Con A-induced IL-2 Production Research Articles

Noni (Morinda citrifolia L.) fruit juice reverses age-related decline in neural-immune interactions in the spleens of old F344 rats

Ethnopharmacological relevanceVarious parts of the tropical plant, Morinda citrifolia L. (Noni), have been widely used in traditional medicine in South and Southeast Asia for several centuries. The therapeutic effects of the noni are believed to be mediated through several phytochemicals such as anthraquinones, iridoid, fatty acid glycosides, alcohols, etc. Aim of the studyThe aim of the study is to investigate the effects of Morinda citrifolia fruit juice (noni fruit juice; NFJ) on neural-immune interactions through the involvement of intracellular signaling pathways both in vitro and in vivo in the splenic lymphocytes of young and old male F344 rats. Material and methodsIn the in vitro study, splenocytes from young and old F344 rats were isolated and treated with 0.0001–1% concentrations of NFJ for a period of 24h, while in the in vivo study, old F344 rats were orally administered (5ml/kg body weight) with NFJ (5%, 10% and 20%) twice daily for 60 days. After the treatment period, concanavalin A (Con A)-induced lymphocyte proliferation, cytokines (IL-2, IFN-γ, IL-6, and TNF-α) production, expression of tyrosine hydroxylase (p-TH), nerve growth factor (NGF), m-TOR, IκB-α, p-NF-κB (p50 and p65), p-ERK, p-Akt, p-CREB and lipid peroxidation, protein carbonyl formation, nitric oxide (NO) production were examined in the splenocytes. ResultsIn vitro NFJ incubation of splenic lymphocytes increased Con A-induced lymphocyte proliferation, IL-2 and IFN-γ production, and expression of p-ERK, p-Akt, and p-CREB in young and old rats. In vivo treatment of old rats with NFJ increased lymphoproliferation, IL-2 and IFN-γ production, the expression of p-TH, NGF, and NO production, and suppressed IL-6 production, lipid peroxidation, protein carbonyl formation, and the expression of IκB-α and p-NF-κB (p50) in the splenocytes. ConclusionTaken together, these results suggest that Morinda citrifolia fruit juice enhanced neural-immune interactions and cell survival pathways while inhibiting inflammatory processes that may be useful in the treatment of age-associated diseases.

Estrogen modulates neural–immune interactions through intracellular signaling pathways and antioxidant enzyme activity in the spleen of middle-aged ovariectomized female rats

Modulation of neural–immune interactions by estrogen in the spleens of ovariectomized (OVX) middle-aged female rats was examined. Con A-induced lymphoproliferation, splenic tyrosine hydroxylase (TH) and nerve growth factor (NGF) expression, levels of p-ERK 1/2, p-CREB, and p-Akt, and activity of superoxide dismutase decreased in OVX rats while estrogen treatment enhanced their expression, levels, and activity. Also, estrogen treatment enhanced Con A-induced IFN-γ production and decreased Con A-induced IL-2 production compared to OVX animals. In contrast, estrogen increased the extent of lipid peroxidation and protein carbonyl formation while OVX induced a decline in protein carbonyl formation. These results suggest that estrogen enhances neural–immune interactions while simultaneously affecting it through generation of free radicals as reflected by increased lipid peroxidation and protein carbonyl formation.

Diverse age-related effects of Bacopa monnieri and donepezil in vitro on cytokine production, antioxidant enzyme activities, and intracellular targets in splenocytes of F344 male rats

Aged people are more prone to developing neurodegenerative and infectious diseases, autoimmune disorders, and cancer due to impairment of neuroendocrine-immune functions. Neuronal degeneration and immunosuppression aided by increased generation of reactive oxygen species combined with loss of antioxidant enzyme activities promote the aging process. Bacopa monnieri (brahmi), an Ayurvedic herb, and donepezil, a reversible acetylcholinesterase inhibitor, have been used to reverse cognitive dysfunctions in several neurodegenerative diseases. The aim of this study was to investigate the effects of in vitro incubation of lymphocytes from spleens of young (3-month-old), early middle-aged (8- to 9-month-old), and old (18-month-old) F344 rats with brahmi (0.001%, 0.01%, 0.05%, 0.1%, and 1%) and donepezil (5, 10, 25, 50, and 100μg/ml) on Concanavalin (Con A)-induced proliferation of T lymphocytes and cytokine production, and the activities of antioxidant enzymes [superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione-S-transferase (GST)]. In addition, the effects of these compounds on the expression of intracellular signaling pathway markers (ERK, p-ERK, CREB, p-CREB, Akt and p-Akt), nitric oxide (NO) production, and the extent of lipid peroxidation were measured in the splenocytes. Age-related decline in Con A-induced proliferation of T lymphocytes was not reversed by treatment with brahmi and donepezil but donepezil alone further reduced the lymphocyte proliferation in young rats. Lower doses of brahmi treatment reversed the age-related decrease in Con A-induced IL-2 and IFN-γ production by the splenocytes while their production by splenocytes was suppressed by treatment with donepezil in the young and early middle-aged rats. An age-associated decline in the activities of SOD, CAT, GPx, and GST was evident in the lymphocytes of spleen. Brahmi enhanced CAT activity of lymphocytes in all the age groups while donepezil increased SOD activity in old rats. Both brahmi and donepezil increased GPx and GST activities in a dose-dependent manner in the lymphocytes of all age groups. There was an age-related decline in NO production and increase in the extent of lipid peroxidation in the splenocytes. Brahmi and donepezil increased NO production in the lymphocytes of early middle-aged and old rats. Brahmi reversed the age-related increase in lipid peroxidation in the splenocytes of both early-middle-aged and old rats while donepezil suppressed lipid peroxidation only in the splenocytes of old rats. The expressions of p-ERK1/2 and p-CREB in the splenocytes were elevated following treatment with brahmi and donepezil in the early middle-aged and old rats while age-related decline in p-Akt expression was reversed by treatment of lymphocytes with brahmi alone in early-middle-aged and old rats. Taken together, these results suggest that both brahmi and donepezil exert distinct age-related effects on the cell-mediated immune responses through selective modulation of antioxidant enzyme activities and intracellular targets that may influence the therapeutic efficacy of these drugs in neurodegenerative diseases.

Advanced age negatively influences mesenteric lymph node T cell responses after burn injury

While the pathophysiology of burn injury is well established in young adults, the factors that contribute to pathogenesis and increased death in elderly burn patients are not defined. The purpose of this study is to determine the effects of burn injury on mesenteric lymph node (MLN) T cell responses in young and aged mice. MLN is a cluster of lymph nodes that drains various parts of the intestine and is known to play role in clearance bacteria originating from the intestinal lumen. Results presented here suggest a significant suppression in Con A-induced MLN cell proliferation and IL-2 production in uninjured aged mice compared with uninjured young mice. Following 24 h after injury, although, a significant decrease in lymph node cell proliferation and IL-2 production was observed in both young and aged mice compared with their respective sham-injured animals, the suppression was more in aged mice. In addition we found a reduction in IFN-γ, a Th-1 cytokine by MLN T cells from aged burned mice relative to young burn ( P<0.05) or sham-injured mice ( P<0.01). The Th-2 cytokine IL-4, on the other hand, was significantly increased in both young and aged burn-injured mice MLN T cells compared with their respective sham-injured mice. These results show that burn injury causes a greater suppression in MLN T cells ability to proliferate and a more pronounced shift to Th-2 phenotype in aged mice as compared with young mice. Such decreases in T cell functions may impair MLN's ability to clear the bacterial pathogens originating from intestine and thereby contribute to increased pathogenesis in injured host.

Alterations in T lymphocyte activity following chemical sympathectomy in young and old Fischer 344 rats

In aged Fischer 344 (F344) rats, sympathetic noradrenergic (NA) innervation of the spleen is markedly diminished compared with young rats. To determine if diminished NA innervation can still provide functional signals to splenic T cells, young (3 months old) and old (17 months old) F344 rats were treated with the NA-selective neurotoxin, 6-hydroxydopamine (6-OHDA) to destroy peripheral NA nerve fibers. In 3-month-old rats, no alterations in spleen cell Con A-induced T cell proliferation, IL-2 or IFN-γ production were observed up to 15 days after sympathectomy, when splenic NE was maximally depleted. By 21 days post-sympathectomy, when NE levels had partially recovered, Con A-induced proliferation and IFN-γ production, but not IL-2 production, were reduced in sympathectomized animals. After day 21 post-sympathectomy, no alterations in T cell functions were observed in sympathectomized animals. In 17-month-old rats, spleen cell Con A-induced proliferation and IL-2 production were reduced 5 days after sympathectomy in the absence of changes in CD5 + T cells or IFN-γ production. Desipramine pretreatment, to block 6-OHDA uptake and prevent sympathectomy, completely blocked the 6-OHDA-induced effects, demonstrating that the destruction of NA nerve fibers is required. After day 5 post-sympathectomy, no sympathectomy-induced alterations in Con A-induced T cell functions were observed in old animals. These differences between young and old rats demonstrate that old animals are more susceptible to loss of sympathetic NA innervation, perhaps because compensatory mechanisms are limited. The sympathectomy-induced reduction in T cell proliferation indicates that splenic NA innervation in old animals, though diminished, can exert a positive regulatory influence on T lymphocyte function. Further study of sympathetic neural–immune interactions in the aged rat may provide a means to improve T cell responsiveness in aging.

Effects of l-deprenyl treatment on noradrenergic innervation and immune reactivity in lymphoid organs of young F344 rats

Sympathetic noradrenergic (NA) neuronal activities in the thymus, spleen and mesenteric lymph nodes (MLN) and immune responses in the spleen were examined in young male F344 rats treated daily with 0, 0.25 mg, or 2.5 mg/kg body weight of l-deprenyl, an irreversible monoamine oxidase-B (MAO-B) inhibitor. Rats were treated daily for 1, 15, or 30 days, and sacrificed 7 days after the last deprenyl treatment. Deprenyl treatment increased norepinephrine (NE) content in the spleen without modifying the pattern and density of NA innervation in the splenic white pulp. The concentration of NE was unaltered in the thymus, but it was increased in the MLN of deprenyl-treated rats. One day of treatment with deprenyl decreased splenic NK cell activity while 15 days of deprenyl treatment enhanced splenic NK cell activity. Deprenyl elevated Con A-induced T lymphocyte proliferation following 30 days of treatment, but did not alter spleen cell Con A-induced IL-2 production or the percentage of CD5+ T cells in the spleen. A moderate decrease in the percentage of sIgM+ B cells was observed in the spleens of 15- and 30-day deprenyl-treated rats. These results suggest that deprenyl has sympathomimetic action on sympathetic NA nerve fibers in the spleen; the enhancement of NA neuronal activity may contribute to the modulation of immune responses in the spleen.

Neurokinin type-1 receptor antagonist inhibits enhancement of T cell functions by substance P in normal and neuromanipulated capsaicin-treated rats

Substance P (SP) plays a major role in the regulation of the interaction between immune and nervous systems. SP administration stimulates Con A-induced proliferation of spleen and peripheral blood lymphocytes from normal and neonatally capsaicin treated rats, which correlated with enhanced IL-2 production and expression of activation antigens such as IL-2 receptor α chain (CD25) and RT1B MHC class II molecule. Moreover, SP markedly increased the percentage of CD5 + and CD4 + T lymphocytes in the peripheral blood of capsaicin-treated rats. Concomitant administration of SP with the non-peptide Neurokinin-1 receptor (NK 1R) antagonist SR140333 completely inhibited the SP-mediated augmentation of Con A-induced PBL proliferation and IL-2 production as well as of CD4 + CD25 + and CD4 + RT1B + T cell numbers in normal and capsaicin-treated rats. SR 140333 also blocked the increased percentage of peripheral blood CD4 + T cells induced by SP in capsaicin-treated rats.

L-deprenyl-induced increase in IL-2 and NK cell activity accompanies restoration of noradrenergic nerve fibers in the spleens of old F344 rats

Previously, we have hypothesized a causal relationship between some measures of immunosenescence and the age-related decline in sympathetic noradrenergic (NA) nerve fibers in spleen and lymph nodes of F344 rats. In the present study, we investigated this interrelationship further by measuring NK cell activity, Con A-induced IL-2 production, norepinephrine (NE) concentration, and morphological localization of NA and neuropeptide-Y (NPY) nerve fibers in the spleens of old (21 months old) male F344 rats after 10 weeks of daily treatment with low doses of l-deprenyl, an irreversible monoamine oxidase-B inhibitor, followed by a 9-day wash-out period. NK cell activity and Con A-induced IL-2 production were increased in deprenyl-treated old rats in comparison to untreated and saline-treated old rats. Deprenyl treatment did not alter the percentage of CD5+ T-cells, but moderately increased the percentage of sIgM + B-cells in the spleens of old rats. In addition to changes in immune responses, NE content and the volume density of NA and NPY nerve fibers were partially augmented in the spleens of deprenyl-treated old rats. In a separate study, various concentrations of deprenyl were added in vitro to spleen cells from young and old F344 rats to examine the direct effects of the drug on Con A-induced IL-2 production. In contrast to in vivo treatment, in vitro addition of deprenyl did not alter the Con A-induced IL-2 production by splenocytes from old rats. Together, these results suggest that the ability of deprenyl to enhance certain immune responses are interlinked to the restoration of sympathetic NA and NPY nerve fibers in the spleens of old rats.

Effects of brazilin on induction of immunological tolerance by sheep red blood cells in C57BL/6 female mice

Brazilin was examined for its effects on the induction of immunological tolerance. Brazilin was administered to C57BL/6 female mice for 2 consecutive days before the immunization with high dose SRBC (10(9) cells) which can produce immunological tolerance. Delayed type hypersensitivity, IgM plaque forming cells, ConA induced IL-2 production and mitogen- or antigen-induced proliferation of lymphocytes were measured as evaluation parameters. Administration of brazilin prior to immunization could keep the DTH and IL-2 production almost optimally immunized levels. Brazilin also inhibited the elevation of non-specific suppressor cell activity. ConA induced proliferation of splenocytes in high dose SRBC immunized mice was significantly decreased by pretreatment of brazilin. And this might be one of the reason for augmentation of DTH by brazilin. However, IgM plaque forming cells were not affected by the treatment of brazilin. These results indicate that brazilin prevents the induction of immunological tolerance caused by high dose SRBC by suppressing the elevation of suppressor cell activity and by inhibiting the decrease in IL-2 production in C57BL/6 female mice.

Cytokine-deficient CD8+ Tc1 cells induced by IL-4: retained inflammation and perforin and Fas cytotoxicity but compromised long term killing of tumor cells.

Abstract After antigenic stimulation, naive CD8+ T cells differentiate into cytotoxic Tc1 cells secreting the cytokines IL-2, IFN-gamma, and TNF, which aid their proliferation and effector functions. We have previously shown that IL-4 acts directly on differentiated Tc1 cells to impair subsequent Con A-induced IL-2 production. As IL-4 may be produced in the vicinity of Tc1 cells during normal immune responses, we have further analyzed the short and long term functions of IL-4-treated Tc1 cells. We now show that these cells also have a defect in the synthesis of IFN-gamma, TNF, and IL-10 in response to antigenic stimulation. IL-2 synthesis was the most sensitive, as stimulation of IL-4-treated Tc1 cells with higher numbers of APCs partially restored IFN-gamma, TNF, and IL-10, but not IL-2, synthesis. Injection of allo-specific Tc1 cells into mice expressing the target Ag revealed reduced cytokine synthesis in vivo by IL-4-treated Tc1 cells. Loss of cytokine synthesis did not impair the short term effector functions of Tc1 cells, as they induced adoptively transferred delayed type hypersensitivity in recipient mice and retained both perforin- and Fas-dependent cytolytic mechanisms in vitro. Long term coculture of tumor targets and tumor-specific Tc1 cells indicated that normal Tc1 cells proliferated and killed tumor cells, whereas IL-4-treated Tc1 cells failed to proliferate and hence were unable to curtail the proliferation of tumor cells. These results suggest that IL-4 synthesis in vivo would not affect immediate effector functions of differentiated Tc1 cells, but would compromise immunity by reducing their long term functional capability.

Immunomodulating and articular protecting activities of a new anti-rheumatic drug, TAK-603

We investigated the pharmacological activities of a newly synthesized anti-rheumatic drug, TAK-603. (1) In vivo: In adjuvant arthritic (AA) rats, TAK-603 inhibited the hind paw swelling and the body weight loss. The minimum effective dose was 3.13 mg/kg/day (p.o.). Histological and radiographic studies showed that TAK-603 suppressed the development of synovial lesions and joint and bone destruction. TAK-603 was also effective in AA rats when administered for the first 7 days after the adjuvant injection. It suppressed type IV allergy (25 mg/kg/day, p.o.) but had no effect on type III allergy. It had little effect in acute inflammation, analgesic and antipyretic models. These data suggest that TAK-603 acts on the immune system, especially on cellular immunity. (2) In vitro: TAK-603 suppressed the mitogen-induced proliferation of mouse lymphocytes and the ConA-induced IFN-γ and IL-2 production by rat lymphocytes at 10−7 to 10−5 M. It also significantly inhibited the IL-1 induced extracellular matrix reduction in rabbit chondrocytes. It had no effects on prostaglandin E2 (PGE2) production in rat peritoneal cells. These data show that TAK-603 has the ability to suppress the immune system and protect cartilage from destruction. TAK-603 is expected to be a promising drug for rheumatoid arthritis.

Butyrate enhances the in vitro anti-SRBC (sheep red blood cell) antibody responses in murine splenocytes.

Butyrate at concentrations of 200-600 microM markedly enhanced the in vitro antibody productions against sheep red blood cells (SRBC) in murine splenocytes. However, other saturated short-chain fatty acids, including acetate, propionate and valeric acid, and 4-carbon compounds such as butanol, acetoacetate and beta- and gamma-hydroxybutyrate had no such effects. The presence of butyrate in the early phase of the cell culture was crucial for enhancement of the response. Butyrate also augmented the antibody production in T-cell-depleted splenocytes supplemented with the culture supernatant of concanavalin A (Con A)-stimulated lymphocytes. Interleukin (IL)-2 secreted from splenocytes in response to SRBC was increased by adding butyrate to the culture, but IL-1 secretion was not affected. On the other hand, Con A or lipopolysaccharide-stimulated proliferation of splenocytes was partly depressed by the addition of butyrate, while Con A-induced IL-2 production was not effected. These findings suggest that butyrate may act on T and B cells to promote their differentiation during the process of antibody production.

IL-1, TNF-α and IL-2 Production by Peritoneal and Spleen Cells from Schistosoma mansoni Infected Mice and Its Potentiation by Preimmunization with schistosomal Antigens and Immunostimulants

In the present study we tested the effect of immunization with schistosome derived antigens such as frozen-thawed schistosomula in combination with either BCG, liposomes or liposomal muramyl tripeptide-phosphatidyl ethanolamine (MTP-PE), on the resistance of mice to infection, and on the function of their macrophages and lymphocytes. Immunization with either F-T schistosomula + BCG or F-T schistosomula + MTP-PE and subsequent infection, resulted in a 2–3-fold increase in adherent peritoneal macrophage-mediated schistosomulicidal activity (SCA). Peritoneal and spleen macrophages from immunostimulant treated and/or immunized animals showed a significant increase in LPS triggered TNF-α production, as compared to non-treated controls. The highest increase in TNF-α production was achieved after immunization with either F-T schistosomula + BCG or F-T schistosomula + MTP-PE. LPS triggered IL-1 production was elevated in spleen and peritoneal macrophages from F-T schistosomula + BCG treated mice, and also in spleen macrophages treated with F-T schistosomula + MTP-PE. Only immunization with F-T schistosomula + BCG increased ConA-induced spleen lymphocyte proliferation and IL-2 production. Immunization of mice with F-T schistosomula + BCG also induced protection against parasite infection, while F-T schistosomula + MTP-PE failed to do so. Potentiation of antischistosomal resistance seems to require both macrophage and lymphocyte activation which was achieved only when BCG served as an immunostimulant.

Effects of MPTP and vitamin E treatments on immune function in mice.

The effects of treatment with the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and vitamin E, an antioxidant, on immune functions were examined. Male C57/B1 mice were fed daily with natural vitamin E for 12 weeks, subsequently injected i.p. with MPTP or its vehicle, and sacrificed 1 week later. Control mice received the stripped corn oil vehicle daily, in place of vitamin E. Oral vitamin E feeding increased cerebral vitamin E content by 60% (P = 0.05). However, MPTP attenuated this rise in cerebral vitamin E content when measured 1 week after treatment with the neurotoxin (P = 0.05). MPTP also produced an 80-90% reduction in striatal dopamine content in both the stripped corn oil control group and the vitamin E-treated group (P = 0.0000). One week after MPTP injection, the numbers of peripheral blood lymphocytes and the percent of spleen T-cells, but not B-cells, were decreased in those groups receiving MPTP alone or MPTP plus vitamin E (P less than 0.05 and 0.02, respectively). The Con A-induced IL-2 production of spleen cells was decreased in all treated groups (P less than 0.005). There was no difference in the mitogenic stimulative response to PHA, Con A or LPS. However, the response to PWM was increased in both MPTP and MPTP plus vitamin E-treated groups (P less than 0.05 and 0.001, respectively). On the other hand, the one-way mixed lymphocyte response of the splenocytes from the MPTP-treated group was increased (P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

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-γ 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-γ 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 (Mφ) 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 Mφ. 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 Mφ was equivalent to the proliferation of aged T cells cultured with aged Mφ. 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.

Effects of a novel anti-inflammatory retinoid-like 2,4,6,8-nonatetraenoic acid on the immunological changes associated with adjuvant-induced arthritis

Proliferative responses to the T-cell mitogen, Con A, were markedly suppressed in spleen cells isolated from rats 12–16 days following induction of adjuvant-induced arthritis (AA). These responses were only partially restored following removal of plastic-adherent cells (AC-depleted). Prophylactic treatment of AA rats with a novel anti-inflammatory retinoid-like 2,4,6,8-nonatetraenoic acid, Ro 23-6457, increased mitogen-induced proliferative responses in spleen cells, particularly in AC-depleted cultures. Treatment of AA rats with Ro 23-6457 significantly increased Con A-induced IL-2 production by both unseparated and AC-depleted spleen cells. Although exogenous IL-2 did not restore proliferative responses to Con A-stimulated spleen cells from vehicle-treated AA rats, responses in AC-depleted cells from Ro 23-6457-treated AA rats were further enhanced by the addition of IL-2. Following stimulation with LPS, supernatants from cultures of adherent spleen cells isolated from AA rats contained more IL-1 (expressed as units/ml) than cultures from normal rats. Treatment of AA rats with a high dose of Ro 23-6457 normalised IL-1 levels in these cultures. Treatment of normal rats with Ro 23-6457 had no significant effects on any parameter tested. These data suggest that Ro 23-6457's modulation of certain disease-associated alterations in immune function in AA rats may contribute to its anti-inflammatory activity.

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

Abstract 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.

The effects of pertussis toxin and cholera toxin on mitogen-induced interleukin-2 production: Evidence for G protein involvement in signal transduction

GTP-binding proteins, known as G proteins, play important roles in transducing signals generated by the binding of specific ligands to cell surface receptors. We examined the possibility that a G protein is involved in transducing the concanavalin A (Con A) signal for IL-2 production using a T-cell hybridoma, FS6-14.13, and the bacterial toxins, pertussis toxin (PTX) and cholera toxin (CTX). These toxins are known to interact with and modify the functions of G proteins. High concentrations of PTX (25–50 μg/ml) stimulated IL-2 production in the FS-6 cells in the absence of Con A, presumably due to the ability of its B subunit to crosslink membrane proteins. However, in the presence of Con A, PTX inhibited IL-2 production at concentrations ranging from 0.05 to 50 μg/ml. It is unlikely that this inhibition was due to a competitive interaction between Con A and PTX for binding sites at the cell surface, since high concentrations of PTX only minimally reduced Con A-FITC binding, evaluated by FACS analysis. In addition, concentrations of PTX which were not able to stimulate IL-2 production in the absence of Con A, retained their ability to inhibit IL-2 production in the presence of Con A. These data suggest the involvement of the PTX A subunit in this activity. In support of this possibility, PTX catalyzed ADP-ribosylation of a M r = 41,000-Da protein in FS-6 membranes. This strongly suggests that a PTX substrate is involved in transducing the Con A signal for IL-2 production in FS-6 cells. CTX also inhibited Con A-induced IL-2 production, an effect mimicked by the addition of dibutyryl-cAMP. This suggests that a CTX substrate linked to the adenylyl cyclase-cAMP pathway is probably not involved in transducing the stimulatory Con A signal, but may play a role in downregulating T-cell activation.

The AKR thymoma BW5147 is able to produce lymphokines when stimulated with calcium ionophore and phorbol ester.

We produced the T cell hybridoma D9C1.12.17 by fusing an IL-4-producing T cell clone D9.1Hi with the AKR thymoma BW5147. The resulting hybridoma produced IL-2 as well as IL-4 even though none of the parental cells produced IL-2 after stimulation with Con A. The production of IL-2 was confirmed at the mRNA level by using an S1 nuclease protection assay. Further analysis indicated that Con A-induced IL-2 production was a common phenomenon among T cell hybridomas derived from this fusion. Although BW5147 does not produce detectable lymphokines after Con A stimulation, this line was able to produce IL-2, granulocyte-macrophage colony stimulating factor, and small amounts of IL-3 and IFN-gamma when stimulated with calcium ionophore and phorbol ester. The latter agents are thought to mimic the activating signal(s) delivered through the Ag:MHC TCR. This observation indicates that BW5147 has the ability to produce lymphokines but may lack component(s) which couple the extracellular signal to lymphokine production, and suggests that in T cell hybridomas, part of the spectrum of lymphokines produced may be contributed by BW5147.

Lyt-2+ cells. Requirements for concanavalin A-induced proliferation and interleukin 2 production.

The requirements for inducing Lyt-2+ T cell proliferation in response to concanavalin A (Con A) were examined. Purified Lyt-2+ or L3T4+ spleen cells of C57BL/6 origin were stimulated with Con A and syngeneic macrophages (MO) in the presence of monoclonal antibodies to T cell markers or to polymorphic determinants on major histocompatibility complex molecules, and assessed for the ability to proliferate and to produce interleukin (IL) 2. alpha I-Ab failed to inhibit the Con A response of Lyt-2+ cells at dilutions that significantly inhibited the response of L3T4+ cells. In contrast, alphaKb/Db or alpha Lyt-2.2 specifically inhibited the response of Lyt-2+ cells, but not L3T4+ cells. The ability of alpha Kb/Db and of alpha Lyt-2.2 to inhibit the response of Lyt-2+ cells was dependent upon the concentration of Con A. These data demonstrate that optimal triggering of T cell subsets to proliferate and to produce IL-2 in response to Con A requires interactions with the appropriate restricting major histocompatibility complex molecule. The role of accessory cells in Lyt-2+ Con A-induced proliferation and IL-2 production was also investigated. Purified Lyt-2+ cells and purified L3T4+ cells failed to respond to Con A in the absence of MO. IL-1 reconstituted the response when MO were limiting, but failed to restore the response of either Lyt-2+ or L3T4+ cells when T cells were rigorously purified to remove all MO. These results demonstrate that triggering Lyt-2+ T cells, like L3T4+ T cells, requires accessory cells, and that this does not merely reflect a requirement for IL-1 production. Thus, Con A-induced proliferation and IL-2 production by Lyt-2+ T cells requires intimate contact with accessory cells and interactions dependent upon the class I-restricting element.