ABSTRACT
Genetic and in vivo evidence suggests that aberrant recognition of RNA-containing autoantigens by Toll-like receptors (TLRs) 7 and 8 drives autoimmune diseases. Here we report on the preclinical characterization of MHV370, a selective oral TLR7/8 inhibitor. In vitro, MHV370 inhibits TLR7/8-dependent production of cytokines in human and mouse cells, notably interferon-α, a clinically validated driver of autoimmune diseases. Moreover, MHV370 abrogates B cell, plasmacytoid dendritic cell, monocyte, and neutrophil responses downstream of TLR7/8. In vivo, prophylactic or therapeutic administration of MHV370 blocks secretion of TLR7 responses, including cytokine secretion, B cell activation, and gene expression of, e.g., interferon-stimulated genes. In the NZB/W F1 mouse model of lupus, MHV370 halts disease. Unlike hydroxychloroquine, MHV370 potently blocks interferon responses triggered by specific immune complexes from systemic lupus erythematosus patient sera, suggesting differentiation from clinical standard of care. These data support advancement of MHV370 to an ongoing phase 2 clinical trial.
Subject(s)
Autoimmune Diseases , Lupus Erythematosus, Systemic , Humans , Mice , Animals , Toll-Like Receptor 7/metabolism , Toll-Like Receptor 7/therapeutic use , Lupus Erythematosus, Systemic/drug therapy , Lupus Erythematosus, Systemic/metabolism , Hydroxychloroquine/pharmacology , Hydroxychloroquine/therapeutic use , InterferonsABSTRACT
IL-1 receptor-activated kinase 1 (IRAK1) is involved in signal transduction downstream of many TLRs and the IL-1R. Its potential as a drug target for chronic inflammatory diseases is underappreciated. To study its functional role in joint inflammation, we generated a mouse model expressing a functionally inactive IRAK1 (IRAK1 kinase deficient, IRAK1KD), which also displayed reduced IRAK1 protein expression and cell type-specific deficiencies of TLR signaling. The serum transfer model of arthritis revealed a potentially novel role of IRAK1 for disease development and neutrophil chemoattraction exclusively via its activity in nonhematopoietic cells. Consistently, IRAK1KD synovial fibroblasts showed reduced secretion of neutrophil chemoattractant chemokines following stimulation with IL-1ß or human synovial fluids from patients with rheumatoid arthritis (RA) and gout. Together with patients with RA showing prominent IRAK1 expression in fibroblasts of the synovial lining, these data suggest that targeting IRAK1 may be therapeutically beneficial. As pharmacological inhibition of IRAK1 kinase activity had only mild effects on synovial fibroblasts from mice and patients with RA, targeted degradation of IRAK1 may be the preferred pharmacologic modality. Collectively, these data position IRAK1 as a central regulator of the IL-1ß-dependent local inflammatory milieu of the joints and a potential therapeutic target for inflammatory arthritis.
Subject(s)
Arthritis, Rheumatoid , Interleukin-1 Receptor-Associated Kinases , Neutrophils , Synovial Membrane , Animals , Arthritis, Rheumatoid/metabolism , Cells, Cultured , Disease Models, Animal , Interleukin-1 Receptor-Associated Kinases/metabolism , Interleukin-8/metabolism , Mice , Neutrophils/metabolism , Synovial Membrane/metabolismABSTRACT
Inappropriate activation of TLR7 and TLR8 is linked to several autoimmune diseases, such as lupus erythematosus. Here we report on the efficient structure-based optimization of the inhibition of TLR8, starting from a co-crystal structure of a small screening hit. Further optimization of the physicochemical properties for cellular potency and expansion of the structure-activity relationship for dual potency finally resulted in a highly potent TLR7/8 antagonist with demonstrated in vivo efficacy after oral dosing.
ABSTRACT
The cytosolic metalloenzyme leukotriene A4 hydrolase (LTA4H) is the final and rate-limiting enzyme in the biosynthesis of pro-inflammatory leukotriene B4 (LTB4). Preclinical studies have validated this enzyme as an attractive drug target in chronic inflammatory diseases. Despite several attempts, no LTA4H inhibitor has reached the market, yet. Herein, we disclose the discovery and preclinical profile of LYS006, a highly potent and selective LTA4H inhibitor. A focused fragment screen identified hits that could be cocrystallized with LTA4H and inspired a fragment merging. Further optimization led to chiral amino acids and ultimately to LYS006, a picomolar LTA4H inhibitor with exquisite whole blood potency and long-lasting pharmacodynamic effects. Due to its high selectivity and its ability to fully suppress LTB4 generation at low exposures in vivo, LYS006 has the potential for a best-in-class LTA4H inhibitor and is currently investigated in phase II clinical trials in inflammatory acne, hidradenitis suppurativa, ulcerative colitis, and NASH.
Subject(s)
Aminobutyrates/therapeutic use , Anti-Inflammatory Agents/pharmacology , Enzyme Inhibitors/therapeutic use , Epoxide Hydrolases/antagonists & inhibitors , Pyridines/therapeutic use , Aminobutyrates/chemical synthesis , Aminobutyrates/pharmacokinetics , Animals , Anti-Inflammatory Agents/chemical synthesis , Anti-Inflammatory Agents/pharmacokinetics , Arthritis, Experimental/drug therapy , Dogs , Drug Discovery , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacokinetics , Female , Humans , Inflammation/drug therapy , Male , Mice, Inbred C57BL , Molecular Structure , Pyridines/chemical synthesis , Pyridines/pharmacokinetics , Rats, Wistar , Structure-Activity RelationshipABSTRACT
Inappropriate activation of endosomal TLR7 and TLR8 occurs in several autoimmune diseases, in particular systemic lupus erythematosus (SLE). Herein, the development of a TLR8 antagonist competition assay and its application for hit generation of dual TLR7/8 antagonists are reported. The structure-guided optimization of the pyridone hit 3 using this biochemical assay in combination with cellular and TLR8 cocrystal structural data resulted in the identification of a highly potent and selective TLR7/8 antagonist (27) with in vivo efficacy. The two key steps for optimization were (i) a core morph guided by a TLR7 sequence alignment to achieve a dual TLR7/8 antagonism profile and (ii) introduction of a fluorine in the piperidine ring to reduce its basicity, resulting in attractive oral pharmacokinetic (PK) properties and improved TLR8 binding affinity.
Subject(s)
Lupus Erythematosus, Systemic/drug therapy , Pyridones/chemistry , Pyridones/pharmacology , Toll-Like Receptor 7/antagonists & inhibitors , Toll-Like Receptor 8/antagonists & inhibitors , Animals , Cells, Cultured , Drug Discovery , Humans , Indazoles/chemistry , Indazoles/pharmacokinetics , Indazoles/pharmacology , Lupus Erythematosus, Systemic/metabolism , Male , Mice, Inbred C57BL , Models, Molecular , Pyridones/pharmacokinetics , Rats, Sprague-Dawley , Toll-Like Receptor 7/chemistry , Toll-Like Receptor 7/metabolism , Toll-Like Receptor 8/chemistry , Toll-Like Receptor 8/metabolismABSTRACT
GPR4 is a pH-sensing G protein-coupled receptor highly expressed in vascular endothelial cells and can be activated by protons in the inflamed tissue microenvironment. Herein, we report that acidosis-induced GPR4 activation increases paracellular gap formation and permeability of vascular endothelial cells through the Gα12/13/Rho GTPase signaling pathway. Evaluation of GPR4 in the inflammatory response using the acute hindlimb ischemia-reperfusion mouse model revealed that GPR4 mediates tissue edema, inflammatory exudate formation, endothelial adhesion molecule expression, and leukocyte infiltration in the inflamed tissue. Genetic knockout and pharmacologic inhibition of GPR4 alleviate tissue inflammation. These results suggest GPR4 is a pro-inflammatory receptor and could be targeted for therapeutic intervention.
ABSTRACT
Inflammatory bowel disease (IBD) is characterized by chronic, recurring inflammation of the digestive tract. Current therapeutic approaches are limited and include biologics and steroids such as anti-TNFα monoclonal antibodies and corticosteroids, respectively. Significant adverse drug effects can occur for chronic usage and include increased risk of infection in some patients. GPR4, a pH-sensing G protein-coupled receptor, has recently emerged as a potential therapeutic target for intestinal inflammation. We have assessed the effects of a GPR4 antagonist, 2-(4-((2-Ethyl-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl)methyl)phenyl)-5-(piperidin-4-yl)-1,3,4-oxadiazole (GPR4 antagonist 13, also known as NE-52-QQ57) in the dextran sulfate sodium (DSS)-induced acute colitis mouse model. The GPR4 antagonist 13 inhibited intestinal inflammation. The clinical parameters such as body weight loss and fecal score were reduced in the GPR4 antagonist 13 treatment group compared to vehicle control. Macroscopic disease indicators such as colon shortening, splenic expansion, and mesenteric lymph node enlargement were all reduced in severity in the GPR4 antagonist 13 treated mice. Histopathological features of active colitis were alleviated in GPR4 antagonist 13 treatment groups compared to vehicle control. Finally, inflammatory gene expression in the colon tissues and vascular adhesion molecule expression in the intestinal endothelia were attenuated by GPR4 antagonist 13. Our results indicate that GPR4 antagonist 13 provides a protective effect in the DSS-induced acute colitis mouse model, and inhibition of GPR4 can be explored as a novel anti-inflammatory approach.
Subject(s)
Colitis/drug therapy , Colitis/metabolism , Intestinal Mucosa/drug effects , Intestinal Mucosa/metabolism , Oxadiazoles/pharmacology , Receptors, G-Protein-Coupled/metabolism , Animals , Colitis/pathology , Disease Models, Animal , E-Selectin/metabolism , Female , Gene Expression Regulation/drug effects , Inflammation/drug therapy , Inflammation/metabolism , Intestinal Mucosa/pathology , Male , Mice , Mice, Inbred C57BL , Oxadiazoles/therapeutic use , Piperidines/pharmacology , Piperidines/therapeutic use , Pyrazoles/pharmacology , Pyrazoles/therapeutic use , Receptors, G-Protein-Coupled/antagonists & inhibitors , Vascular Cell Adhesion Molecule-1/metabolismABSTRACT
GPR4, a G-protein coupled receptor, functions as a proton sensor being activated by extracellular acidic pH and has been implicated in playing a key role in acidosis associated with a variety of inflammatory conditions. An orally active GPR4 antagonist 39c was developed, starting from a high throughput screening hit 1. The compound shows potent cellular activity and is efficacious in animal models of angiogenesis, inflammation and pain.
Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Drug Design , Inflammation/drug therapy , Receptors, G-Protein-Coupled/antagonists & inhibitors , Administration, Oral , Animals , Anti-Inflammatory Agents, Non-Steroidal/administration & dosage , Anti-Inflammatory Agents, Non-Steroidal/chemistry , Arthritis/drug therapy , Arthritis/metabolism , COS Cells , Chlorocebus aethiops , Dose-Response Relationship, Drug , Female , HEK293 Cells , HeLa Cells , Humans , Inflammation/metabolism , Mice , Molecular Structure , Pain/drug therapy , Pain/metabolism , Rats , Rats, Sprague-Dawley , Receptors, G-Protein-Coupled/metabolism , Structure-Activity RelationshipABSTRACT
A novel, selective, and efficacious GPR4 antagonist 13 was developed starting from lead compound 1a. While compound 1a showed promising efficacy in several disease models, its binding to a H3 receptor as well as a hERG channel prevented it from further development. Therefore, a new round of optimization addressing the key liabilities was performed and led to discovery of compound 13 with an improved profile. Compound 13 showed significant efficacy in the rat antigen induced arthritis as well as in the hyperalgesia and angiogenesis model at a well-tolerated dose of 30 mg/kg.
Subject(s)
Inflammation/prevention & control , Neovascularization, Physiologic/drug effects , Nociception/drug effects , Receptors, G-Protein-Coupled/antagonists & inhibitors , Administration, Oral , Animals , Drug Design , Female , HEK293 Cells , Humans , Rats , Rats, Sprague-Dawley , Receptors, Histamine H3/metabolismABSTRACT
Systemic candidiasis is a leading cause of nosocomial bloodstream infection with a high mortality rate despite treatment. Immune-based strategies are needed to improve outcomes. We previously reported that genetic deficiency in the chemokine receptor CCR1 improves survival and ameliorates tissue damage in Candida-infected mice. Here, we found that treatment of immunocompetent Candida-infected mice with the CCR1-selective antagonist BL5923 improves survival, decreases the kidney fungal burden, and protects from renal tissue injury.
Subject(s)
Acute Kidney Injury/prevention & control , Candida albicans/drug effects , Candidiasis, Invasive/drug therapy , Hemorrhage/prevention & control , Piperazines/pharmacology , Receptors, CCR1/antagonists & inhibitors , Acute Kidney Injury/immunology , Acute Kidney Injury/microbiology , Acute Kidney Injury/mortality , Animals , Candida albicans/growth & development , Candida albicans/pathogenicity , Candidiasis, Invasive/immunology , Candidiasis, Invasive/microbiology , Candidiasis, Invasive/mortality , Drug Administration Schedule , Female , Gene Expression , Hemorrhage/immunology , Hemorrhage/microbiology , Hemorrhage/mortality , Host-Pathogen Interactions , Mice , Mice, Inbred C57BL , Receptors, CCR1/genetics , Receptors, CCR1/immunology , Survival AnalysisABSTRACT
Systemic lupus erythematosus is a chronic inflammatory autoimmune disease, the development of which is characterized by a progressive loss of renal function. Such dysfunction is associated with leukocyte infiltration in the glomerular and tubulointerstitial compartments in both human and experimental lupus nephritis. In this study, we investigated the role of the Ccr1 chemokine receptor in this infiltration process during the progression of nephritis in the lupus-prone New Zealand Black/New Zealand White (NZB/W) mouse model. We found that peripheral T cells, mononuclear phagocytes, and neutrophils, but not B cells, from nephritic NZB/W mice were more responsive to Ccr1 ligands than the leukocytes from younger prenephritic NZB/W mice. Short-term treatment of nephritic NZB/W mice with the orally available Ccr1 antagonist BL5923 decreased renal infiltration by T cells and macrophages. Longer Ccr1 blockade decreased kidney accumulation of effector/memory CD4(+) T cells, Ly6C(+) monocytes, and both M1 and M2 macrophages; reduced tubulointerstitial and glomerular injuries; delayed fatal proteinuria; and prolonged animal lifespan. In contrast, renal humoral immunity was unaffected in BL5923-treated mice, which reflected the unchanged numbers of infiltrated B cells in the kidneys. Altogether, these findings define a pivotal role for Ccr1 in the recruitment of T and mononuclear phagocyte cells to inflamed kidneys of NZB/W mice, which in turn contribute to the progression of renal injury.
Subject(s)
Lupus Nephritis/therapy , Myeloid Cells/immunology , Neutrophil Infiltration , Receptors, CCR1/antagonists & inhibitors , T-Lymphocyte Subsets/immunology , Age Factors , Animals , B-Lymphocytes/immunology , B-Lymphocytes/pathology , Chemokine CCL3/biosynthesis , Chemokine CCL3/deficiency , Chemokine CCL3/genetics , Chemokine CCL3/physiology , Chemokine CCL5/biosynthesis , Chemokine CCL5/genetics , Chemokine CCL5/physiology , Chemotaxis, Leukocyte , Disease Progression , Drug Evaluation, Preclinical , Humans , Kidney/immunology , Kidney/pathology , Ligands , Lupus Nephritis/immunology , Lupus Nephritis/pathology , Mice , Mice, Inbred NZB , Myeloid Cells/drug effects , Myeloid Cells/metabolism , Myeloid Cells/pathology , Neutrophil Infiltration/drug effects , RNA, Messenger/biosynthesis , Random Allocation , Receptors, CCR1/biosynthesis , Receptors, CCR1/genetics , Receptors, CCR1/physiology , Spleen/immunology , Spleen/pathology , Splenomegaly/etiology , Splenomegaly/immunology , Splenomegaly/pathology , T-Lymphocyte Subsets/drug effects , T-Lymphocyte Subsets/metabolism , T-Lymphocyte Subsets/pathologyABSTRACT
Recent reports have suggested critical roles of myeloid cells in tumor invasion and metastasis, although these findings have not led to therapeutics. Using a mouse model for liver dissemination, we show that mouse and human colon cancer cells secrete CC-chemokine ligands CCL9 and CCL15, respectively, and recruit CD34(+) Gr-1(-) immature myeloid cells (iMCs). They express CCL9/15 receptor CCR1 and produce matrix metalloproteinases MMP2 and MMP9. Lack of the Ccr1, Mmp2, or Mmp9 gene in the host dramatically suppresses outgrowths of disseminated tumors in the liver. Importantly, CCR1 antagonist BL5923 blocks the iMC accumulation and metastatic colonization and significantly prolongs the survival of tumor-bearing mice. These results suggest that CCR1 antagonists can provide antimetastatic therapies for patients with disseminated colon cancer in the liver.
Subject(s)
Cell Differentiation , Colonic Neoplasms/pathology , Liver Neoplasms/secondary , Myeloid Cells/pathology , Receptors, CCR1/metabolism , Animals , Chemokines, CC/metabolism , Colonic Neoplasms/metabolism , Disease Models, Animal , Humans , Ligands , Liver/metabolism , Liver/pathology , Liver Neoplasms/enzymology , Liver Neoplasms/pathology , Macrophage Inflammatory Proteins/metabolism , Matrix Metalloproteinase 2/metabolism , Matrix Metalloproteinase 9/metabolism , Mice , Myeloid Cells/metabolism , Receptors, CCR1/antagonists & inhibitors , Signal Transduction , Survival AnalysisABSTRACT
The chemokine receptors CCR2 and CCR5 represent potential novel therapeutic targets to treat important inflammatory and infectious diseases, including atherosclerosis and HIV infection. To study the functions of both receptors in vivo, we aimed to generate Ccr2/Ccr5 double-deficient mice. As these genes are separated by <20 kb, they were inactivated consecutively by two rounds of gene targeting in embryonic stem (ES) cells. Thereby neomycin and hygromycin selection cassettes flanked by four identical loxP recognition sequences for Cre recombinase were integrated into the ES cell genome together with EGFP and DsRed2 reporter genes. Both selection cassettes could be deleted in vitro by transiently transfecting ES cells with Cre expression vectors. However, after blastocyst microinjection these cells yielded only weak chimeras, and germline transmission was not achieved. Therefore, Ccr2/Ccr5 double-deficient mice were generated from ES cells still carrying both selection cassettes. Microinjection of zygotes with a recombinant fusion protein consisting of maltose-binding protein and Cre (MBP-Cre) allowed the selective deletion of both cassettes. All sequences in between and both reporter genes were left intact. Deletion of both selection cassettes resulted in enhanced DsRed2 reporter gene expression. Cre protein microinjection of zygotes represents a novel approach to perform complex recombination tasks.
Subject(s)
Genes, Reporter , Integrases/administration & dosage , Receptors, CCR2/genetics , Receptors, CCR5/genetics , Zygote , Animals , Base Sequence , DNA Primers , Embryonic Stem Cells/metabolism , Gene Deletion , Genetic Vectors , Germ Cells , In Situ Hybridization, Fluorescence , Integrases/genetics , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Microinjections , Recombination, Genetic , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Diabetic nephropathy is associated with interstitial macrophage infiltrates, but their contribution to disease progression is unclear. We addressed this question by blockade of chemokine receptor (CCR)1 because CCR1 mediates the macrophage recruitment to the renal interstitium. In fact, when CCR1 was blocked with BL5923, a novel orally available CCR1 antagonist, the interstitial recruitment of ex vivo labeled macrophages was markedly decreased in uninephrectomized male db/db mice with advanced diabetic nephropathy. Likewise, BL5923 (60 mg/kg, twice a day) orally administered from months 5 to 6 of life reduced the numbers of interstitial macrophages in uninephrectomized db/db mice. This was associated with reduced numbers of Ki-67 proliferating tubular epithelial and interstitial cells, tubular atrophy, and interstitial fibrosis in uninephrectomized db/db mice. Glomerular pathology and proteinuria were not affected by the CCR1 antagonist. BL5923 reduced renal mRNA expression of Ccl2, Ccr1, Ccr2, Ccr5, transforming growth factor-beta1, and collagen I-alpha1 when compared with untreated uninephrectomized male db/db mice of the same age. Thus, we identified a previously unrecognized role for interstitial macrophages for tubulointerstitial injury, loss of peritubular microvasculature, interstitial inflammation, and fibrosis in type 2 diabetic db/db mice. These data identify oral treatment with the CCR1 antagonist BL5923 as a potential therapy for late-stage diabetic nephropathy.
Subject(s)
Diabetes Mellitus, Type 2/complications , Diabetic Neuropathies/pathology , Kidney/drug effects , Macrophages/pathology , Administration, Oral , Animals , Antigens, Differentiation/analysis , Cell Line , Cells, Cultured , Diabetic Neuropathies/etiology , Gene Expression/drug effects , Immunohistochemistry , Kidney/metabolism , Kidney/pathology , Kidney Glomerulus/drug effects , Kidney Glomerulus/metabolism , Kidney Glomerulus/pathology , Kidney Tubules/drug effects , Kidney Tubules/metabolism , Kidney Tubules/pathology , Macrophages/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Obese , Microscopy, Fluorescence , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptors, CCR1 , Receptors, CCR2 , Receptors, Chemokine/antagonists & inhibitors , Receptors, Chemokine/genetics , Receptors, Chemokine/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Transforming Growth Factor beta1/geneticsABSTRACT
OBJECTIVE: Chemokines such as CCL2/monocyte chemotactic protein 1 (MCP-1) play a key role in leukocyte migration and are potential targets in the treatment of chronic inflammatory disorders. The objective of this study was to evaluate the effects of human anti-CCL2/MCP-1 monoclonal antibody (ABN912) treatment in patients with rheumatoid arthritis (RA). METHODS: Patients with active RA were enrolled in a randomized, placebo-controlled, dose-escalation study of ABN912. Infusions were administered on day 1 and day 15. In the dose-escalation phase, 4 cohorts of 8 patients each underwent serial arthroscopic biopsy of synovial tissue. Immunohistochemistry and digital image analysis were used to characterize biomarkers in synovial tissue. Laboratory evaluation included pharmacokinetic analysis and immunotypic studies of peripheral blood mononuclear cells. To assess the clinical effects of treatment with ABN912, an additional 21 patients were treated with the highest dose tolerated. RESULTS: The total study population comprised 45 patients: 33 patients received ABN912, and 12 patients received placebo. ABN912 treatment was well tolerated. Unexpectedly, there was a dose-related increase in ABN912-complexed total CCL2/MCP-1 levels in peripheral blood, up to 2,000-fold. There was no detectable clinical benefit of ABN912 compared with placebo, nor did treatment with the study drug result in a significant change in the levels of biomarkers in synovial tissue and peripheral blood. CONCLUSION: ABN912 treatment did not result in clinical or immunohistologic improvement and may have been associated with worsening of RA in patients treated with the highest dose. The results might be related to the greatly increased level of total CCL2/MCP-1 in serum that was observed following treatment with ABN912. This observation may be relevant for a variety of antibody-based therapies.
Subject(s)
Antibodies, Monoclonal/therapeutic use , Antirheumatic Agents/therapeutic use , Arthritis, Rheumatoid/drug therapy , Chemokine CCL2/immunology , Immunologic Factors/therapeutic use , Adult , Aged , Antibodies, Monoclonal/pharmacokinetics , Antirheumatic Agents/pharmacokinetics , Arthritis, Rheumatoid/metabolism , Arthritis, Rheumatoid/physiopathology , Biomarkers/metabolism , Biopsy , Dose-Response Relationship, Drug , Female , Humans , Image Processing, Computer-Assisted , Immunohistochemistry , Immunologic Factors/pharmacokinetics , Male , Middle Aged , Synovial Membrane/drug effects , Synovial Membrane/metabolism , Synovial Membrane/pathology , Treatment OutcomeABSTRACT
Cinnamides as novel CCR1 antagonist chemotypes are described with high affinity to human and rodent receptors. A1B1 and A4B7 showed oral activity in the mouse collagen induced arthritis.
Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/chemistry , Anti-Inflammatory Agents, Non-Steroidal/therapeutic use , Arthritis, Experimental/drug therapy , Receptors, Chemokine/antagonists & inhibitors , Administration, Oral , Animals , Anti-Inflammatory Agents, Non-Steroidal/administration & dosage , Humans , Mice , Rats , Receptors, CCR1 , Structure-Activity RelationshipABSTRACT
BACKGROUND: Pig-to-human xenotransplantation is hampered by strong humoral and cellular immune responses, including acute vascular rejection (AVR). Infiltration of vascular xenografts by recipient polymorphonuclear neutrophils (PMN) is an early feature of AVR. Since little is known about the initiation of PMN recruitment, the present study investigated whether activated porcine endothelial cells (EC) release factors that induce human PMN recruitment. METHODS: Primary and immortalized porcine aortic EC cultures were stimulated with phorbol-myristate acetate/ionomycin, lipopolysaccharide, tumor-necrosis factor-alpha, or interferon-gamma. The interleukin (IL)-8 concentration of porcine EC supernatants was tested by ELISA. Human and porcine PMN were isolated from peripheral blood by Ficoll sedimentation and centrifugation, characterized by morphology and flow cytometry, and analyzed for chemotaxis using Boyden chambers or Transwells. PMN chemokine receptor desensitization was determined by intracellular calcium-flux measurements. RESULTS: Porcine EC supernatants contained significant amounts of porcine IL-8 and triggered chemotaxis in both human and porcine PMN. Chemotaxis of porcine, but not human, PMN was inhibited by anti-porcine IL-8 antibodies and recombinant porcine IL-8 induced strong chemotaxis only in porcine PMN. Porcine EC supernatants desensitized human PMN CXC-chemokine receptor (CXCR) 2, but not CXCR1, a receptor for human IL-8. Human PMN chemotaxis induced by porcine EC supernatants was significantly inhibited by blocking CXCR2 and platelet-activating factor (PAF). CONCLUSIONS: Both chemokines acting via CXCR2 and PAF are released by porcine EC inducing efficient chemotaxis of human PMN. These mechanisms responsible for the recruitment of human PMN to porcine endothelium during cell-mediated rejection of xenografts represent potential targets for preventive strategies.
Subject(s)
Chemokines/physiology , Neutrophil Infiltration/physiology , Platelet Activating Factor/physiology , Receptors, Interleukin-8B/physiology , Swine/metabolism , Animals , Aorta/cytology , Cell Line, Transformed , Chemokines/metabolism , Chemotactic Factors/metabolism , Chemotaxis, Leukocyte/drug effects , Chemotaxis, Leukocyte/physiology , Endothelial Cells/metabolism , Humans , Interleukin-8/metabolism , Interleukin-8/pharmacology , Receptors, Interleukin-8B/metabolism , Recombinant Proteins/pharmacologyABSTRACT
Effective immune surveillance is essential for maintaining protection and homeostasis of peripheral tissues. However, mechanisms controlling memory T cell migration to peripheral tissues such as the skin are poorly understood. Here, we show that the majority of human T cells in healthy skin express the chemokine receptor CCR8 and respond to its selective ligand I-309/CCL1. These CCR8(+) T cells are absent in small intestine and colon tissue, and are extremely rare in peripheral blood, suggesting healthy skin as their physiological target site. Cutaneous CCR8(+) T cells are preactivated and secrete proinflammatory cytokines such as tumor necrosis factor-alpha and interferon-gamma, but lack markers of cytolytic T cells. Secretion of interleukin (IL)-4, IL-10, and transforming growth factor-beta was low to undetectable, arguing against a strict association of CCR8 expression with either T helper cell 2 or regulatory T cell subsets. Potential precursors of skin surveillance T cells in peripheral blood may correspond to the minor subset of CCR8(+)CD25(-) T cells. Importantly, CCL1 is constitutively expressed at strategic cutaneous locations, including dermal microvessels and epidermal antigen-presenting cells. For the first time, these findings define a chemokine system for homeostatic T cell traffic in normal human skin.
Subject(s)
Receptors, Chemokine/immunology , Receptors, Lymphocyte Homing/immunology , Skin/immunology , T-Lymphocytes/immunology , Antigens, CD/blood , Antigens, CD/immunology , Cells, Cultured , Cytokines/biosynthesis , Cytokines/immunology , Humans , Immunologic Memory/immunology , Immunophenotyping , Receptors, CCR8 , T-Lymphocytes/cytologyABSTRACT
The surge in interest in chemokines is explained by the recognition that numerous aspects of immunity are intimately related to leukocyte traffic. Chemokines are leukocyte attractants but also contribute to immune processes that do not directly involve leukocyte migration. Recent progress is most evident in the areas of lymphocyte development, immune response initiation and immune pathology. Important observations have also been reported on chemokine-receptor interactions, signal transduction and cellular responses. New insights into the role of chemokines in leukocyte attraction and relocation will be discussed, with emphasis on the distinct levels of leukocyte migration control that ultimately determine the performance of our immune defense system.
Subject(s)
Chemokines/physiology , Chemotaxis, Leukocyte/physiology , Cell Differentiation/physiology , Cell Movement/immunology , Chemokines/classification , Chemokines/immunology , Chemotactic Factors/physiology , Chemotaxis, Leukocyte/immunology , Gene Expression Regulation , Humans , Leukocytes/cytology , Leukocytes/immunology , Leukocytes/physiology , Lymphopoiesis/physiology , Models, Immunological , Signal Transduction/immunology , T-Lymphocytes/immunology , T-Lymphocytes/physiologyABSTRACT
We studied the role of Rho kinase and extracellular signal-regulated kinase (ERK)-2 in the polarization and migration of T lymphocytes in response to the CCR7 ligands EBI1 ligand chemokine (ELC; CCL19) and secondary lymphoid-tissue chemokine (SLC; CCL21). Both Rho kinase protein isoforms are expressed in T lymphocytes. Inhibition of the Rho kinases with Y-27632 strongly inhibited SLC- and ELC-induced polarized morphology and chemotaxis of T lymphocytes. Although the chemokines induced ERK-2 activation, the blockade of this signaling pathway showed no effect on polarization and migration. This study indicates an important role of Rho kinase in CCR7-mediated polarization and migration of T lymphocytes, whereas ERK-2 is not involved in these processes.