Citations

The Rac1 guanine nucleotide exchange factor, Vav, is activated in hematopoietic cells in response to a large variety of stimuli. The downstream signaling events derived from Vav have been primarily characterized as leading to transcription or transformation. However, we report here that Vav and Rac1 in natural killer (NK) cells regulate the development of cell-mediated killing. There is a rapid increase in Vav tyrosine phosphorylation during the development of antibody-dependent cellular cytotoxicity and natural killing. In addition, overexpression of Vav, but not of a mutant lacking exchange factor activity, enhances both forms of killing by NK cells. Furthermore, dominant-negative Rac1 inhibits the development of NK cell–mediated cytotoxicity by two mechanisms: (a) conjugate formation between NK cells and target cells is decreased; and (b) those NK cells that do form conjugates have decreased ability to polarize their granules toward the target cell. Therefore, our results suggest that in addition to participating in the regulation of transcription, Vav and Rac1 are pivotal regulators of adhesion, granule exocytosis, and cellular cytotoxicity.

The current knowledge of CD4 function is limited to its role as a necessary coreceptor in TCR-initiated signaling. We have investigated whether CD4 regulates additional T cell functions. Using human primary resting CD4+ T cells, we demonstrate that CD4 activation is sufficient to induce lymphocyte death. Immediately after CD4 cross-linking, CD4+ T cells are rendered susceptible to apoptosis mediated by TNF or FasL. This, together with the concomitant induction of FasL within the same population, results in significant CD4+ T cell death in vitro. The CD4-dependent induction of susceptibility to apoptosis that is mediated by TNF or FasL is protein synthesis independent but phosphorylation dependent. After CD4 activation, PKC regulates susceptibility to apoptosis mediated by FasL but not the induction of susceptibility to TNF-dependent apoptosis. Moreover, significant differences between CD3 and CD4 activation were observed with regards to the kinetics of induction of CD4+ T cell susceptibility to FasL- and TNF-mediated apoptosis. Altogether, these results provide a model with which to study the molecular mechanisms regulating lymphocyte survival after CD4 activation, and highlight the potential role of CD4 in controlling lymphocyte apoptosis under physiological conditions or in disease states such as HIV infection.

Natural killer (NK) cells are named based on their natural cytotoxic activity against a variety of target cells. However, the mechanisms by which sensitive targets activate killing have been difficult to study due to the lack of a prototypic NK cell triggering receptor. Pharmacologic evidence has implicated protein tyrosine kinases (PTKs) in natural killing; however, Lck-deficient, Fyn-deficient, and ZAP-70–deficient mice do not exhibit defects in natural killing despite demonstrable defects in T cell function. This discrepancy implies the involvement of other tyrosine kinases. Here, using combined biochemical, pharmacologic, and genetic approaches, we demonstrate a central role for the PTK Syk in natural cytotoxicity. Biochemical analyses indicate that Syk is tyrosine phosphorylated after stimulation with a panel of NK-sensitive target cells. Pharmacologic exposure to piceatannol, a known Syk family kinase inhibitor, inhibits natural cytotoxicity. In addition, gene transfer of dominant-negative forms of Syk to NK cells inhibits natural cytotoxicity. Furthermore, sensitive targets that are rendered NK-resistant by major histocompatibility complex (MHC) class I transfection no longer activate Syk. These data suggest that Syk activation is an early and requisite signaling event in the development of natural cytotoxicity directed against a variety of cellular targets.

To investigate the cellular dynamics of ZAP-70, we have studied the distribution and regulation of its intracellular location using a ZAP-70 green fluorescent protein chimera. Initial experiments in epithelial cells indicated that ZAP-70 is diffusely located throughout the quiescent cell, and accumulates at the plasma membrane upon cellular activation, a phenotype enhanced by the coexpression of Lck and the initiation of ZAP-70 kinase activity. Subsequent studies in T cells confirmed this phenotype. Intriguingly, a large amount of ZAP-70, both chimeric and endogenous, resides in the nucleus of quiescent and activated cells. Nuclear ZAP-70 becomes tyrosine phosphorylated upon stimulation via the T cell receptor, indicating that it may have an important biologic function.

We have generated transgenic mice that constitutively express murine interleukin (IL)-5 in the lung epithelium. Airway expression of this cytokine resulted in a dramatic accumulation of peribronchial eosinophils and striking pathologic changes including the expansion of bronchusassociated lymphoid tissue (BALT), goblet cell hyperplasia, epithelial hypertrophy, and focal collagen deposition. These changes were also accompanied by eosinophil infiltration of the airway lumen. In addition, transgenic animals displayed airway hyperresponsiveness to methacholine in the absence of aerosolized antigen challenge. These findings demonstrate that lung-specific IL-5 expression can induce pathologic changes characteristic of asthma and may provide useful models to evaluate the efficacy of potential respiratory disease therapies or pharmaceuticals.

Apoptosis of bystander uninfected CD4+ T lymphocytes by neighboring HIV-infected cells is observed in cell culture and in lymphoid tissue of HIV-infected individuals. This study addresses whether antigen-presenting cells such as human macrophages mediate apoptosis of CD4+ T cells from HIV-infected individuals. Uninfected human macrophages, and to a larger degree, HIV-infected macrophages mediate apoptosis of T cells from HIV-infected, but not from uninfected control individuals. This macrophage-dependent killing targets CD4+, but not CD8+ T lymphocytes from HIV-infected individuals, and direct contact between macrophages and lymphocytes is required. Additional analyses indicated that the apoptosis-inducing ligands, FasL and tumor necrosis factor (TNF), mediate this macrophage-induced apoptosis of CD4+ T cells. These results support a role for macrophage-associated FasL and TNF in the selective depletion of CD4+ T cells in HIV-infected individuals.

Interaction between inducible costimulator (ICOS) and its ligand is implicated in the induction of cell-mediated and humoral immune responses. However, the molecular details of this interaction are unknown. We report here a mutagenesis analysis of residues in ICOS that are critical for ligand binding. A three-dimensional model of the extracellular immunoglobulin-like domain of ICOS was used to map the residues conserved within the CD28 family. This analysis identified a surface patch containing the characteristic “PPP” sequence and is conserved in human and mouse ICOS. Mutations in this region of human ICOS reduce or abolish ligand binding. Our results suggest that the ligand binding site in ICOS maps to a region overlapping yet distinct from the CD80/CD86 binding sites in CD28 and cytotoxic T lymphocyte antigen (CTLA)-4. Thus, the analysis suggests that differences in ligand binding specificity between these related costimulatory molecules have evolved by utilization of overlapping regions with different patterns of conserved and nonconserved residues. Two site-specific mutants generated in the course of our studies bound ICOS ligand with higher avidity than wild-type ICOS. An S76E mutant protein of ICOS blocked T cell costimulatory function of ICOS ligand and inhibited T cell response to allogeneic antigens superior to wild-type ICOS. Our studies thus identified critical residues involving in ICOS receptor–ligand interaction and provide new modulators for immune responses.

Rheumatoid arthritis (RA) is a heterogeneous syndrome of which a subset of patients develops vascular inflammation. The genetic determinants that confer risk for rheumatoid vasculitis are not known, but patients with vascular complications are known to have an expansion of CD4+CD28null T cells, a cell population potentially involved in endothelial damage. CD4+CD28null T cell clones isolated from RA patients with vasculitis were found to express killer cell immunoglobulin–like receptors (KIRs) with the stimulatory KIR2DS2 often present in the absence of opposing inhibitory receptors with related specificities. To test the hypothesis that the KIR2DS2 gene is involved in the development of vasculitis, association studies were performed. The KIR2DS2 gene was significantly enriched among patients with rheumatoid vasculitis compared with normal individuals (odds ratio 5.56, P = 0.001) and patients with RA but no vasculitis (odds ratio 7.96, P = 0.001). Also, the distribution of human histocompatibility leukocyte antigen (HLA)-C, the putative ligand for KIRs, was significantly different in patients with rheumatoid vasculitis in comparison with the control populations. These data suggest that HLA class I–recognizing receptors and HLA class I genes are genetic risk determinants that modulate the pattern of RA expression. Specifically, KIR2DS2 in conjunction with the appropriate HLA-C ligand may have a role in vascular damage by regulating CD4+CD28null T cells.

Previous pharmacologic and genetic studies have demonstrated a critical role for the low molecular weight GTP-binding protein RhoA in the regulation of cell-mediated killing by cytotoxic lymphocytes. However, a specific Rho family guanine nucleotide exchange factor (GEF) that activates this critical regulator of cellular cytotoxicity has not been identified. In this study, we provide evidence that the Rho family GEF, Vav-2, is present in cytotoxic lymphocytes, and becomes tyrosine phosphorylated after the cross-linking of activating receptors on cytotoxic lymphocytes and during the generation of cell-mediated killing. In addition, we show that overexpression of Vav-2 in cytotoxic lymphocytes enhances cellular cytotoxicity, and this enhancement requires a functional Dbl homology and Src homology 2 domain. Interestingly, the pleckstrin homology domain of Vav-2 was found to be required for enhancement of killing through some, but not all activating receptors on cytotoxic lymphocytes. Lastly, although Vav and Vav-2 share significant structural homology, only Vav is able to enhance nuclear factor of activated T cells–activator protein 1–mediated gene transcription downstream of the T cell receptor. These data demonstrate that Vav-2, a Rho family GEF, differs from Vav in the control of certain lymphocyte functions and participates in the control of cell-mediated killing by cytotoxic lymphocytes.

Adenosine deaminase (ADA) is a purine catabolic enzyme that manages levels of the biologically active purines adenosine and 2′-deoxyadenosine in tissues and cells. ADA-deficient mice die at 3 wk of age from severe respiratory distress. This phenotype is progressive and is linked to perturbations in pulmonary purine metabolism. The inflammatory changes found in the lungs of ADA-deficient mice included an accumulation of activated alveolar macrophages and eosinophils. These changes were accompanied by a pronounced enlargement of alveolar spaces and increases in mucus production in the bronchial airways. The alveolar enlargement was found to be due in part to abnormal alveogenesis. Lowering adenosine and 2′-deoxyadenosine levels using ADA enzyme therapy decreased the pulmonary eosinophilia and resolved many of the lung histopathologies. In addition, genetically restoring ADA to the forestomach of otherwise ADA-deficient mice prevented adenine metabolic disturbances as well as lung inflammation and damage. These data suggest that disturbances in purinergic signaling mediate the lung inflammation and damage seen in ADA-deficient mice.