Regulation of T cell autocrine growth. T4+ cells become refractory to interleukin 2

During the course of investigating the regulation of IL-2-dependent T cell proliferation, we found that the subset of human T cells expressing the T4 surface glycoprotein become refractory to IL-2 growth promotion earlier than T8+ cells. Since T4+ cells proliferate in an autocrine fashion to endogenous IL-2, whereas most T8+ cells respond in a paracrine fashion to IL-2 derived from T4+ cells, we thought it likely that a unique mechanism was operative to restrict T4+ cell IL-2-dependent autocrine proliferation. Moreover, we anticipated that the T4+ cell IL-2-refractory state related either to suppression by T8+ cells, or to expression of T4+ cell IL-2-R. However, several experimental approaches did not support either of these mechanisms as being responsible for the loss of T4+ cell IL-2 responsiveness. Isolated T4+ cells ceased to respond to IL-2 well before T8+ cells, and before the disappearance of adequate levels of IL-2-R. Moreover, a detailed comparison of IL-2-R expression by T4+ vs. T8+ cells revealed no differences in the number, affinity, rate of expression, or functional activity of high-affinity IL-2-R expressed by the two subsets. Accordingly, T4+ cell autocrine IL-2 responsiveness is restricted by a mechanism that is independent of IL-2-R, and which ultimately results in cessation of both T4+ and T8+ cell IL-2-dependent clonal expansion.     

Human interleukin 1 induces interleukin 1 gene expression in human vascular smooth muscle cells

The recognition that cells of the vascular wall can secrete cytokines such as IL-1 suggests new mechanisms for initiating or sustaining inflammatory responses in blood vessels. We report that purified human monocyte-derived IL-1 or recombinant human IL-1 (rIL-1 beta and rIL-1 alpha) induce cultured human smooth muscle cells derived from veins or arteries to synthesize IL-1 beta mRNA and produce and release biologically active IL-1. rIL-1 beta also stimulated the production of PGE2 by smooth muscle cells. Exposure to rIL-1 beta (1-100 ng/ml), or rIL-1 alpha (0.01-10 ng/ml) increased IL-1 beta mRNA levels within 30 min. Actinomycin D (1 microgram/ml) prevented the induction of IL-1 beta mRNA by rIL-1. IL-1 alpha mRNA was detected in SMC treated with cycloheximide (1 microgram/ml) and rIL-1 beta, or cycloheximide alone. rIL-1 alpha and rIL-1 beta produced maximal levels of IL-1 beta mRNA after 4 h, and intracellular IL-1 biological activity after 6 h of exposure. Release of IL-1 activity in the extracellular medium began after 1 h of incubation with rIL-1 beta or rIL-1 alpha, and continued for up to 24 h. Anti-TNF antiserum that neutralized the biological activity of rTNF did not affect rIL-1-induced production of IL-1 beta mRNA or IL-1 release, suggesting that the release of TNF does not mediate these processes. Several experimental approaches indicated that the release of IL-1 by smooth muscle cells was not due to endotoxin contamination of the IL-1 preparations. Anti-IL-1 antiserum blocked the induction of smooth muscle cell IL-1 gene expression by rIL-1 beta. Polymyxin B did not prevent IL-1-induced IL-1 expression by these cells, but blocked the effect of endotoxin. Heat treatment destroyed the stimulatory capacity of rIL-1 beta, but did not affect the ability of bacterial endotoxin to induce IL-1 expression. The production of IL-1 by human vascular smooth muscle cells was not due to contamination of the cell cultures with blood monocytes, inasmuch as treatment with an antimonocyte antibody (anti-Mo2) and complement did not alter IL-1 beta mRNA content or the amount of IL-1 released from the cells in response to endotoxin, rIL-1 alpha, or rIL-1 beta.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of interleukin 1 on human thymocytes and purified human T cells

Human Interleukin 1 (IL-1) purified by molecular weight fractionation, isoelectric focusing, and gel electrophoresis has been tested on human thymocytes and highly purified human T cells. IL-1 prepared in this manner could not support the long-term growth of T cells yet would augment lectin-stimulated mitogenesis. The IL-1 preparations were shown to possess the lectin-augmenting activity at dilutions containing less than 1 ng of the measurable protein. These data are in agreement with the model that IL-1 stimulates production of IL-2 from lectin- stimulated lymphocytes.     

High-affinity receptor-mediated internalization and degradation of interleukin 2 in human T cells

Receptor-mediated internalization and degradation of IL-2 were investigated in cell lines carrying human T cell leukemia/lymphoma (lymphotrophic) virus type I (HTLV-I) and PHA-treated normal PBL. The HTLV-I-carrying cell lines ILT-Yan and TL-Mor, and the PBL expressed both high- and low-affinity IL-2-R. However, another HTLV-I-carrying T cell line, MT-1, expressed mainly low-affinity receptors. Greater than 50% of the IL-2 bound to high-affinity receptors was internalized within 10 min when these cells were incubated at 37 degrees C. The internalized IL-2 was rapidly degraded and the products were excreted into the culture fluid. The t1/2 of IL-2 degradation in these cells was estimated as 60-80 min at 37 degrees C. The internalization and degradation of IL-2 were both temperature dependent. Light-microscopic autoradiography with 3H-labeled IL-2 confirmed the internalization of IL-2, and suggested that some IL-2 might be carried to the nucleus.     

Interleukin 2 induction of pore-forming protein gene expression in human peripheral blood CD8+ T cells

Our studies have analyzed pore-forming protein (PFP) mRNA expression in resting and stimulated human peripheral blood CD3- large granular lymphocytes (LGL), CD3+ T cells, and their CD4+ or CD8+ subsets. Signals that stimulate T cells to develop cytotoxic activity (i.e., IL- 2 or OKT-3 mAb) led to the induction of PFP mRNA in T cells. The data indicated that IL-2 directly increased PFP mRNA in the CD8+ subset of T cells, in the absence of new DNA or protein synthesis. Abrogation of IL- 2-induced PFP mRNA expression and cytotoxic potential of T cells by the anti-p75 IL-2 receptor mAb suggested that low numbers of p75 IL-2 receptors on CD8+ T cells were capable of transducing signals responsible for these IL-2-induced effects. The induction of T cell PFP mRNA via CD3, using OKT-3 mAb, was less rapid but greater than that caused by IL-2; however, a combination of PMA and ionomycin, which bypasses crosslinking of the TCR/CD3 complex, could not mimic this increase in PFP mRNA levels in T cells. The role of second messenger systems in regulating PFP mRNA expression remains to be determined. In contrast, high constitutive PFP mRNA expression was observed in CD3- LGL and these mRNA levels could not be enhanced by stimulation with IL- 2. The cytotoxic potential of peripheral blood T cells and LGL induced in response to IL-2 correlated with IL-2-induced PFP mRNA levels in these cells and was consistent with PFP being one of several important molecules involved in the effector function of cytotoxic lymphocytes.     

Selective induction of rheumatoid factors by superantigens and human helper T cells.

Production of autoantibodies specific for the Fc region of autologous IgG, called rheumatoid factors (RF), is a characteristic finding in patients with rheumatoid arthritis (RA). To study the requirements regulating the synthesis of these autoantibodies, we have cloned human helper T cells and co-cultured them with purified B cells. To mimic cognate T-B cell interaction, we have used bacterial superantigens that function by cross-linking HLA molecules on the B cell with selected T cell receptor (TCR) molecules expressing a particular polymorphism of the V beta gene segment. Data presented here demonstrate that the staphylococcal enterotoxin D (SE D), but not other bacterial superantigens, exhibits an ability to induce IgM, IgG, and especially RF production, in B cells from RA patients and normal individuals. Comparison with the polyclonal antibody production in B cell cultures driven by anti-CD3-stimulated T cell clones confirmed that SE D shifted the repertoire of secreted antibodies toward immunoglobulins with Fc binding specificity, suggesting that SE D preferentially stimulates RF+ B lymphocytes. B cells with the potential to secrete RF were highly frequent in RA patients, requiring as few as 150 peripheral B cells/culture to detect RF in the culture supernatants. SE D-induced RF synthesis was strictly dependent on the presence of selected CD4+T helper cells and required a direct membrane contact between B cells and T helper cells. Here, we propose a model that SE D selectively induces RF production depending on the availability of SE D responsive T cells in the TCR repertoire of the responder.     

Expansion of natural killer cells but not T cells in human interleukin 2/interleukin 2 receptor (Tac) transgenic mice

Transgenic mice expressing both human IL-2 and the L chain of IL-2-R constitutively had an unusual expansion of Thy-1+/CD3-4-8- large granular lymphocytes, which bore the elevated NK activity. Unexpectedly, the transgenic mice had neither T cell expansion nor autoreactive antibodies. The increase in number and activity of NK cells seems to be responsible for both the severe interstitial pneumonia and lymphocyte depletion in the spleen that we found in these transgenic mice. In addition, we found the selective loss of Purkinje cells in the cerebellum of the mice, which gave rise to their disturbed gait. All the transgenic mice died by 4 wk of age.     

Regulation of T lymphocyte proliferation. Interleukin 2-mediated induction of c-myb gene expression is dependent on T lymphocyte activation state

We previously reported that with time, after antigenic stimulation of antigen-regulated murine T lymphocyte clones, total IL-2-R expression decayed 10-50-fold, commensurate with a decline in the ability of the cells to proliferate to IL-2. However, late after antigenic stimulation, when the cells were refractory to the IL-2-proliferative stimulus, high levels of high affinity IL-2-R remained. In this report we further explore the basis of unresponsiveness to IL-2 in the quiescent clones. We show that the proto-oncogene c-myc is induced in the late cell population by IL-2 to comparable levels observed early after antigen stimulation. IL-2-dependent c-myb induction, however, is seen only early after activation but not in the late-activated population. Analysis of the IL-2-dependent expression of c-myb mRNA with time after antigenic stimulation showed that steadystate c-myb expression declines dramatically with kinetics closely paralleling a decay in IL-2-dependent proliferative ability. In contrast, steadystate c-myc expression remains high throughout this period. Expression of c-myb is critical for proliferation of these cells since antisense oligodeoxy-nucleotide to c-myb can inhibit their IL-2-dependent proliferation. We present evidence for a pathway of c-myb induction via the TCR that is independent of the IL-2/IL-2-R interaction. In addition, the inhibition of IL-2-R-induced c-myb expression by 2-aminopurine and enhanced induction of c-myb via the TCR demonstrate that TCR activation and IL-2-R activation lead to induction of c-myb by different mechanisms.     

Expression of interleukin 2 receptors and binding of interleukin 2 by gamma interferon-induced human leukemic and normal monocytic cells

Gamma interferon induced surface expression of interleukin 2 (IL-2) receptors on normal human monocytes and the monocytoid cell lines U937 and HL60. These receptors were detected by anti-IL-2 receptor monoclonal antibodies, and U937 IL-2 receptors were indistinguishable from T lymphocyte IL-2 receptors by immunoprecipitation. Also, U937 IL- 2 receptors bound biologically active IL-2. These results suggest a role for monocyte IL-2 receptors in T cell/monocyte interaction during an immune response.     

Differential requirements for the induction of interleukin 2 responsiveness in L3T4+ and Lyt-2+ T cell subsets

Minimal requirements for the induction of interleukin 2 (IL-2) responsiveness in purified subsets of murine T lymphocytes have been investigated. Whereas Lyt-2+ cells could be induced to IL-2-dependent growth by lectin, phorbol ester, or calcium ionophore, none of these stimuli was by itself sufficient for L3T4+ cells. The latter cells could, however, be induced to respond to IL-2 by combinations of lectin plus phorbol ester or ionophore plus phorbol ester (but not lectin plus ionophore). Under optimal conditions, growth of L3T4+ cells (like Lyt-2+ cells) was independent of accessory cells and cell-cell contact.     

CD28-induced costimulation of T helper type 2 cells mediated by induction of responsiveness to interleukin 4

Type 1 and type 2 cloned T helper (Th) cells are believed to require different antigen-presenting cell (APC)-derived costimuli for proliferation. In the case of Th1-cloned T cells, CD28 signaling costimulates production of autocrine interleukin 2 (IL-2). Th2 cells produce their autocrine growth factor, IL-4, without costimulation, but require APC-derived costimuli, or IL-1, to respond to IL-4. Here we demonstrate that engagement of CD28 on Th2 cells with anti-CD28 antibody or with APC-associated B7 costimulates Th2 responsiveness to IL-4 but does not affect IL-4 or IL-2 production by Th2 cells. Costimulation of Th2 cells via CD28 appears to involve the induction of IL-1 production by Th2 cells, which acts in an autocrine fashion to induce IL-4 responsiveness. These results suggest that CD28-induced costimulation plays an important role in responses mediated by both types of Th cells.