have confirmed that histamine is chemotactic and that agonists of the known histamine receptors (H1, H2, and H3) do not induce such a response. Furthermore, studies employing histamine-receptor antagonists have shown an inhibition of chemotaxis only by the H3 antagonists clobenpropit and thioperamide. Since these compounds are also antagonists of hH4 we postulate that the receptor mediating histaminergic chemotaxis is this novel histamine H4 receptor.

The T-cell-specific receptor, CTLA-4, has been demonstrated to be a potent negative regulator of lymphocyte activation, the functional significance of which has been demonstrated in murine tumor models using blocking antibodies. However, the mechanism(s) involved in enhancing tumor regression has not been identified. In this study, we determined whether IFN gamma was playing a role in this activity. In vitro, anti-CTLA-4 enhanced IFN gamma production by lymph node cells obtained from tumor-bearing mice (351 pg/ml vs 77 pg/ml). Additionally, fibrosarcoma-challenged animals treated with anti-CTLA-4 had elevated levels of the IFN-inducible enzyme 2-5-OAS in draining lymph nodes (850 pM vs 260 pM for controls) and an increased amount of IFN gamma in tumor lysates (at day 7, 620 pg/100 micrograms vs 160 pg/100 micrograms in controls). The importance of IFN gamma was demonstrated by the ability of neutralizing antibodies to completely abrogate the anti-tumor effects of anti-CTLA-4. Moreover, fibrosarcoma cells were shown to be exquisitely sensitive to IFN gamma-mediated class I upregulation and histological examination of tumors from anti-CTLA-4-treated mice revealed a trend toward increased tumor cell apoptosis and decreased angiogenesis. These studies have demonstrated that one mechanism for the anti-tumor effects of anti-CTLA-4 relates to its ability to augment IFN gamma production, resulting in an increased expression of class I on the tumor, enhanced apoptosis, and a decrease in blood vessel growth.

We describe the genomic organization of a recently identified CC chemokine, MIP3alpha/CCL20 (HGMW-approved symbol SCYA20). The MIP-3alpha/CCL20 gene was cloned and sequenced, revealing a four exon, three intron structure, and was localized by FISH analysis to 2q35-q36. Two distinct cDNAs were identified, encoding two forms of MIP-3alpha/CCL20, Ala MIP-3alpha/CCL20 and Ser MIP-3alpha/CCL20, that differ by one amino acid at the predicted signal peptide cleavage site. Examination of the sequence around the boundary of intron 1 and exon 2 showed that use of alternative splice acceptor sites could give rise to Ala MIP-3alpha/CCL20 or Ser MIP-3alpha/CCL20. Both forms of MIP-3alpha/CCL20 were chemically synthesized and tested for biological activity. Both flu antigen plus IL-2-activated CD4(+) and CD8(+) T lymphoblasts and cord blood-derived dendritic cells responded to Ser and Ala MIP-3alpha/CCL20. T lymphocytes exposed only to IL-2 responded inconsistently, while no response was detected in naive T lymphocytes, monocytes, or neutrophils. The biological activity of Ser MIP-3alpha/CCL20 and Ala MIP-3alpha/CCL20 and the tissue-specific preference of different splice acceptor sites are not yet known., (Copyright 2001 Academic Press.)

Recent studies have suggested a pivotal role for secondary lymphoid chemokine (SLC) in directing dendritic cell trafficking from peripheral to lymphoid tissues. As an extension of these studies, we examined the consequences of anti-SLC Ab treatment during Ag priming on T cell function in an inflammatory response. We used a model of T cell-mediated inflammation, contact hypersensitivity (CHS), where priming of the effector T cells is dependent upon epidermal dendritic cell, Langerhans cells, and migration from the hapten sensitization site in the skin to draining lymph nodes. A single injection of anti-SLC Ab given at the time of sensitization with FITC inhibited Langerhans cell migration into draining lymph nodes for at least 3 days. The CHS response to hapten challenge was inhibited by anti-SLC Ab treatment in a dose-dependent manner. Despite the inhibition of CHS, T cells producing IFN-gamma following in vitro stimulation with anti-CD3 mAb or with hapten-labeled cells were present in the skin-draining lymph nodes of mice treated with anti-SLC Ab during hapten sensitization. These T cells were unable, however, to passively transfer CHS to naive recipients. Animals treated with anti-SLC Ab during hapten sensitization were not tolerant to subsequent sensitization and challenge with the hapten. In addition, anti-SLC Ab did not inhibit CHS responses when given at the time of hapten challenge. These results indicate an important role for SLC during sensitization for CHS and suggest a strategy to circumvent functional T cell priming for inflammatory responses through administration of an Ab inhibiting dendritic cell trafficking.

Macrophage inflammatory protein (MIP-1 alpha), a member of the CC chemokine subfamily, has been shown to attract T cells and monocytes in vitro and to be expressed at sites of inflammation. Although the in vitro activities of MIP-1 alpha have been well documented, the in vivo biological activities of MIP-1 alpha in humans have not been studied. To address this, we challenged human subjects by intradermal injection with up to 1000 pmol of MIP-1 alpha and performed biopsies 2, 10, and 24 h later. Although no acute cutaneous or systemic reactions were noted, endothelial cell activation, as indicated by the expression of E-selectin, was observed. In agreement with its in vitro activity, monocyte, lymphocyte, and, to a lesser degree, eosinophil infiltration was observed, peaking at 10-24 h. Surprisingly, in contrast to its reported lack of in vitro neutrophil-stimulating activity, a rapid infiltration of neutrophils was observed in vivo. This neutrophil infiltration occurred as early as 2 h, preceding the appearance of other cells, and peaked at 10 h. Interestingly, we found that neutrophils in whole blood, but not after isolation, expressed CCR1 on their cell surface. This CCR1 was thought to be functional as assessed by neutrophil CD11b up-regulation following whole-blood MIP-1 alpha stimulation. These studies substantiate the biological effects of MIP-1 alpha on monocytes and lymphocytes and uncover the previously unrecognized activity of MIP-1 alpha to induce neutrophil infiltration and endothelial cell activation, underscoring the need to evaluate chemokines in vivo in humans.

The in vitro chemotactic activity of chemokines have been well documented. However, study of their in vivo effects where components of rolling, adherence and diapedesis are pre-requisites to leukocyte infiltration have not been examined in higher species. In this study, we examined the biological activity of the CC chemokine, MIP-1alpha, in rhesus monkeys. Following an intradermal injection, a significant cellular infiltrate and an increase in the number of inflamed vessels were observed. This response peaked at 24 h and was sustained for up to 48 hours after injection. Phenotypically, the specific infiltrate consisted exclusively of CD68+ monocytes with no increase in other cell types over the saline injected controls. These studies represent the first examination of the in vivo effects of MIP-1alpha in higher species and indicate that MIP-1alpha is a selective monocyte recruiting agent in vivo.

Experimental allergic encephalomyelitis is a murine model of preclinical autoimmune disease that has pathologic similarities to multiple sclerosis (MS). Although CD4+ T cells have been shown to play a crucial role in the development of disease, we recently demonstrated a link between the development of paralysis and eosinophil infiltration into the spinal cord. As such, CD4+ cells may initiate disease, but eosinophils may be the actual effector cells responsible for causing damage to myelin and causing paralysis. Because MS patients sometimes experience early visual problems, ie, optic neuritis, we explored whether an early eosinophil infiltrate was also observed in the optic nerves of SJL mice after the passive transfer of encephalitogenic T cells. Seven days after the passive transfer of myelin basic protein (MBP)-reactive T cell blasts, we observed a significant infiltration of eosinophils into the optic nerves of the mice. This infiltration persisted during the early phases of paralysis, then declined to baseline values by the peak of limb paralysis on Day 10, and remained at baseline during the remission phase of the disease. Remyelination of optic nerves was observed at this time. These results suggest that eosinophil infiltration into the optic nerve is one of the earliest events occurring after the passive transfer of encephalitogenic T cells in murine experimental allergic encephalomyelitis.

Superantigens such as staphylococcal enterotoxin A and B (SEA and SEB) activate the immune system by stimulating a large proportion of T lymphocytes through specific Vbeta regions of the TCR and activating macrophages by binding to MHC class II molecules. While the mechanisms by which superantigens activate T lymphocytes have been elucidated, their role in the generation of local immune responses to bacterial invasion is still unclear. In this study we have examined the ability of the superantigens SEA and SEB to elicit an inflammatory reaction in vivo, in s.c. air pouches in the mouse. Upon injection into the s.c. air pouch, the two superantigens stimulated a time-dependent increase in the number of leukocytes appearing in the pouch exudate. The leukocytes migrating into the pouch exudate were predominantly neutrophils, with some mononuclear phagocytes and eosinophils present. No T lymphocytes were detected either in the pouch lining tissue or in the exudate cells. Injection of SEA resulted in increased ICAM-1 expression, as detected by immunohistochemistry, on endothelial cells in the tissue surrounding the air pouch and accumulation of TNF-alpha and the chemokines macrophage inflammatory protein-2 (MIP-2), MIP-1alpha, and JE in the pouch exudate. In addition, pretreatment of mice with Abs raised against ICAM-1, TNF-alpha, MIP-2, MIP-1alpha, KC, or JE inhibited leukocyte accumulation induced by SEA. These data demonstrate that bacterial superantigens may promote inflammation at extravascular sites in vivo, and that this response is secondary to the generation of inflammatory mediators, including chemokines.

Chemokines are essential mediators of normal leukocyte trafficking as well as of leukocyte recruitment during inflammation. We describe here a novel non-ELR CXC chemokine identified through sequence analysis of cDNAs derived from cytokine-activated primary human astrocytes. This novel chemokine, referred to as I-TAC (interferon-inducible T cell alpha chemoattractant), is regulated by interferon (IFN) and has potent chemoattractant activity for interleukin (IL)-2-activated T cells, but not for freshly isolated unstimulated T cells, neutrophils, or monocytes. I-TAC interacts selectively with CXCR3, which is the receptor for two other IFN-inducible chemokines, the IFN-gamma-inducible 10-kD protein (IP-10) and IFN-gamma- induced human monokine (HuMig), but with a significantly higher affinity. In addition, higher potency and efficacy of I-TAC over IP-10 and HuMig is demonstrated by transient mobilization of intracellular calcium as well as chemotactic migration in both activated T cells and transfected cell lines expressing CXCR3. Stimulation of astrocytes with IFN-gamma and IL-1 together results in an approximately 400,000-fold increase in I-TAC mRNA expression, whereas stimulating monocytes with either of the cytokines alone or in combination results in only a 100-fold increase in the level of I-TAC transcript. Moderate expression is also observed in pancreas, lung, thymus, and spleen. The high level of expression in IFN- and IL-1-stimulated astrocytes suggests that I-TAC could be a major chemoattractant for effector T cells involved in the pathophysiology of neuroinflammatory disorders, although I-TAC may also play a role in the migration of activated T cells during IFN-dominated immune responses.

Antigen stimulation of T cells results in a series of biochemical events including the interaction of both SH2 domains of ZAP-70 with phosphorylated ITAMS on the T cell receptor. In order to study the physiological relevance of decreasing native ZAP-70-SH2 interaction in vivo, we generated transgenic mice expressing a T cell-specific, dominant negative form of ZAP-70 consisting of only the tandem SH2 domains (ZAP-NC). Phenotypically, these animals had a comparable distribution of lymphocyte subsets in the thymus and spleen compared with the wild-type (WT) controls. However, examination of peripheral blood revealed a slow but progressive decrease in the number of lymphocytes, particularly CD4+ cells, with age (17% reduction by 3 months, 58% reduction by 6 months). Allogeneic responses were then evaluated in vitro as well as in vivo using a subcutaneous sponge matrix implant. Although spleen cells cultured for 4 days in vitro with alloantigen developed normal functional responses, allogeneic responses generated in vivo within a subcutaneous sponge matrix were impaired. This was characterized by a depression in cytotoxic T lymphocyte (CTL) activity, a 82% reduction in the frequency of helper T cells, and a 78% reduction in the capacity of sponge-infiltrating lymphocytes to produce IL-2 in response to secondary antigen stimulation. These results indicate that although overt lymphocyte development and in vitro function were unremarkable, expression of a truncated ZAP-70 affected the in vivo survival of peripheral lymphocytes and altered the in vivo generation of functional activity to alloantigen.

In this study, we have evaluated the role of specific chemotactic cytokines in leukocyte recruitment to s.c. tissue in response to TNF-alpha in vivo. Injection of TNF-alpha into s.c. air pouches led to a rapid, transient accumulation of leukocytes. Maximal accumulation of leukocytes in the air pouch was observed at between 2 and 4 h after injection of TNF-alpha. The cellular exudate comprised predominantly neutrophils, with smaller numbers of eosinophils and mononuclear phagocytes also being recruited. However, lymphocyte recruitment was not observed. TNF-alpha injection induced a time-dependent increase in the levels of immunoreactive macrophage inflammatory protein (MIP)-2, MIP-1alpha, and JE in the pouch exudate as well as increased steady-state mRNA levels of KC, MIP-2, MIP-1alpha, and JE in the tissue lining the s.c. pouch and of MIP-2, MIP-1alpha, and JE in the exudate cell population. Passive immunization with specific Abs directed against each of these chemokines significantly inhibited the accumulation of neutrophils, mononuclear phagocytes, and eosinophils in response to TNF-alpha. Taken together, these data demonstrate the existence of a chemokine network in vivo involving at least four individual chemokines that regulates recruitment of the major peripheral blood granulocytes and mononuclear phagocytes to s.c. sites during acute inflammation. To our knowledge, these data are also the first demonstration that the C-C chemokine JE is involved in neutrophil recruitment in a physiologic system in vivo.

Monocyte chemoattractant protein-1 (MCP-1) is a CC chemokine that attracts monocytes and T lymphocytes in vitro; however, its in vivo functions are poorly understood. To address this question, we constructed transgenic mice expressing MCP-1 controlled by an insulin promoter. These mice developed a chronic insulitic infiltrate composed of F4/80+ monocytes with minor populations of CD4+, CD8+, and B220+ cells. Despite persistent transgene expression, the insulitis never progressed, and blood glucose levels remained normal. Thus, MCP-1 alone is sufficient to elicit a monocytic infiltrate, but not to activate elicited cells. These results differ from those obtained with another transgenic model using the mouse mammary tumor virus long terminal repeat, in which mice expressed substantial MCP-1 in several organs but had no infiltrates. However, mice expressing both transgenes had minimal insulitis, indicating that high systemic levels of MCP-1 prevented monocytes from responding to local MCP-1. Thus, the ability of MCP-1 to elicit monocytic infiltration depends on its being expressed at low levels in an anatomically restricted area.

In our previous work, DNase hypersensitivity mapping was used to identify an enhancer within the human CD8 alpha (hCD8 alpha) gene which allowed T cell-specific expression of a reporter construct in transiently transfected cell lines. To study the role of this intronic enhancer in vivo, transgenic mice were made using human CD8 genomic constructs. We found that while a 14 kb wild-type human CD8 alpha (WThCD8) genomic construct did not lead to expression in mature peripheral CD8+ T cells, this transgene was consistently expressed in small populations of T cells and B cells, and in a subset of mouse NK cells. While murine CD8 is not normally expressed on resting NK cells, expression of the human CD8 transgene on mouse NK cells is appropriate since CD8 is expressed on a subset of human NK cells. Deletion of the intronic enhancer resulted in a complete loss of transgene expression in most lines and a loss of expression only in NK cells in one line. Our results indicate, firstly, that cis-acting sequences within the 14 kb genomic fragment are sufficient for NK cell-specific expression. In addition, our results suggest that the enhancer may have dual roles in regulation of transgene expression. It may enhance general expression of the transgene and may also be required for NK cell-specific expression.

Leukotriene B4 (LTB4) is a chemotactic and cell-activating factor present at inflammatory sites in a variety of autoimmune diseases including multiple sclerosis (MS). In this study, we used a murine model of MS, experimental allergic encephalomyelitis (EAE), to assess the potential role of LTB4 on cell infiltration and paralysis. Injection of encephalogenic T cells into naive animals induced paralysis and weight loss that was completely inhibited by treatment with the selective LTB4 receptor antagonist CP-105,696 (ED50= 8.6 mg/kg orally). Although migration of lymphocytes into the central nervous system was unaffected, the efficacious effects of CP-105,696 correlated with up to a 97% decrease in eosinophil infiltration into the lower spinal cord as determined by light and electron microscopy and quantitated by levels of the specific enzyme marker eosinophil peroxidase. These results demonstrate that eosinophil recruitment in EAE is dependent on LTB4 receptor ligation and further reveal a previously unrecognized role for eosinophils in the pathogenesis of this disease.

The activity of trovafloxacin against 22 clinical Legionella isolates was determined by broth microdilution susceptibility testing. The trovafloxacin concentration required to inhibit 90% of strains tested was < or = 0.004 micrograms/ml, in contrast to 0.032 micrograms/ml for ofloxacin. In guinea pig alveolar macrophages, trovafloxacin achieved intracellular levels up to 28-fold over the extracellular concentration, which was similar to the levels obtained with erythromycin. Trovafloxacin (0.25 micrograms/ml) reduced bacterial counts of two L. pneumophila strains grown in guinea pig alveolar macrophages by > 2 log10 CFU/ml, without regrowth, under drug-free conditions over a 3-day period; trovafloxacin was significantly more active than ofloxacin or erythromycin (0.25 to 1 microgram/ml) in this assay. Single-dose (10 mg of prodrug CP-116,517-27 per kg of body weight given intraperitoneally [i.p.], equivalent to 7.5 mg of trovafloxacin per kg) pharmacokinetic studies performed in guinea pigs with L. pneumophila pneumonia revealed peak serum and lung trovafloxacin levels to be 3.8 micrograms/ml and 5.0 micrograms/g, respectively, at 0.5 h and 4.2 micrograms/ml and 2.9 micrograms/g, respectively, at 1 h. Administration of a lower prodrug dose (1.4 mg of trovafloxacin equivalent per kg i.p.) gave levels in lung and serum of 0.4 microgram/g and 0.4 microgram/ml, respectively, 1 h after drug administration. The terminal half-lives of elimination from serum and lung were 0.8 and 1.1 h, respectively. All 15 infected guinea pigs treated for 5 days with CP-116,517-27 once daily (10 mg/kg/day i.p., equivalent to 7.5 mg of trovafloxacin per kg/day) survived for 10 days after antimicrobial therapy, as did all 15 guinea pigs treated with ofloxacin once daily (10 mg/kg/day i.p.) for 5 days. None of 13 animals treated with saline survived. In a second experiment with animals, trovafloxacin (1.4 mg/kg/day i.p. for 5 days) protected all 16 guinea pigs from death, whereas all 15 animals treated with saline died. Trovafloxacin is an effective antimicrobial agent against Legionella in vitro and in vivo, with the ability to concentrate in macrophages and kill intracellular organisms.

We have constructed transgenic mice in which the mouse mammary tumor virus long terminal repeat controls the expression of murine monocyte chemoattractant protein-1 (MCP-1). Several independently derived lines of transgenic mice constitutively expressed MCP-1 protein in a variety of organs. Protein extracts from these organs had substantial in vitro monocyte chemoattractant activity that was neutralized by an anti-MCP-1 Ab, indicating that transgenic MCP-1 protein is biologically active. However, no transgenic mouse at any age displayed monocyte infiltrates in MCP-1-expressing organs. Two transgenic lines had circulating MCP-1 levels of 13 to 26 ng/ml, which is a concentration sufficient to induce maximal monocyte chemotaxis in vitro. These transgenic lines showed a 1 to 1.5 log greater sensitivity to infection with Listeria monocytogenes and Mycobacterium tuberculosis. A third transgenic line had lower serum levels of MCP-1 and was resistant to L. monocytogenes. The results suggest that this transgenic model is one of monocyte nonresponsiveness to locally produced MCP-1 due to either receptor desensitization or neutralization of a chemoattractant gradient by high systemic concentrations of MCP-1. Regardless of the mechanism, the data indicate that constitutively high levels of MCP-1 expression do not induce monocytic infiltrates, and that MCP-1 is involved in the host response to intracellular pathogens.

A heterodimer containing the mouse 35 kDa and human 40 kDa subunit of IL-12 was expressed in COS cells (cIL-12). Administration of 25-200 U of the cIL-12-COS supernatant to mice twice daily for 2 days augmented spleen cell IFN-gamma production in response to IL-2 and peritoneal macrophage activity (superoxide and nitrites) as compared to animals receiving mock transfected supernatants. cIL-12 also increased levels of IFN-gamma in serum but most dramatically following an LPS injection (50-fold over controls). Animals pretreated with cIL-12 suffered enhanced mortality following challenge with the Gram negative organism E. coli but enhanced survival or clearance following infection with the Gram positive organisms L. monocytogenes and S. aureus. Although daily treatment of mice with cIL-12 following an intranasal influenza A infection elevated levels of IFN-gamma in the bronchioalveolar lavage fluid three-fold over controls, neither prophylactic or therapeutic treatment with the same dose level decreased viral titres in the lung. In addition, no effect was observed in animals infected with encephalomyocarditis virus or respiratory syncytial virus. Therefore, cIL-12 is a potent in vivo augmentor of IFN-gamma production. It has differential effects on infectious disease depending on the invading organism and time of administration; being efficacious for intracellular bacteria but ineffective at the same dose levels against viral diseases.

Renal vasoconstriction has been implicated as a major contributing factor for the nephrotoxic effects of cyclosporine. In an attempt to assess the relative contribution of thromboxane (Tx), the effects of coadministering CsA with the selective Tx synthetase inhibitor, Dazmegrel (DAZ) (Pfizer, Inc.), were determined. Rats were treated orally with 50 mg/kg of DAZ plus 50 mg/kg of CsA and various indicators of nephrotoxicity and efficacy were assessed. Animals treated with CsA + DAZ had a normalization of renal TxB2 synthesis as compared with animals treated with CsA alone (160 vs. 338 pg/ml). Kidney proximal tubule damage following CsA treatment alone was also reduced in animals coadministered DAZ, as indicated by reduced urinary excretion of N-acetyl-beta-D-glucosaminidase (NAG) (9.7 vs. 14.1 U/g creatinine) and by histological examination of kidney sections. However, DAZ did not affect blood levels of CsA nor its efficacious activity in a model of experimental allergic encephalomyelitis (EAE). These studies suggest a major role of elevated thromboxane production in the acute nephrotoxic effects of CsA and demonstrate a reduction in this toxicity by DAZ without altering CsA's efficacious activity.

Azithromycin was shown to achieve high concentrations in human skin fibroblasts. Intracellular penetration occurred rapidly (10 micrograms/mg of cellular protein after 3 h) and then increased progressively over a 3-day period; azithromycin accumulated up to 21 times more than erythromycin (61.1 versus 2.9 micrograms/mg of protein). Uptake was dependent on the extracellular concentration, was inhibited at 4 degrees C, did not occur in nonviable cells, and was reduced by a low pH. Intracellular accumulation was not affected by the metabolic inhibitor 2,4-dinitrophenol or sodium fluoride or by the nucleoside transport inhibitor 2-chloradenosine. Once concentrated in cells, azithromycin remained intracellular and was released slowly in the absence of extracellular drug, compared with erythromycin (17 versus 78% released after 1 h). After 48 h of incubation in drug-free medium, 27% of the initial amount of azithromycin remained cell associated. The release of azithromycin was not affected by various monokines reported to stimulate fibroblasts (interleukin-1 or tumor necrosis factor) or by exposure to bacteria. Incubation of azithromycin-loaded fibroblasts with human polymorphonuclear leukocytes resulted in a higher intracellular accumulation of azithromycin in polymorphonuclear leukocytes than in cells incubated with free nonintracellular azithromycin for the same time (8.3 versus 2.2 micrograms/ml after 2 h), suggesting a more efficient or rapid uptake through cell-to-cell interaction. The widespread distribution of fibroblasts in tissues suggests a potential for these cells, and possibly other lysosome-containing tissue cells, to serve as a reservoir for azithromycin, slowly releasing it for activity against extracellular organisms at sites of infection and passing it to phagocytes for activity against intracellular pathogens and potential transport to sites of infection.

Azithromycin, a novel azalide antibiotic, concentrated in human and mouse polymorphonuclear leukocytes (PMNs), murine peritoneal macrophages, and mouse and rat alveolar macrophages, attaining intracellular concentrations up to 226 times the external concentration in vitro. In murine peritoneal macrophages, azithromycin achieved concentration gradients (internal to external) up to 26 times higher than erythromycin. The cellular uptake of azithromycin was dependent on temperature, viability, and pH and was decreased by 2,4-dinitrophenol. Azithromycin did not decrease phagocyte-mediated bactericidal activity or affect PMN or macrophage oxidative burst activity (H2O2 release or Nitro Blue Tetrazolium reduction, respectively). Azithromycin remained in cells for several hours, even after extracellular drug was removed. However, its release was significantly enhanced by phagocytosis of Staphylococcus aureus (82 versus 23% by 1.5 h). In vivo, 0.05 micrograms of azithromycin was found in peritoneal fluids of mice 20 h after oral treatment with a dose of 50 mg/kg. Following caseinate-induced PMN infiltration, there was a sixfold increase in peritoneal cavity azithromycin to 0.32 micrograms, most of which was intracellular. Therefore, the uptake, transport, and later release of azithromycin by these cells demonstrate that phagocytes may deliver active drug to sites of infection.