Walter Newman

The β-Chemokine Receptors CCR3 and CCR5 Facilitate Infection by Primary HIV-1 Isolates

We examined the ability of chemokine receptors and related G protein-coupled receptors to facilitate infection by primary, clinical HIV-1 isolates. CCR5, when expressed along with CD4, the HIV-1 receptor, allowed cell lines resistant to most primary HIV-1 isolates to be infected. CCR3 facilitated infection by a more restricted subset of primary viruses, and binding of the CCR3 ligand, eotaxin, inhibited infection by these isolates. Utilization of CCR3 and CCR5 on the target cell depended upon the sequence of the third variable (V3) region of the HIV-1 gp120 exterior envelope glycoprotein. The ability of various members of the chemokine receptor family to support the early stages of HIV-1 infection helps to explain viral tropism and beta-chemokine inhibition of primary HIV-1 isolates.

Circulating adhesion molecules in disease.

The demonstration of soluble isoforms of adhesion molecules has added a layer of complexity of our understanding of lymphoid-endothelial cell interactions. This is especially true in the light of observations which show levels of these isoforms to be raised during disease processes. Here, Andrew Gearing and Walter Newman review the evidence that increased levels of circulating, soluble adhesion molecules may be a key to understanding the prognosis and pathology of certain diseases.

Cloning of the human eosinophil chemoattractant, eotaxin. Expression, receptor binding, and functional properties suggest a mechanism for the selective recruitment of eosinophils.

The CC chemokine eotaxin, identified in guinea pigs and also recently in mice, may be a key element for the selective recruitment of eosinophils to certain inflamed tissues. Using a partial mouse eotaxin CDNA probe, the human eotaxin gene was cloned and found to be 61.8 and 63.2% identical at the amino acid level to guinea pig and mouse eotaxin. Human eotaxin protein was a strong and specific eosinophil chemoattractant in vitro and was an effective eosinophil chemoattractant when injected into the skin of a rhesus monkey. Radiolabeled eotaxin was used to identify a high affinity receptor on eosinophils (0.52 nM Kd), expressed at 4.8 x 10(4) sites per cell. This receptor also bound RANTES and monocyte chemotactic protein-3 with lower affinity, but not macrophage inflammatory protein-1 alpha. Eotaxin could desensitize calcium responses of eosinophils to RANTES and monocyte chemotactic protein-3, although RANTES was able to only partially desensitize eosinophil calcium responses to eotaxin. Immunohistochemistry on human nasal polyp with antieotaxin mAbs showed that certain leukocytes as well as respiratory epithelium were intensely immunoreactive, and eosinophil infiltration occurred at sites of eotaxin upregulation. Thus eotaxin in humans is a potent and selective eosinophil chemoattractant that is expressed by a variety cell types in certain inflammatory conditions.

Lymphocyte interactions with endothelial cells

Adhesion of lymphocytes to endothelium is vital to lymphocyte migration into lymphoid tissue and into inflammatory sites. In this review, Yoji Shimizu and colleagues identify the molecules that mediate lymphocyte-endothelial cell adhesion, describe the underlying principles of lymphocyte migration, and discuss a model of the sequence of events that allow a lymphocyte to successfully attach to endothelium and migrate into the surrounding tissue.

Role of HMGB1 in apoptosis-mediated sepsis lethality

Severe sepsis, a lethal syndrome after infection or injury, is the third leading cause of mortality in the United States. The pathogenesis of severe sepsis is characterized by organ damage and accumulation of apoptotic lymphocytes in the spleen, thymus, and other organs. To examine the potential causal relationships of apoptosis to organ damage, we administered Z-VAD-FMK, a broad-spectrum caspase inhibitor, to mice with sepsis. We found that Z-VAD-FMK–treated septic mice had decreased levels of high mobility group box 1 (HMGB1), a critical cytokine mediator of organ damage in severe sepsis, and suppressed apoptosis in the spleen and thymus. In vitro, apoptotic cells activate macrophages to release HMGB1. Monoclonal antibodies against HMGB1 conferred protection against organ damage but did not prevent the accumulation of apoptotic cells in the spleen. Thus, our data indicate that HMGB1 production is downstream of apoptosis on the final common pathway to organ damage in severe sepsis.

Discrete Steps in Binding and Signaling of Interleukin-8 with Its Receptor

The mechanisms by which chemokines bind and signal through their receptors are complex and poorly understood. In the present study, we sought to dissect these processes and to map important functional domains of the two CXC chemokine (interleukin-8) receptors, CXCR1 (formally IL-8RA) and CXCR2 (formally IL-8RB), using blocking monoclonal antibodies (mAbs) to the receptors and a series of chimeras between CXCR1 and CXCR2. A panel of specific mAbs against CXCR1 or CXCR2, generated by immunizing mice with transfectants expressing either receptor, were shown to effectively block IL-8- and/or growth-related oncogene α (GROα) -mediated ligand binding, chemotaxis, elastase release, and VCAM-1 binding in CXCR1 and CXCR2 transfectants and/or human neutrophils. Of particular interest was an anti-CXCR1 mAb, 7D9, that inhibited chemotaxis, elastase release, and VCAM-1 binding but had no detectable effects on ligand binding. The epitopes of these blocking mAbs were mapped by using a series of CXCR1/2 chimera transfectants and synthetic peptides. Most of the anti-CXCR1 antibodies, except 7D9, mapped to the amino acid sequence WDFDDL (CXCR1 residues 10-15), and all the anti-CXCR2 antibodies mapped to the amino acid sequence FEDFW (CXCR2 residues 6-10). The epitope of mAb 7D9 mainly involved a region within the first 45 residues of CXCR1, and it appeared to be conformation-sensitive. These results support a model in which the binding and signaling of IL-8 with its receptor occur in at least two discrete steps involving distinct domains of the receptor. This model is consistent with the notion that discrete conformational changes of the receptor secondary to ligand binding are required to trigger various biological responses. Moreover, the ligand binding and chemotaxis properties of each CXCR1/2 chimeric receptor to IL-8 and GROα were determined. It was found that each is distinct in its ability to confer ligand binding and chemotactic response to IL-8 and GROα, and two conclusions could be made. 1) The N-terminal segment of CXCR1 is a dominant determinant of receptor subtype selectivity, consistent with previous studies using rabbit/human CXCR1/2 chimeras; and 2) the specificity determinant for GROα binding in CXCR2 involves sequences in the N terminus, distal to the first 15 residues, as well as other parts of the receptor.

Primary in vitro sensitisation of human T cells

METHODS for the in vitro development of primary immune responses have been crucial for the understanding of mechanisms of cooperation between T and B cells and between T cells and macrophages1–3. Few efforts to date have been reported for primary in vitro sensitisation of human lymphocytes, most of which have involved sensitisation to allo- or xeno-antigens4–6 or plaque-forming responses to sheep red blood cells (SRBC)7,8. Development of killer T cells against HLA-identical9, or autochthonous tumour cells10,11 have usually required coculture with third party allogeneic cells for development of cytotoxicity. Given the ethical constraints on in vivo studies of human T-cell function, and noting the development for the first time of a system for the sensitisation of guinea pig T cells in vitro to a variety of antigens12, we were encouraged to develop an in vitro system for the priming and boosting of human T cells to well defined haptenic determinants without the requirement for a third party cell.

Genetic and Functional Studies of Continuous Macrophage-Like Cell Lines

The mechanisms by which macrophage functions are regulated represent a fascinating area of cell biology because of the wide variety of cellular and immunological activities carried out by these highly differentiated cells, the most significant being phagocytosis, antigen presentation, resistance to intracellular parasites, killing and growth inhibition of tumor cells, and tissue damage. Definition of macrophage functions has been aided in recent years by the careful study of primary, resident or induced murine peritoneal exudate cells exposed to a variety of stimuli and pharmacological agents. There are, however, a number of inherent limitations on the analysis of macrophage functions imposed by the study of primary cells: 1) most sources of primary macrophages are heterogeneous cell populations; 2) primary cell populations contain macrophages at various stages and states of differentiation; and 3) primary macrophages under standard conditions fail to grow in tissue culture. The availability of cloned populations of macrophage-like continuous cell lines offers the possibility to select stable variants and mutants in individual macrophage functions. The goal of these studies is to isolate a variety of mutants in individual macrophage functions, ultimately to permit formulation of an integrated scheme for the complex mechanisms of macrophage functions. In addition, it has recently become possible to construct somatic cell hybrids which retain selected macrophage functions, thus expanding the possibilities for producing continuous macrophage-like cell lines.