T lymphocytes of young and aged rats. II. Functional defects and the role of interleukin-2

Splenic T lymphocytes of aged Lewis rats respond to Con A and PHA with diminished 3H-TdR uptake compared with splenic T lymphocytes of young Lewis rats. After immunization with allogeneic tumor cells, uptake of 3H-TdR in mixed lymphocyte-tumor cultures and T cell cytotoxicity against tumor target cells are significantly lower with spleen cells of aged rats compared with those of young rats. The culture of spleen cells of aged rats with Con A results in a diminished conversion of Ia-positive T cells from Ia-negative precursors compared with similar cultures of spleen cells of young rats. Spleen cells of both young and aged rats produce high amounts of IL-2 in response to Con A stimulation. "Old" T cells, however, bind relatively little IL-2, do not utilize it in culture, and do not respond to exogenous IL-2 with enhanced 3H-TdR uptake as do "young" T cells. In allogeneic MLTC, "old" T lymphocytes produce little IL-2 compared with "young" cells, and both "young" and "old" cells respond to exogenous IL-2 with enhanced 3H-TdR uptake and increased cytotoxic activity. The data suggest defects in the synthesis and/or recognition of IL-2 as well as defects in the regulation of Ia antigen expression may be responsible, in part, for the reduced T cell function in aged animals.     

Splenic and inguinal lymph node T cells of aged mice respond differently to polyclonal and antigen-specific stimuli.

Numerous changes have been reported to occur in T cell responsiveness of mice with increasing age. However, most of these studies have examined polyclonal stimulation of spleen cells from a limited number of mouse strains. This study investigated the influence of genetic background, source of lymphocytes, and type of stimulus on age-associated changes in T cells response. Con A-induced proliferation and IL-2 and IFN-gamma production by splenic lymphocytes (SL) was significantly greater in CBA/Ca mice compared to C57BL/6 mice, regardless of age. SL of both strains exhibited the predicted age-dependent decline in proliferative response and an increase in IFN-gamma production in response to Con A. In contrast, however, only SL from C57BL/6 mice demonstrated the predicted age-dependent decline in Con A-induced IL-2 production; Con A-induced SL of young and aged CBA/Ca mice produced comparable amounts of IL-2. Differences in age-associated responses to Con A were also observed between SL and inguinal lymph node (ILN) cells of CBA/Ca mice. In contrast to SL, ILN cells demonstrated an increased proliferative response to Con A. However, lymphokine production by Con A-stimulated ILN cells from aged CBA/Ca mice was similar to that of Con A-stimulated SL from aged CBA/Ca mice. To determine if aged ILN T cells respond similarly to polyclonal and antigen-specific stimuli, keyhole limpet hemocyanin (KLH) responses of T cells isolated from ILN of aged and young CBA/Ca mice were examined. KLH-specific T cells from aged mice cultured with KLH-pulsed macrophages (M phi) from aged mice were significantly reduced in their ability to proliferate compared to KLH-specific T cells of young mice cultured with young KLH-pulsed M phi. In contrast to the expected results, the defect was not at the level of the T cells; proliferation of young T cells cultured with aged KLH-pulsed M phi was equivalent to the proliferation of aged T cells cultured with aged M phi. These results suggest that aging has differential effects on polyclonal and antigen-specific T cell proliferation and on polyclonal stimulation of T cells isolated from different lymphoid organs and from different strains of mice.     

In situ hybridization analysis of the age-associated decline in IL-2 mRNA expressing murine T cells

Only a small decrease in the number of L3T4- cells was observed in the Con A-stimulated splenocyte cultures of old mice as compared to young, which cannot account for the threefold decrease in IL-2 production. Northern and dot blot analysis of RNA from splenocytes containing equivalent numbers of L3T4+ cells from young and old mice showed that cells from old mice express less IL-2 mRNA after mitogenic stimulation than cells from young mice. Direct analysis by in situ hybridization of stimulated splenocytes from young and old mice then showed approximately a threefold decrease in the percentage of IL-2 mRNA expressing cells in the spleens of old mice as compared to young (8.7 +/- 4.1% old; 28.7 +/- 11.7% young). The average level of expression of IL-2 mRNA was not significantly different between cells from young and old mice; however, there were approximately 40% fewer cells expressing an intermediate to high amount of IL-2 mRNA in old mice as compared to young (26.3% vs 41.8%). These data suggest that the decrease in IL-2 production with age is associated primarily with a decrease in the frequency of IL-2 mRNA-expressing cells in old mice, especially in those cells expressing intermediate to high levels of IL-2 mRNA.     

Both cloned interleukin 2 and purified interleukin 1 are required for optimal growth of purified L3T4+ and Lyt 2+ lymphocytes initiated by concanavalin A

Concanavalin A (Con A), cloned interleukin 2 (IL-2), purified interleukin 1 (IL-1) or two different crude preparations containing IL-1 activity alone, did not induce proliferation of rigorously accessory cell (AC)-depleted splenic L3T4+ or Lyt 2+ lymphocytes. Con A together with saturating concentrations of cloned IL-2 (100 U/ml) promoted less than 40% of the proliferative responses observed in AC-supplemented L3T4+ and Lyt 2+ T-cell cultures. The three preparations of IL-1 used supported minimal proliferation of Con A-treated purified L3T4+ or Lyt 2+ lymphocytes. However, all these IL-1 preparations promoted significant growth of the T-cell populations if AC (1%) were included in the cultures. Cloned IL-2 combined with purified IL-1 promoted proliferation of Con A-treated L3T4+ and Lyt 2+ lymphocytes achieving approximately 75% of the responses observed in AC-supplemented T-cell cultures. The additive effect of IL-1 was apparent in the presence of saturating concentrations of cloned IL-2. Finally, Con A alone induced a detectable number of both L3T4+ and Lyt 2+ lymphocytes to express IL-2 receptors as determined with the anti-mouse IL-2 receptor antibody 7D4 by immunofluorescence and FACS analysis. Purified IL-1 neither induced detectable number of L3T4+ or Lyt 2+ T cells to express IL-2 receptors nor increased the number of Con A-treated T cells bearing IL-2 receptors. We have interpreted these findings to indicate the following: Con A alone is sufficient to induce highly purified L3T4+ and Lyt 2+ lymphocytes to express IL-2 receptors. Cloned IL-2 and purified IL-1 are required for optimal growth of L3T4+ and Lyt 2+ lymphocytes and these cytokines together efficiently replace AC in growth of T cells initiated by Con A. IL-1 alone does not replace AC in Con A-induced activation of mouse T cells. IL-1 exerts potentiation on IL-2-driven growth of Con A-treated L3T4+ and Lyt 2+ lymphocytes. The additive activity of IL-1 on growth of normal T cells is not due to increased production of IL-2 in the cultures or induction of normal T cells to expression of IL-2 receptors by IL-1. We propose that IL-1 optimizes the action and/or interaction of IL-2 with its receptors on the T-cell membrane (by, i.e., increasing affinity of the IL-2 receptor for its ligand and/or stabilizing the IL-2 receptor).     

Retrovirus-mediated immunosuppression. II. FeLV-UV alters in vitro murine T lymphocyte behavior by reversibly impairing lymphokine secretion

Murine splenocytes and cloned murine T cells were used to study the in vitro immunosuppressive effects of UV-inactivated feline leukemia virus (FeLV-UV) on lymphokine secretion. FeLV-UV can significantly depress the accumulation of IL 2 in cultures of Con A-stimulated C57BL/6 splenocytes and in cultures containing the alloreactive helper T cell clone B6D/2-2m plus Con A. Inhibition of lymphokine accumulation in these cultures could not be attributed to absorption or inactivation of IL 2 by the FeLV-UV or to the FeLV-UV-induced production of substances which interfere with the IL 2 bioassay. Thus, FeLV-UV appears to block production and/or secretion of IL 2 by a direct inhibitory effect on IL 2-secreting murine T lymphocytes. Additional studies indicate that FeLV-UV impairs IL 2 production only if added very soon after lymphocyte contact with lymphokine-inducing agents and that IL 2 secretion resumes when FeLV-UV is removed from the culture. FeLV-UV also impairs accumulation of MAF (interferon-gamma?) in cultures of Con A-stimulated C57BL/6 splenocytes and in cultures containing the alloreactive cytotoxic T lymphocyte clone B6D/2-7c plus Con A. The latter observation again suggests that FeLV-UV impairs lymphokine secretion by a direct effect on lymphokine-producing T lymphocytes. Furthermore, it suggests that FeLV-UV does not selectively impair production of IL 2 nor does it have selective inhibitory effects on helper T cells. Rather, FeLV-UV appears to have a general inhibitory effect on lymphokine production by T lymphocytes. Finally, concentrations of FeLV-UV which suppress MAF production by the CTL clone have little influence on the cytolysis mediated by the same cloned T cell population. Thus, the immunosuppressive influence of FeLV-UV is selective for phenomena associated with induction of new T lymphocyte functions, such as lymphokine secretion, and spares other immune functions already expressed by the same cells.     

Lymphokine production by mitogen and antigen activated mouse intraepithelial lymphocytes

Although most intraepithelial lymphocytes (IEL) in mouse small intestine bear surface markers classically associated with T lymphocytes, the T-cell nature of these cells remains controversial. In the present study IEL from normal mice, or from mice infected with the gut nematode Trichinella spiralis, were therefore tested for their ability to produce T-cell-derived lymphokines in response to in vitro stimulation with concanavalin A (Con A) or with specific worm antigens. The data show that Con A-stimulated IEL produce minimal amounts of IL-2, and intermediate levels of IFN-gamma and IL-3 in comparison to the levels produced by spleen T cells. The FDC-P2 cell line, which proliferates in response to both IL-3 and GM-CSF, was identified as the most sensitive and reproducible indicator of lymphokine activity in supernatants from mitogen-stimulated IEL from normal mice. IEL isolated from mice infected with T. spiralis also produced high levels of FDC-P2 growth factors when challenged in vitro with Trichinella-derived antigens; however, normal IEL did not respond to this stimulus. The data thus provide evidence that antigen-sensitive T cells can arise in (or migrate to) the gut epithelium during gut infection.     

Abnormal regulation of IFN-gamma secretion in vitamin A deficiency

T lymphocytes from vitamin A-deficient (A-) mice show a decreased ability to stimulate B lymphocytes for Ag-specific secondary IgG1 responses in vivo and in vitro. Experiments reported here traced the molecular basis for this functional defect to an overproduction of IFN-gamma by A- CD4+ T cells compared with cells from A-sufficient (A+) mice. Secretion of IL-2 and IL-4 by cells from A- and A+ mice was equivalent. Retinoic acid supplementation in vitro decreased IFN-gamma secretion from A- T cells, indicating that IFN-gamma production is retinoid-responsive. Adding IFN-gamma neutralizing antibodies to cultures established with cells from immune A- mice substantially increased IgG1 production, whereas IL-4 addition moderately increased IgG1 production. Adding retinoic acid to the cultures either at initiation, or 48 h later, fully restored IgG1 production by A- cultures to the level of A+ control cultures. These results are consistent with a role for vitamin A in negatively regulating IFN-gamma secretion.     

Defective activation of T suppressor cell function in nonobese diabetic mice. Potential relation to cytokine deficiencies

Nonobese diabetic (NOD) is an inbred mouse strain susceptible to development of T cell-mediated autoimmune diabetes. The strain is characterized by high percentages of T lymphocytes in lymphoid organs. The syngeneic mixed lymphocyte reaction (SMLR), a T cell response to self MHC class II Ag, is reportedly involved in the generation of a number of immunoregulatory cells, including suppressor inducers. A severely depressed SMLR characteristic of certain other autoimmune strains was found in NOD but not in nonautoimmune SWR/Bm mice. Moreover, IL-2 produced by NOD T cells at day 6 in an SMLR was at least one hundredfold reduced compared with SWR, and NOD T cells harvested from an SMLR at day 6 were functionally defective when tested for ability to induce suppression of an allogeneic MLR. However, functionally competent suppressor T cells were generated in NOD splenic leukocyte cultures in response to Con A, and IL-2 release from these was equivalent to that released by Con A-stimulated SWR splenocytes. A deficiency in cytokine release was not limited to IL-2, because peritoneal exudate cells from NOD exhibited a greatly diminished sensitivity to LPS-stimulated IL-1 release in comparison to SWR mice. IL-2 supplementation both in vitro and in vivo restored the ability of NOD T cells to respond in a SMLR, with production of cells capable of inducing suppression. Like SMLR-activated T cells from untreated SWR controls, SMLR blasts from IL-2-treated NOD mice were enriched for the L3T4 phenotype. IL-1 supplementation in vitro resulted in partial restoration of T suppressor activation in a SMLR. The depressed SMLR exhibited by NOD mice was apparently a stimulator cell dysfunction, because NOD stimulator cells failed to activate T cells from (SWR x NOD)F1 mice, whereas stimulators from SWR or F1 mice were capable of doing so. Collectively, these results suggest a defect in suppressor cell activation rather than an absence of this immunoregulatory cell population.     

Comparison of immunological effects of cholera toxin on autoimmune MRL/lpr and BXSB mice.

MRL/lpr and BXSB mice were treated weekly or biweekly with cholera toxin (CT) in intravenous dose of 2 micrograms/mouse. CT treatment notably alleviated proteinuria in MRL/lpr mice, but did not influence the course of lupus nephritis in BXSB male mice. Flow cytometric analysis showed that anomalous B220+ T cells in spleen and thymus were reduced in CT-treated MRL/lpr mice while no significant change in lymphocyte populations was induced in BXSB male mice by this treatment. The suppressive effect of CT treatment on Con A response and the augmentative action on LPS response were observed in MRL/lpr mice. The latter may reflect increased B cells in relative number in the peripheral lymphoid organs. Mitogenic responses in CT-treated BXSB male mice remained unchanged in comparison with those of untreated group. Increased production of IL-6 by spleen cells was demonstrated in MRL/lpr mice treated with CT while in BXSB mice the level of IL-6 was not changed by the treatment with CT. Production of IFN gamma was suppressed by CT treatment in both strains of mice. This may be attributed to the inhibitory effect of CT on IFN gamma-producing Th1 cells as reported previously (Munoz et al, J. Exp. Med. 172: 95-103, 1990). However, CT treatment did not inhibit anti-DNA antibody production in BXSB mice, whereas the autoantibodies were markedly decreased in MRL/lpr mice treated with CT.     

Effect(s) of lipopolysaccharide on lectin-induced T-cell activation

The present study focuses on the effect of lipopolysaccharide (LPS) on the cellular events leading to T-cell activation by concanavalin A (Con A). Interleukin 2 (Il-2) production is much reduced in Con A-stimulated cultures of spleen cells derived from LPS-treated mice. This depressed Il-2 synthesis is not related to the eventual activity of LPS-activated suppressive B cells. Rather, it reflects an ineffective collaboration between adherent cells and T lymphocytes. The low level of Il-2 produced by LPS-sensitized spleen cells is sufficient for lectin-induced T-cell proliferation. Moreover, acquisition of responsiveness to Il-2 is unaltered by LPS. No strict correlation was found between the deficiency in Il-2 production and the inability of LPS-sensitized spleen cells to generate a thymus-dependent response. Less time (5 hr) is needed for LPS to exert its inhibitory effect on an anti-sheep red blood cell response than on Il-2 synthesis (at least 24 hr). Results are discussed in terms of cellular interactions implicated in a polyclonal T-cell response and with regard to the contribution of Il-2 to the LPS-induced immune unresponsiveness.     

A spleen-derived maturational factor allows immature thymocytes, prepared as cells bearing low amounts of surface sialic acid, to become cytotoxic T cells

A population of immature mouse thymocytes bears low levels of surface sialic acid and can be separated from the more mature high sialic acid-bearing thymocytes by selective agglutination with the sialic acid-specific lectin, lobster agglutinin 1. These immature thymocytes do not proliferate in response to concanavalin A (Con A). They do not produce interleukin 2 (IL-2), do not provide T cell help to B cells for an in vitro antibody response, and as shown here, do not become cytotoxic T lymphocytes when polyclonally stimulated with Con A + IL-2. We describe here a spleen-derived maturational factor which stimulates these immature thymocytes, in the presence of Con A and IL-2, to become cytotoxic T lymphocytes. The maturational factor is a protein secreted by Con A-stimulated mouse or rat spleen cells; it is apparently neither interleukin 1, IL-2, interleukin 3, gamma-interferon, nor combinations of these cytokines, because these materials do not replace the maturational factor. The active material in Con A-stimulated mouse spleen cell supernatant was recovered from a G-75 column in the 33,000-48,000 m.w. range. These experiments suggest that within the lobster agglutinin 1-negative thymocyte population there are cells which can mature under the influence of a spleen-derived factor. It is possible that these cells represent the small subpopulation of immature cells destined to become immunocompetent peripheral T cells. On the other hand, the factor may be rescuing cells destined to die in the thymus.     

Interleukin 1 secretion is not required for human macrophage support of T-cell proliferation

Interleukin 1 (IL-1) is a soluble factor secreted by stimulated monocytes (Mo) and animal macrophages (Mx). We have previously demonstrated that human Mo cultured in vitro for 1-6 days transform to Mx, and retain their ability to support concanavalin A (Con A)-driven T-cell proliferation. We have also shown that, paradoxically, these Mx do not secrete IL-1, when stimulated by endotoxin (LPS). In this study we examined two alternative hypotheses: T cells plus mitogen induce Mx IL-1 production, and human Mx deliver a second signal to T cells via a non-IL-1 mechanism. IL-1 was assayed in a mouse CD-1 thymocyte system without concanavalin A. Mo/Mx were cultured with T cells at low (2 X 10(4)/200 microliters) or high (1 X 10(5)/200 microliters) concentrations for 2 or 4 days, in the presence of Con A. Six hours prior to quantitation of proliferation, 50 microliters of supernatant was removed and assayed for IL-1. As expected both Mo and Mx enhanced T-cell proliferation eight- to tenfold. Mo secreted large amounts of IL-1; there was no demonstrable IL-1 activity present in supernatants from cultures containing either T cells and Mx, or Mx alone. Similar results were obtained by preincubating the cells (Mo, Mx, and T cells) with Con A for 12 hr and removing Con A prior to a 36-hr coculture. We examined the possibility that a small amount of IL-1 may be able to support Con A-stimulated T-cell proliferation and yet may not induce thymocyte proliferation. The highest dilutions of Mo supernatant (1:125) which supported T-cell proliferation also caused a fivefold increase in thymocyte proliferation. Supernatants from Mx failed to stimulate thymocyte proliferation or support Con A-driven T-cell proliferation. However, Mo and Mx lysates contain Il-1 activity. We conclude that human Mx support Con A-induced T-cell proliferation in the absence of IL-1 secretion. Mx may support T-cell proliferation by cell-bound IL-1 or by a non-IL-1 mechanism.     

Macrophage-mediated suppression of con A-induced IL-2 production in spleen cells from syphilitic rabbits

Blast transformation studies have indicated a diminished T cell response in spleen cell preparations from rabbits infected with Treponema pallidum. IL-2 synthesis by T lymphocytes is required for proliferation of these cells. Thus, Con A-induced IL-2 generation was measured in syphilitic animals infected for 9 to 14 days. IL-2 production in the infected rabbits was only one-half that observed for uninfected rabbits. This marked decrease in IL-2 was not caused by decreased IL-1 secretion by adherent cells from infected animals because similar levels were found in both infected and uninfected splenic cultures. This decrease was also not caused by an increase in infected spleen cell adsorption of IL-2; similar numbers of receptors for this IL were present in Con A-stimulated infected and uninfected splenic preparations. The inhibited IL-2 production in infected spleen cells was reversed upon removal of the adherent cells and also elevated upon addition of indomethacin to the cultures. PGE levels were also elevated in splenic cultures from infected animals. Finally, IL-2 synthesis, when evaluated at various days postinfection, showed that at 4 days, splenic cells generated twice as much IL-2 as uninfected cells. At 9 to 14 days, IL-2 levels were dramatically decreased (50% lower than that observed in uninfected cultures), and suppression of IL-2 by adherent cells was observed as late as 35 days post-infection. We propose that premature down regulation (suppression) of IL-2 secretion is mediated by adherent cells via a cyclo-oxygenase product, most likely PGE. These results may explain why most, but not all, treponemes are cleared during infection, and why the secondary manifestations of the disease occur.     

Decline in the production of interleukin-3 with age in mice

Previously, we and others have found that the ability to produce interleukin-1 (IL-1) and interleukin-2 (IL-2) declines with age in mice. The purpose of this study was to determine the influence of age on the capacity of mice to produce interleukin-3 (IL-3). Splenic cells (5 X 10(6)/ml) from young (3-4 months) and old (24-32 months) C57BL/6 mice were first assessed for their IL-3-producing capacities in response to varying doses of concanavalin A (Con A; 2-20 micrograms/ml) in a time-dependent manner. The results showed that the production of IL-3 by both young and old C57BL/6 mice was maximal on Days 3 and 4 in response to 20 micrograms/ml of Con A, and that of IL-2 was minimal (activity was less than 0.1 unit) on Day 4. Consequently, Day 4, was selected to assess the effect of age on IL-3 production by splenic cells. The results showed a twofold reduction in IL-3 production with age (P less than 0.05). Young-old splenic cell mixture experiments at ratios of 1:0, 3:1, 1:1, 1:3, and 0:1 indicated that the decrease in IL-3 production with age was not due to an increase in suppressor cell activity. Experiments based on mixtures of nylon wool-enriched splenic T-cell and adherent cells and on anti-MAC-1 plus complement-treated spleen cells indicated that (a) adherent cells are not required for T-cell production of IL-3, unlike IL-2 production, and (b) the decrease in IL-3 production with age is due solely to alteration in IL-3-producing T cells. Finally, a strong correlation was demonstrated between the production of IL-2 and IL-3 by spleen cells of individual young and old mice (r = 0.92, P less than 0.01). That production of both IL-2 and IL-3 is affected in a similar manner by age would suggest that a single class of helper T cells may be responsible for production of both lymphokines.     

Concanavalin A triggers T lymphocytes by directly interacting with their receptors for activation

Anti-HLA-DR antibodies did not inhibit concanavalin A-(Con A) induced T cell proliferation or the generation of suppressor cells capable of inhibiting immunoglobulin synthesis in autologous mononuclear cells after pokeweed mitogen stimulation. Nylon-wool purified T cells (pretreated with anti-HLA-DR antibody and C) exposed to Con A acquired responsiveness to interleukin 2 (IL 2) and were able to absorb this growth factor, whereas nonlectin-treated cells did not respond to IL 2 and could not absorb it. In the presence of interleukin 1 (IL 1), Con A stimulated the synthesis of IL 2 in purified OKT4+ lymphocytes but not OKT8+ cells. However, in the absence of IL 1, neither resting OKT4+ nor Con A-treated OKT4+ cells produced IL 2. Con A by itself did not directly stimulate macrophages to synthesize IL 1, although it could do so in the presence of OKT4+ but not OKT8+ lymphocytes. In addition, Con A induced proliferation of purified T cells provided IL 1 was supplied to the cultures. Cyclosporin A rendered Con A-treated T cells unresponsive to IL 2, made lectin-stimulated OKT4+ lymphocytes unable to respond to IL 1, and inhibited the synthesis of IL 2. Furthermore, this drug abrogated the Con A-stimulated synthesis of IL 1 by acting on OKT4+ lymphocytes and not on macrophages. Finally, cyclosporin-A suppressed the proliferative response and the generation of suppressor T cells induced by Con A. The following are concluded: 1) HLA-DR antigens do not seem to play any role in the triggering of T cells by Con A, and macrophages participate in lectin-induced activation of T cells mainly by providing IL 1. 2) Cyclosporin-A inhibits activation of T cells by interfering with the mechanism by which Con A stimulates T lymphocytes. 3) Con A triggers T lymphocytes by directly interacting with their receptors for activation.     

Activated T lymphocytes regulate hyaluronan binding to monocyte CD44 via production of IL-2 and IFN-gamma

Interactions of the cell surface proteoglycan CD44 with the extracellular matrix glycosaminoglycan hyaluronan (HA) are important during inflammatory immune responses. Our previous studies indicated that monocyte HA binding could be induced by TNF-alpha. Moreover, monocyte HA binding could be markedly up-regulated by culturing PBMC with anti-CD3 (TCR complex) mAbs. The present study was undertaken to identify soluble factors and/or cell surface molecules of activated T lymphocytes that might regulate HA binding to monocytes. Abs to IL-1 alpha, IL-1 beta, IL-2, IL-3, IL-10, IL-15, GM-CSF, IFN-gamma, and TNF-alpha were tested for their effects on anti-CD3 mAb-, Con A-, and PMA/ionomycin-mediated monocyte HA binding in PBMC cultures. Anti-TNF-alpha, anti-IL-2, and anti-IFN-gamma Abs, when added together to PBMC cultures, completely blocked Con A- and partially blocked anti-CD3- and PMA/ionomycin-induced monocyte HA binding. Furthermore, when added together to PBMC cultures, IL-2 and TNF-alpha induced high levels of monocyte HA binding. Likewise, IFN-gamma augmented TNF-alpha-induced monocyte HA binding. To investigate the role of T cell-monocyte direct contact in induction of monocyte HA binding, we studied PMA/ionomycin-activated, paraformaldehyde-fixed CD4(+) T cells in these assays. Fixed, PMA/ionomycin-activated CD4(+) T lymphocytes induced monocyte HA binding, but direct T cell-monocyte contact was not required. Moreover, anti-IFN-gamma and anti-TNF-alpha Abs blocked fixed PMA/ionomycin-activated CD4(+) T cell-induced monocyte HA binding. Taken together, these studies indicate roles for soluble T lymphocyte-derived factor(s), such as IL-2 and IFN-gamma, and a role for monocyte-derived TNF-alpha in Con A-, TCR complex-, and PMA/ionomycin-induced HA binding to monocyte CD44.     

Radiation sensitivity of T-lymphocytes from immunodeficient "wasted" mice.

Mice with the autosomal recessive gene "wasted" (wst/wst) exhibit neurologic disorders, reduced mucosal immune responses, and abnormal DNA repair mechanisms. The wst/wst mouse has been proposed as a murine model for the human disorder ataxia telangiectasia. Experiments were designed to examine the sensitivity of T-cells from wasted mice to ionizing radiation. Results demonstrated that T-cell clones derived from wasted mice are more sensitive to the killing effects of gamma-rays than similar T-cell clones from control mice. Bulk thymocyte and splenic cell cultures demonstrated similar radiation sensitivity. Both thymic and splenic lymphocytes from wasted mice also expressed low proliferative responses to mitogenic stimulation with concanavalin A (Con A) that could not be attributed to an absence or reduction in T-cell number. However, following activation with Con A, cell cultures exhibited a marked decrease in the percentage of Thyl + cells in wasted mice, in contrast to cultures from control mice in which significant increases in Thyl + cells were observed. Furthermore, when cells were treated with gamma-rays in combination with Con A, Thyl + cells were decreased in control spleen and thymus, but were elevated in similarly treated wasted cultures. These changes were accompanied by an increase in cell volume in T-cells from wasted but not from control mice. These results describe the sensitivity of T-cells from wasted mice to ionizing radiation; in addition, they suggest that the wst/wst abnormality may be associated with cell cycle aberrancies.     

Nitric oxide production is required for murine resident peritoneal macrophages to suppress mitogen-stimulated T cell proliferation. Role of IFN-gamma in the induction of the nitric oxide-synthesizing pathway

Lymphocyte proliferation in Con A- or LPS-stimulated murine splenic cell (SC) cultures was suppressed by the addition of excess macrophages. In Con A-stimulated cultures, suppression was associated with the expression of nitric oxide-synthesizing pathway (NOSP) activity as demonstrated by the accumulation of nitrite, a degradation product of nitric oxide (NO), in the culture supernatants. That NO, a cytotoxic and anti-proliferative metabolite of l-arginine, or other reactive nitrogen intermediates generated through the NOSP mediated the suppressive effect was suggested by the reversal of suppression brought about by the addition of a specific inhibitor of the NOSP (NG-monomethyl-l-arginine acetate) to the culture media. No NOSP activity was detectable in LPS-stimulated SC/macrophage cocultures. The role of T cell-derived IFN-gamma in the induction of the NOSP was investigated by the use of anti-IFN-gamma-mAb. Antibody-treated Con A supernatants failed to induce the NOSP in macrophages, and the addition of the mAb to Con A-stimulated SC/macrophage cocultures obviated the suppressive effects. Indomethacin and catalase only partially restored proliferation in Con A-stimulated SC/macrophage cocultures but were remarkably efficient in preventing macrophage-dependent suppression when LPS was used as the mitogenic stimulus. These results demonstrate a regulatory system of potential relevance in sites of predominant macrophage infiltration by which T cell-derived IFN-gamma activates the production of the mediator, NO, that suppresses T cell proliferation. In addition, these data demonstrate that, although the suppressive effects of excess macrophages appear to be expressed nonspecifically toward both T and B cells, suppression is mediated through a different mechanism in each case.     

Vasoactive intestinal peptide stimulates T lymphocytes to release IL-5 in murine schistosomiasis mansoni infection.

In murine schistosomiasis, granulomas form around ova deposited in the liver and intestines of infected mice. The granulomas have eosinophils that produce vasoactive intestinal peptide (VIP) and T cells that display VIP receptors. IL-5 is a lymphokine important for the development and maturation of eosinophils. It seemed plausible that VIP, released from eosinophils, may interact with lymphocyte VIP receptors and modulate IL-5 production as part of a feedback regulatory circuit. Thus, we determined whether granuloma T cells make IL-5 and whether VIP modulates IL-5 production. Isolated granuloma cells enriched for T lymphocytes spontaneously released IL-5. Culture of these cells in the presence of VIP increased IL-5 secretion. Spleen cells were also studied. Spleen cells from infected mice did not spontaneously release IL-5 or express IL-5 mRNA and VIP did not stimulate these resting spleen cells to produce this IL. However, these cells did express IL-5 mRNA and secreted IL-5 in response to Con A or soluble egg Ag. VIP could not appreciably modulate IL-5 release when cells were cultured with VIP and the Ag or mitogen. Spleen cells washed free of Con A ceased IL-5 secretion within 24 h. These preactivated splenic T cells resumed vigorous IL-5 secretion in response to either Con A or VIP. Yet only Con A prominently induced IL-5 mRNA expression. VIP was an effective stimulus at concentrations equal to or above the kDa of the VIP receptor on both splenic and granuloma T cells (10(-8) M). It is concluded that, in murine schistosomiasis, VIP invokes IL-5 release from activated T cells that are not undergoing immediate TCR stimulation.