Gammalinolenic acid, an unsaturated fatty acid with anti-inflammatory properties, blocks amplification of IL-1 beta production by human monocytes.

Administration of gammalinolenic acid (GLA), an unsaturated fatty acid, reduces joint inflammation in patients with rheumatoid arthritis. Addition of GLA in vitro suppresses release of IL-1beta from human monocytes stimulated with LPS. LPS-induced IL-1beta release is followed by IL-1-induced IL-1beta release, an amplification process termed autoinduction. We show here with peripheral blood monocytes from normal volunteers and from patients with rheumatoid arthritis by using IL-1R antagonist to block autoinduction and IL-1alpha stimulation to simulate autoinduction that approximately 40% of IL-1beta released from LPS-stimulated cells is attributable to autoinduction and that GLA reduces autoinduction of IL-1beta while leaving the initial IL-1beta response to LPS intact. Experiments with cells in which transcription and protein synthesis were blocked suggest that GLA induces a protein that reduces pro-IL-1beta mRNA stability. IL-1beta is important to host defense, but the amplification mechanism may be excessive in genetically predisposed patients. Thus, reduction of IL-1beta autoinduction may be protective in some patients with endotoxic shock and with diseases characterized by chronic inflammation.

Induction of global anergy rather than inhibitory Th2 lymphokines mediates posttrauma T cell immunodepression.

Depressed mitogen-induced IL-2 and IFN-gamma responses after severe mechanical or thermal injury are postulated to result from an expansion of Th2 lymphocytes with concomitant excessive production of IL-4 and/or IL-10. Here, we simultaneously assessed proliferation and Th1 (IFN-gamma) versus Th2 (IL-10, IL-4) lymphokine production in trauma patients' isolated T cells stimulated in a costimulation sufficient, antigen presenting cell independent system (anti CD3 + anti-CD4). T cells with depressed proliferation and IL-2 production simultaneously lost IL-4, IL-10, and IFN-gamma protein and mRNA responses. Exogenous IL-12 addition did not restore IFNgamma responses, but exogenous IL-2 partially restored IL-4, IFN-gamma, and IL-10 production. Although initially partially restored by exogenous IL-2 or stimulation with PMA + ionomycin, patient T cells with persisting anergy progressively lost even these lymphokine and proliferative responses. Development of global T cell anergy was not a result of lost T cell viability or protein synthesis, since it corresponded to predominance of anergic T cells with upregulated expression of CD11b, but downregulated CD28 and CD3 expression. Thus, the subset of posttrauma patients whose isolated T cells become unresponsive experienced progressively worsening global anergy, mediated not by an increased production of Th2 lymphokines, but possibly by T cell incapacity to be activated through TCR triggering or Ca(2+) mobilization.

Relationships between T lymphocyte apoptosis and anergy following trauma.

BACKGROUND: Severely injured trauma patients experience T cell depletion. A subset of these patients also develop T cell unresponsiveness (anergy), as characterized by the failure of their T cells to proliferate or to produce T lymphokines in response to a direct stimulus through the T cell receptor. We hypothesized that T cell apoptosis plays a role in the development of posttrauma T cell depletion and/or T cell anergy by deleting an activated T cell population. We found that moderately increased T cell depletion posttrauma is not innately deleterious or immediately responsible for anergy, but may predispose to later development of T cell anergy, possibly due to a more stringent requirement for activation of the remaining naive T cells. METHODS: A total of 30 blunt trauma and burn patients were assessed twice weekly for the following parameters: (1) clinical outcome expressed as severity of organ dysfunction as measured by the multiple organ dysfunction syndrome score, (2) proliferative response of highly purified T cells to anti-CD3/anti-CD4, (3) level of apoptosis as determined by flow cytometric analysis of propidium iodide-stained monocyte reduced peripheral blood mononuclear cells, either unstimulated or in response to mitogenic challenge or Fas (CD95) stimulation. RESULTS: A wide range of apoptosis levels are seen in the patients' T cells. Apoptosis is increased when all trauma patients' T cells are compared to T cells of normal volunteers. However, at the time a patients' T cells are anergic, there is no increased level of apoptosis. In fact, the point of maximum anergy (lowest proliferative response) correlates to diminished apoptotic response. Increased T cell apoptosis can be stimulated by anti-Fas antibody in trauma patients' responsive T cells but not in maximally anergic T cells. These data suggest that patients' T cell anergy is not an immediate result of apoptotic T cell depletion upon stimulation. However, patients who later develop T cell anergy have increased T cell apoptosis earlier in their clinical course than patients who never experience T cell anergy. CONCLUSIONS: Increased levels of apoptosis are not directly associated with negative trauma patient outcome nor the immediate cause of T cell anergy. However, unusually high levels of apoptosis and development of severe T cell depletion occurring before complete activation and expansion of the posttrauma T cell response may presage anergy and subsequent organ failure.

Increased monocyte TNF-alpha message stability contributes to trauma patients' increased TNF production.

Post-trauma elevation of tumor necrosis factor alpha (TNF-alpha) appears to be critical in mediating many symptoms of systemic inflammatory response syndrome (SIRS), resulting in late mortality. Although increased monocyte (mphi) TNF-alpha production plays a pivotal role in this TNF-alpha elevation, the molecular mechanisms leading to increased mphi TNF-alpha production have yet to be elucidated. We demonstrate that, although TNF-alpha mRNA levels are increased in all trauma patients' mphi, which produce elevated levels of TNF-alpha protein, in the majority of patients, these increased TNF-alpha mRNA levels are under normal transcriptional and posttranscriptional control. Consequently, the increased TNF-alpha production by these patients' mphi is probably due to preactivation of these mphi by trauma-released mediators. However, a small minority of patients, whose mortality rate was 57%, produce TNF-alpha of primarily the membrane-associated type. The mphi TNF-alpha mRNA accumulation of these patients in response to in vitro stimulation is significantly augmented. All of these patients experienced SIRS. In this subset of patients' mphi, TNF-alpha mRNA stability was aberrantly increased. Such an increase in TNF-alpha mRNA stability could lead to devastatingly prolonged production of TNF-alpha protein. This demonstration of increased TNF-alpha mRNA stability in post-trauma mphi represents a novel correlation of elevated membrane-associated TNF-alpha protein, increased mortality, and a mechanism for this occurrence.

Biochemical identity and characterization of the mouse interleukin-2 receptor beta and gamma c subunits.

Although the mouse IL-2 receptor (IL-2R) beta and gamma c subunits have been identified by molecular cloning, the biochemical identity of these subunits has not yet been established. In the present study, the mouse IL-2R was biochemically characterized from cell lines expressing normal and aberrant IL-2R. Using novel monoclonal antibodies specific for the beta or gamma c subunits, we established that the M(r) of the beta chain is 90,000-100,000 and that of the gamma c subunit is 75,000-80,000. Analysis of transfected EL4 cells that expressed alpha, gamma c, and truncated beta subunits or mutant EL4 cells, which selectively lacked cell surface gamma c, revealed that no other material migrated to a position on SDS-PAGE characteristic of IL-2/IL-2R beta and IL-2/IL-2R gamma c cross-linked complexes, respectively. Thus, the beta and gamma c subunits appear to be the sole IL-2R constituents of these IL-2 cross-linked complexes. The IL-2/IL-2R gamma c, but not the IL-2/IL-2R beta, complex exhibited enhanced mobility after SDS-polyacrylamide gel electrophoresis under nonreducing conditions, suggesting a more compact structure for gamma c as a result of intrachain disulfide bonds. The primary posttranslational modification of the mouse beta and gamma c subunits is N-linked glycosylation. These biochemical studies reconcile past uncertainties concerning the subunit composition of the mouse IL-2R and are consistent with a model of the IL-2R containing only three subunits.

Expression and function of the gamma c subunit of the IL-2, IL-4, and IL-7 receptors. Distinct interaction of gamma c in the IL-4 receptor.

IL-2R, IL-4R, and IL-7R share a common subunit referred to as gamma c and the IL-13R has been proposed to contain gamma c as a subunit. In this report we have used two novel mAbs (3E12 and 4G3) to distinct epitopes of mouse gamma c to determine its lymphoid cell distribution and to examine whether gamma c uses similar epitopes to interact with different cytokines and cytokine receptors. FACS analysis revealed that gamma c is expressed in most lymphocytes, myeloid cells, embryonic thymocytes, and lymphoid cell lines. Results from radiolabeled ligand binding studies, biochemical analysis of ligand-receptor cross-linked complexes, and cytokine bioassays indicate that the epitope defined by mAb 4G3 closely defines the IL-7 binding region of gamma c and overlaps the IL-2 binding region of gamma c. These studies also indicate that gamma c interacts with IL-4 in the context of the IL-4R in a manner that is distinct from its role in the IL-2R and IL-7R and suggest that the 3E12 epitope defines a region of gamma c that intimately interacts with the IL-4R. The B9 plasmacytoma, which proliferates in response to IL-4 and IL-13, was shown to not express gamma c. Thus, at least in some circumstances, gamma c is dispensable for signaling via the IL-4R and is not a required subunit of the IL-13R.

Selection of internalization-deficient cells by interleukin-2-Pseudomonas exotoxin chimeric protein: the cytoplasmic domain of the interleukin-2 receptor beta chain does not contribute to internalization of interleukin-2.

To study the structural basis of ligand-induced receptor-mediated internalization of interleukin-2 (IL-2), a strategy has been developed to generate variant T cells that are deficient in internalization of this cytokine. IL-2 receptor (IL-2R) alpha- and beta-bearing EL4 cells, that express high-affinity IL-2R and internalize IL-2, were treated with low doses of IL-2-Pseudomonas exotoxin chimeric protein (IL-2-PE40). This treatment resulted in isolation of a variant (CX1) that was unable to express high-affinity IL-2R or internalize IL-2. Transfection of CX1 with the IL-2R beta cDNA led to surface expression of IL-2R beta and high-affinity IL-2R as well as the ability to internalize IL-2. This finding indicates that the absence of the beta subunit was the sole defect in CX1 responsible for its failure to internalize IL-2. By transfecting CX1 with mutated beta cDNA, several CX1 transfectants were produced that expressed a beta-subunit that lacked all amino acids of the intracytoplasmic region. These transfectants expressed high-affinity IL-2R and internalized IL-2 at a rate comparable to cells expressing wild-type beta-chain. These results demonstrate that internalization of IL-2 is independent of any signals contained in the intracytoplasmic tail of the beta subunit and raise the possibility that such signals may be entirely contained within the gamma subunit.