Distinct but overlapping epitopes for the interaction of a CC-chemokine with CCR1, CCR3 and CCR5.

Chemokines play an important role in inflammation. The mechanism via which they bind to more than one receptor and activate them is not well understood. The chemokines are thought to interact with their receptors via two distinct sites, one necessary for binding and the other for activation of signal transduction. In this study we have used alanine scanning mutagenesis to identify residues on RANTES that specifically interact with its receptors CCR1, CCR3, and CCR5 for binding and activation. Residues within a potential receptor binding site known as the N-loop (residues 12-20) and near the N-terminus of RANTES were individually mutated to alanine. The results of this study show that, within the N-loop, the side chain of R17 is necessary for RANTES binding to CCR1, F12 for binding to CCR3, and F12 and I15 for binding to CCR5, thus forming distinct but overlapping binding epitopes. In addition, our finding that P2 is necessary for binding to CCR5 is the first to show that a residue near the N-terminus of a CC-chemokine is involved in binding to a receptor. We have also found that P2, D6, and T7 near the N-terminus are involved in activating signal transduction via CCR1, P2 and Y3 via CCR3, and Y3 and D6 via CCR5. These results indicate that RANTES interacts with each of its receptors in a distinct and specific manner and provide further evidence to support the two-site model of interaction between chemokines and their receptors.

Subversion of chemokine receptors by HIV: how can we exploit this?

The recent discovery that the chemokines RANTES (regulated upon activation, normal T-cell-expressed and secreted), macrophage inflammatory protein-1alpha (MIP-1a), and macrophage inflammatory protein-1beta (MIP-1b) are important modulators of HIV-1 infection and the subsequent identification of the essential role that chemokine receptors play as co-receptors for HIV-1 infection have provided new insight into the pathogenesis of HIV-1. On May 10, 1996, Ed Berger's group of the NIH announced the identity of one of the elusive ;co-receptors' for HIV-1. This, and subsequent papers showing that the expression of specific seven transmembrane (7TM) chemokine receptors and CD4 confers susceptibility to infection by HIV-1, has led to rapid and exciting advances in both chemokine and HIV research. During the year since then, a number of elegant studies by different groups have identified additional co-receptors for HIV-1 and the receptor involved in HIV-2 infection, as well as extended our understanding of the mechanism HIV utilises to enter cells. A number of researchers at academic institutes, pharmaceutical and biotech companies are gambling that this will translate into new therapeutics for the treatment of HIV infection and AIDS.

Extracellular matrix proteins and leukocyte function.

Extracellular matrix (ECM) proteins profoundly affect physiological functioning at the cellular level. Cell growth and differentiation, as well as cell shape and migration via the cytoskeleton, are all affected by ECM proteins. Leukocyte interactions with matrices have recently become an exciting field of research because a number of different leukocyte functions are significantly affected by their binding to ECM proteins. This may be especially important in inflammatory responses where leukocytes are primed for inflammatory mediator and cytokine production by binding to ECM proteins during extravasation. Because activated leukocytes produce potentially damaging substances, the progress of an inflammatory response can be profoundly affected by the ECM proteins encountered by leukocytes during their migration from within the peripheral circulation to sites of inflammation. This review summarizes recent publications describing components of the ECM that influence leukocyte function, the receptors involved in leukocyte binding to ECM proteins, and focuses on the effects of ECM proteins on the production of inflammatory mediators and cytokines by human peripheral blood leukocytes.

Trypanosoma cruzi affects nitric oxide production by murine peritoneal macrophages.

Macrophages from mice that are infected with various intracellular pathogens including Leishmania major, Trypanosoma cruzi, and Salmonella typhimurium are stimulated to produce large quantities of nitric oxide (NO). Both viable and heat-treated L. major amastigotes have been shown to be effective co-signals for NO production in vitro. NO produced by macrophages has anti-microbial and immunosuppressive functions in an immune response. We have shown previously that NO plays a complicated role in T. cruzi infections since macrophages are important both in mediating an immune response against the parasite as well as in mediating immunosuppression. In this study we examined how T. cruzi affects NO production by macrophages from C3HeB/FeJ and C57BL/6 mice in vitro. We found that live trypomastigotes neither stimulate nor decrease NO production by interferon (IFN)-gamma-activated macrophages. However, heat-treated or glutaraldehyde-fixed trypomastigotes of T. cruzi significantly decrease NO production by IFN-gamma-activated macrophages and as a result decrease macrophage-mediated trypanocidal and immunosuppressive activity. We have determined that this decrease in NO production by T. cruzi is not due to stimulation of transforming growth factor-beta production and involves tumor necrosis factor-alpha only in C3HeB/FeJ macrophages. This study demonstrates the complexity of the T. cruzi-macrophage interaction as well as confirms previously demonstrated differences between macrophages from 2 strains of mice.

Interleukin-2 receptors in experimental Chagas' disease.

Mammals infected with the protozoan parasite Trypanosoma cruzi develop suppressed cellular and humoral immune responses. This immunosuppression has been correlated with reduced T-cell responses involving deficient interleukin-2 (IL-2) production and is apparently mediated primarily by suppressor macrophages. Various forms of immunosuppression in other systems have been associated with increased levels of soluble IL-2 receptors (sIL-2R), and in the present study levels of sIL-2R in the sera of T. cruzi-infected mice during the course of infection were examined in enzyme-linked immunosorbent assays. It was found that serum levels of sIL-2R were elevated only during the third week of acute infection, a time of intense immunosuppression. In addition, IL-2R on the surface of T cells were examined by flow cytometric analyses to determine whether there is an alteration in the number of IL-2R-positive cells and whether there is a change in expression of these receptors as infection progresses. The results revealed no significant change in the percentage of cells expressing IL-2R, nor did T cells become suppressed in their ability to express IL-2R in response to concanavalin A during the course of infection.