Kuldeep Neote

Differential expression of three T lymphocyte-activating CXC chemokines by human atheroma-associated cells

Activated T lymphocytes accumulate early in atheroma formation and persist at sites of lesion growth and rupture, suggesting that they may play an important role in the pathogenesis of atherosclerosis. Moreover, atherosclerotic lesions contain the Th1-type cytokine IFN-gamma, a potentiator of atherosclerosis. The present study demonstrates the differential expression of the 3 IFN-gamma-inducible CXC chemokines--IFN-inducible protein 10 (IP-10), monokine induced by IFN-gamma (Mig), and IFN-inducible T-cell alpha chemoattractant (I-TAC)--by atheroma-associated cells, as well as the expression of their receptor, CXCR3, by all T lymphocytes within human atherosclerotic lesions in situ. Atheroma-associated endothelial cells (ECs), smooth muscle cells (SMCs), and macrophages (MO) all expressed IP-10, whereas Mig and I-TAC were mainly expressed in ECs and MO, as detected by double immunofluorescence staining. ECs of microvessels within lesions also expressed abundant I-TAC. In vitro experiments supported these results and showed that IL-1beta, TNF-alpha, and CD40 ligand potentiated IP-10 expression from IFN-gamma-stimulated ECs. In addition, nitric oxide (NO) treatment decreased IFN-gamma induction of IP-10. Our findings suggest that the differential expression of IP-10, Mig, and I-TAC by atheroma-associated cells plays a role in the recruitment and retention of activated T lymphocytes observed within vascular wall lesions during atherogenesis.

CP-481,715, a Potent and Selective CCR1 Antagonist with Potential Therapeutic Implications for Inflammatory Diseases

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.

Identification of G-protein binding sites of the human interleukin-8 receptors by functional mapping of the intracellular loops.

Interleukin 8 (IL‐8) is considered to be a major mediator of the inflammatory response. Recent evidence indicates that a direct physical association occurs between IL‐8 receptors and the α subunit of guanine nucleotide regulatory protein (Giα2) upon stimulation of human neutrophils by IL‐8. In the present study, we identified by site‐directed mutagenesis key residues within the three intracellular loops of the IL‐8RA receptor involved in the interaction with Giα2. We first systematically mutated, in groups of two to four, all the residues in the three intracellular loops of the IL‐8 type A receptor to alanine and analyzed the mutant receptors transiently expressed in 293 cells. Four residues in the second intracellular loop (Y136, L137, I139, V140) and one residue in the third intracellular loop (M241) were shown to be crucial for mediating calcium signaling in response to IL‐8. Other residues in the second and third intracellular loops were also found to affect IL‐8RA‐mediated signaling, but to a lesser extent. These effects were not due to lower expression or low IL‐8 binding affinities to the mutated receptors. Mutagenesis of the residues in the first intracellular loop had only weak effects on the mobilization of calcium induced by IL‐8. We then used a coimmunoprecipitation protocol with anti‐Giα2 antibodies to determine the involvement of the two regions defined above in Giα2 coupling to IL‐8 type A receptors. Whereas the anti‐Giα2 antibodies coimmunoprecipitated IL‐8 receptors in the wild‐type cells, this interaction was lost in cells expressing mutated receptors that affected intracellular calcium mobilization. The peptides corresponding to the regions of the type A receptor found to be critical for Giα2 coupling and induction of intracellular calcium mobilization were next introduced into cells expressing wild‐type IL‐8RA or IL‐8RB to assess their role in coupling Giα2 to both IL‐8 receptors. The results obtained in the latter experiments suggest that the same regions of the second intracellular loop (Y136, L137, I139, V140) and of the third intracellular loop (M241) are critically involved in the coupling of both IL‐8RA and IL‐8 RB to Giα2 as well as to a downstream effector (or effectors) involved in calcium mobilization.—Damaj, B. B., McColl, S. R., Neote, K., Songqing, N., Ogborn, K. T., Hébert, C. A., Naccache, P. H. Identification of G‐protein binding sites of the human interleukin‐8 receptors by functional mapping of the intracellular loops. FASEB J. 10, 1426‐1434 (1996)

Diverging Signal Transduction Pathways Activated by Interleukin 8 (IL-8) and Related Chemokines in Human Neutrophils IL-8 AND Gro-α DIFFERENTIALLY STIMULATE CALCIUM INFLUX THROUGH IL-8 RECEPTORS A AND B

Interleukin 8 (IL-8) and Gro-α are members of the CXC branch of a family of cytokines recently designated the “chemokine” superfamily. Recent evidence indicates that, contrary to previously held beliefs, IL-8 and Gro-α may not be perceived equivalently by neutrophils. In this study, we have evaluated the effects of IL-8 and Gro-α on the rate of calcium influx in human neutrophils and in 293 cells transfected with type A or type B IL-8 receptors. Of these two chemokines, only Gro-α induced an influx of calcium in neutrophils as judged by the sensitivity of the mobilization of calcium to the extracellular calcium chelator EGTA and to the nonselective divalent cation channel inhibitor SK&F 96365, as well as by manganese quenching experiments. IL-8 was similarly without effect on the rate of Mn2+ influx in 293 cells transfected with IL-8 receptor A (IL-8RA) or IL-8RB. On the other hand, Gro-α induced an SK&F 96365-sensitive increase of the rate of Mn+2 influx in IL-8RB-, but not in IL-8RA-transfected 293 cells. These results indicate not only that neutrophils respond differently to IL-8 than they do to Gro-α but, furthermore, that the consequences of the binding of IL-8 and Gro-α to IL-8RB are distinct.

The Human Specific CCR1 Antagonist CP-481,715 Inhibits Cell Infiltration and Inflammatory Responses in Human CCR1 Transgenic Mice

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-γ 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.

Expression of rat I-TAC/CXCL11/SCYA11 during central nervous system inflammation: comparison with other CXCR3 ligands.

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.

Expression of IFN-Inducible T Cell α Chemoattractant by Human Endothelial Cells Is Cyclosporin A-Resistant and Promotes T Cell Adhesion: Implications for Cyclosporin A-Resistant Immune Inflammation

IFN-inducible T cell α chemoattractant (I-TAC) is a recently discovered member of the CXC chemokine family. It is a potent T cell chemoattractant expressed by IFN-γ-treated astrocytes, monocytes, keratinocytes, bronchial epithelial cells, and neutrophils. In this study, we show that I-TAC is also expressed by IFN-γ-treated endothelial cells (EC), both at the mRNA and protein levels. Induction of the I-TAC message is rapid and sustained over 24 h. TNF-α does not induce I-TAC mRNA alone, but does act synergistically with IFN-γ. Blocking Abs to I-TAC, or to its receptor, CXCR3, reduce T cell adhesion to EC monolayers demonstrating that the expressed protein is functional. Finally, the expression of I-TAC by EC is resistant to the immunosuppressive drug cyclosporin A, suggesting that I-TAC may contribute to the chronic immune inflammation characteristic of graft arteriosclerosis.

Chapter 22. Chemokines as Therapeutic Targets

Publisher Summary It is apparent that the chemokines, whose primary functions are the control of leukocyte chemotaxis and activation, play a central role in leukocyte trafficking. Therefore, they are likely to be very important mediators in disease processes that depend on leukocyte infiltration and damage. This chapter discusses the most recent advances and discoveries regarding chemokines, concentrating on their structure and function and their receptors, in vitro and in vivo biological activities, and how the modulation of their activity might present new therapeutic opportunities. The main focus is on the known human proteins. Chemokines are divided into two distinct families, the C–X–C class and the C–C class, referring to the presence or absence of an amino acid between the first pair of conserved cysteine residues at the amino-terminus. Based on their biological activity, the chemokines have been postulated to play a pivotal role in the extravasation and subsequent infiltration of leukocytes into sites of inflammation and immune reactivity. Given their immune cell infiltrating properties, the chemokines have the potential to be used as therapeutic vaccines against tumors by enhancing immune responses to tumors.

Novel Chemokines Identified in Expressed Sequence Tag Databases via Bioinformatics

The chemokines are the largest family of cytokines known. Initially isolated from activated leukocytes as chemoattractants, MCP-1 and IL-8, the first two chemokines cloned on the basis of biological activity were shown to attract and activate monocytes and neutrophils, respectively. At that time it could not have been realized that the few molecules discovered would prove to be merely part of a large superfamily of genes, however, the chemokines presently comprise approx 40 members. All the members of this superfamily are related by both function and structure. All of the chemokines cloned to date exhibit the ability to mediate chemotaxis of specific and overlapping subsets of leukocytes, hence the term chemokine, a contraction of chemotactic cytokine. The main structural element that is common among chemokines is the presence of cysteine residues. Based on predicted primary amino acid structure, the superfamily is divided into four groups: the CXC or α chemokines that contain four cysteine residues in highly conserved positions where the first pair of cysteines have an intervening amino acid; the CC or β chemokines that have either four or six conserved cysteines where the first two cysteines are juxtaposed; the C or γ chemokine subfamily, currently consisting of only one member, lymphotactin, which has two conserved cysteines at locations that are similar to other chemokines, the CX3C or δ chemokine subfamily, that also presently consists of only one member, a newly discovered chemokine known as fractalkine or neutrotactin, that has three intervening amino acids between the first two cysteines. With the sole exception of fractalkine, which is an integral membrane protein, all chemokines are 8- to 10-kDa proteins that are highly basic.