Impaired cytosolic free calcium response in splenic T-cells from mice fed with ethanol-containing diet

Calcium-dependent signal transduction pathways of T-cell proliferation have been extensively studied in the past years. However, little is known about effects of ethanol on the calcium-dependent signal transduction pathway in T-cell proliferation. Thus, a murine model was used to determine effects of ethanol in vivo on T-cell proliferation and the intracellular free calcium concentration [Ca²⁺]_i in response to Concanavalin A (Con A) and recombinant IL2 (rIL2) in T-cells. Splenic cells from young C57BL/6 mice, that had been fed on 3 different diets (ethanol-, maltose substitute- and standard liquid-diet) for 7–8 weeks were tested for their proliferative responses to Con A and rIL2. Concurrently, measurement was also made of [Ca²⁺]_i in the nylon-wool-enriched resting T-cells induced by Con A and in Con-A-activated blast T-cells induced by rIL2. Our results showed that [Ca²⁺]_i increases were seen in the splenic T-cells from three different groups of mice following Con A, but not rIL2 stimulation. However, this increase was much smaller in the splenic T-cells from ethanol-fed mice as compared to mice on maltose- or standard-diet. Furthermore, we also demonstrated that the impaired [Ca²⁺]_i increase was seen in the T-cells of the same ethanol-fed mice having decreased the proliferative response to Con A. This reduced proliferation did not result from the presence of excessive suppressor T-cell activity. Finally, we also demonstrated that both the number of IL2 binding sites/cell and the K_d values of the low- and high-affinity IL2R on the T-cells from ethanol-fed mice were unaltered. Because evidence indicates that (1) a normal level of [Ca²⁺]_i increase is a prerequisite for the production of IL2 by mitogen-stimulated T-cells, and (2) T-cells from ethanol-fed mice have normal capacities to produce IL2 that is the crucial growth factor controlling T-cells to progress through the cell cycle, these lines of evidence taken together with the results of this study suggest that the impairment in [Ca²⁺]_i increases in T-cells from ethanol-fed mice may not be the primary factor contributing to the diminished T-cell proliferation in the same mice.     

Impaired calcium mobilization and differential tyrosine phosphorylation in intestinal intraepithelial lymphocytes.

Intestinal intraepithelial lymphocytes (iIEL) exhibit a unique activation state characterized by the expression of activation markers and effector functions, but a minimal response to mitogenic signals in vitro. To further characterize this activation status, iIEL were compared with splenic T cells for two key activation signals, calcium mobilization and tyrosine phosphorylation. Calcium mobilization was impaired in iIEL treated with the calcium ionophores ionomycin or A23187, thapsigargin, or by CD3-cross-linking. The calcium mobilization defect is shared by mature and embryonic iIEL. Anti-phosphotyrosine Western blot analysis revealed that the iIEL are able to respond to T-cell receptor (TCR)-mediated signals by tyrosine phosphorylation, although the patterns of phosphorylation differ from those seen in splenic T cells. We conclude that iIEL are unable to mobilize calcium in vitro, which may be due to modulation of TCR-mediated signal transduction pathways by the microenvironment of the intestinal epithelium and/or caused by the standard isolation procedure used to prepare iIEL, which must be considered in future in vitro studies of iIEL function.     

In vivo mutation in gene A of splenic lymphocytes from phiX174 transgenic mice

Single-burst analysis was applied to a forward assay for gene A mutation in splenic lymphocytes of phiX174 transgenic mice for the purpose of optimizing analytical parameters for identifying in vivo mutations. The effect of varying the cutoff value for an in vivo burst on induced mutant frequency, fold increase, and the significance of the difference between control and N-ethyl-N-nitrosourea (ENU)-treated mice was calculated by two different methods. The plating density was reduced to an average of less than 10 background mutant plaques per aliquot in order to separate in vitro bursts. The spectrum of mutations contributing < 60 plaques per aliquot from control animals was not significantly different from the control spectra from E. coli or transgenic phiX174 cells in culture. The mutant spectra from ENU-treated animals was highly different between mutant bursts of > 80 plaques per aliquot compared to mutations contributing < 60 plaques per aliquot (P < 0.000001), the former fitting the spectrum expected for ENU-induced mutations. The latter spectrum was also different from control animals and E. coli (P < 0.000001), suggesting the difference was caused by ex vivo mutation. With the mutations found in this study, the total number of reported target sites for gene A is now 33. The results support the interpretation that, in contrast to results for the lacI transgene, 100% of mutants isolated in gene A from control animals and cells were fixed in E. coli. We attribute the difference between the two genes to hot-spot sites for mutation in gene A and to a testable hypothesis that the mosaic plaque assay for the lacI transgene underestimates the frequency of ex vivo mutants.     

束缚应激小鼠脾淋巴细胞功能的改变

目的：探讨束缚应激（IMS）对小鼠脾淋巴细胞功能的影响。方法：用MTT法检测细胞增殖，用流式细胞术检测膜IL－2受体（mIL-2R)和细胞增殖周期，用荧光指示剂Fura-2/AM检测细胞内[Ca^2 ]，将IMS后的结果与正常组进行比较。结果：IMS后细胞增殖反应的最低点在IMS结束后0，24h后接近正常，在IMS后0h,mIL-2R较正常组显著降低，细胞增殖可能受阻于G2／M期，细胞内[Ca^2 ]显著增高（P＜0.05）。结论；IMS可以引起淋巴细胞功能障碍，从而导致免疫抑制。     

Alterations in the proliferative response of T cells from aged and chimeric mice

Factors involved in the nonspecific and specific proliferative responses of T cells were examined in the A/J strain of mice that were physiologically aged in the specific pathogen-free condition. In aged A/J mice, the production of IL-1 and IL-2 from optimally stimulated peritoneal macrophages and splenic T cells was significantly decreased, while the production of IL-3 by T cells was increased. T cells from old mice showed a higher threshold for concanavalin A stimulation for the induction of IL-2 receptors. The same T cells responded only slightly in the mixed lymphocyte reaction (MLR) stimulated by allogeneic class II antigens, while they showed a high background stimulation without stimulators. The addition of syngeneic accessory cells did not increase the proliferative response of T cells from aged animals. T cells from bone marrow chimeras constituted by transferring stem cells from young A/J into irradiated old B10.A mice (young A/J----old B10.A chimeras) showed a pattern very similar to that of old A/J T cells, whereas those from the old A/J----young B10.A chimeras exhibited a response pattern comparable to that of normal young A/J cells. These results indicated that the proliferative responses in aged animals are abnormal, partially due to the alterations in the interleukin cascade. Some changes are probably due to the effect of environment where T cells undergo early differentiation, because bone marrow stem cells from aged mice can generate T cells with normal functions when differentiated in the young environment.     

Mitogenic responses of splenic B and T lymphocytes in neonatally capsaicin-treated mice

The proliferative response of spleen cells from neonatally capsaicin-treated mice to 5 mitogens (LPS, PWM, dextran sulfate, ConA and PHA) were tested. Both B- and T-lymphocyte responses were essentially unaffected by the capsaicin treatment, which is known to destroy certain small-sized neuropeptide containing primary sensory neurons. However, the capsaicin-treated mice displayed a shift in the dose response to PHA so that the maximal response was obtained with a lower dose of the mitogen. The results exclude major effects of the affected sensory neurons on the development or expression of immune competence, but point to a possible modulatory effect on some step in the response to PHA.     

Depression of proliferative response to fetal lymphocytes of retroplacental blood lymphocytes in human pregnancy

Retroplacental blood lymphocytes (RPL) in human pregnancy were studied for proliferative response to related fetal lymphocytes and it was compared with that of peripheral blood lymphocytes (PBL) of the same donor. RPL proliferated poorly to related cord blood lymphocytes in 9 of 10 cases when compared with autologous PBL. RPL also showed lower proliferative response to unrelated adult lymphocytes in 7 of 12 cases. Proliferation of RPL was weaker to related cord blood lymphocytes than to allogeneic adult lymphocytes. These results indicate that the response of T cells at feto-maternal interface is impaired, especially to fetal antigens.     

The role of non-specific lymphocytes in antigen-induced proliferative response in vitro

The antigen-induced proliferative response of lymph node cells from immunized mice is proportional to the number of primed lymphocytes in the microtiter wells. Addition of normal lymphocytes did not alter the magnitude of the antigen-specific [³H]TdR uptake of immune lymph node cells. In contrast, normal lymphocytes increased the antigen-specific thymidine incorporation of enriched populations of antigen-specific lymphocytes. This enhancing effect was especially pronounced, and proportional to the number of supplementing unsensitized lymphocytes, with a small cell number of enriched lymphocytes, where the specific responsiveness could barely be detected without addition of normal lymphocytes.Enriched populations derived through adherence to antigen-pulsed macrophages (‘selected’ cultures) as well as those obtained by growing immune lymph node cells with antigen-pulsed macrophages (‘supernatant’ cultures) had an improved proliferative response after addition of normal lymphocytes. However, the proliferative response of the ‘selected’ cultures was extremely dependent on normal lymphocytes as they could express only 10% of their full stimulatory capability in the absence of the latter. This indicates that the blastogenic signal delivered by the activated specific T cells recruits normal lymphocytes which do not adhere to the antigen-pulsed macrophages. Under the same conditions the recruiting signal is incapable of inducing the multiplication of antigen-sensitized cells.The potential recruitable uncommitted lymphocytes are present in excess among immune lymph node cells, while depleted from the ‘selected’ cultures. The enriched ‘supernatant’ cultures seem to contain considerable numbers of recruitable lymphocytes although they are present in quantities less than are required to provide for the full amplifying signal.This meaning of these findings for the evaluation of enrichment of antigen-specific T cells and of antigen-induced response based on [³H]TdR uptake is discussed.     

Transfer of immunity to cryptococcosis by T-enriched splenic lymphocytes from Cryptococcus neoformans-sensitized mice.

Splenic enriched T-cells and sera were obtained from inbred CBA/J mice injected 7 or 35 days earlier with either 10(3) viable Cryptococcus neoformans or sterile physiological saline. The transfer of enriched T-cells collected 7 days after immunization or of normal enriched T-cells did not transfer immunity to C. neoformans or delayed-type hypersensitivity responsiveness to cryptococcal culture filtrate (CneF) antigen to the recipients. However, enriched T-cells harvested 35 days after immunization, when transferred to recipient mice, were able to confer immunity as indicated by the reduction in numbers of C. neoformans cells in the tissues, and they also transferred delayed-type hypersensitivity responsiveness to CneF antigens. Sera from either sensitized or normal mice were unable to transfer immunity to recipient animals. These results suggested that there was a time requirement for development of the immune response in the donor mice and that T-cells were crucial in the host defense against a cryptococcal infection. Culturing of day-35 C. neoformans-sensitized T-cells in the presence of homologous antigen (CneF) but not in the presence of heterologous antigen (purified protein derivative or 2, 4-dinitro-1-fluorobenzene) induced the production of migration inhibition factor, thus indicating that lymphocytes from C. neoformans-injected mice were specifically sensitized to CneF antigen.     

Dipyrone attenuates acute sickness response to lipopolysaccharide in mice

Sickness behavior appears to be the expression of a central motivational state that reorganizes the organism's priorities to cope with infectious pathogens. To evaluate the effect of dipyrone in lipopolysaccharide (LPS)-induced sickness behavior, mice were subjected to the forced swim test (FST), tail suspension test (TST), dark-light box test, open field test, sucrose preference intake test and food intake test. LPS administration increased the immobility time in the TST, increased the time spent floating in the FST, and depressed locomotor activity in the open field test. Treatment with LPS decreased the total number of transitions made between the dark and light compartments of the apparatus and induced anhedonia and anorexia. Pre-treatment with dipyrone (10, 50, or 200 mg/kg) attenuated behavioral changes induced by LPS in the FST, TST, open field and light-dark box tests. In addition, dipyrone prevented anhedonia and anorexia in mice challenged with LPS. Considering that dipyrone attenuates LPS-induced behavioral changes, it is proposed that LPS-induced sickness behavior is dependent on the COX pathway.     

Increased proliferative response of lymphocytes from intestinal lymph during long chain fatty acid absorption.

The effect of long chain and medium chain fatty acid absorption on transport and mitogen-induced blast transformation of lymphocytes in intestinal lymphatics was investigated. Intestinal lymph was collected from mesenteric lymph duct cannulated rats maintained in Bollman's cage. Following the intraduodenal administration of oleic acid (long chain fatty acid) or octanoic acid (medium chain fatty acid), only oleic acid produced a significant increase in lymphocyte flux and enhanced proliferative response of lymphocyte in intestinal lymph, without significant alteration of lymphocyte subsets. These changes appeared to be closely correlated with the appearance of radiolabelled oleic acid. Absorption of a medium chain fatty acid, octanoic acid, most of which appeared to be transported to portal blood, did not produce a significant elevation of lymphocyte flux or increased proliferative response of lymphocyte in intestinal lymph. Pluronic L-81, which is a potent inhibitor of the intracellular formation and secretion of chylomicron, significantly attenuated the increased lymphocyte flux and suppressed the enhancement of lymphocyte responsiveness to PHA in intestinal lymph after stimulation by oleic acid administration. There is a possibility that lymphocyte transport and proliferative response in intestinal lymph during oleic acid absorption are closely related to the process of chylomicron formation and secretion to lymphatics in the intestinal mucosa.     

Accessory cell requirement in the proliferative response of T lymphocytes to hemocyanin

T cells were obtained from mice immunized to keyhole limpet hemocyanin (KLH) and tested in culture for their proliferation to KLH or to concanavalin A. The T-cell proliferation to KLH depended on the presence of Ia-bearing macrophages. This was shown only after extensive manipulations of T cells depleting accessory cells by adherence to dishes and passage through nylon-wool columns. The macrophage dependency required homology with macrophages from strains of mice sharing the left side of H-2. The macrophage dependency was not bypassed by conditioned media from macrophages. In contrast, the proliferative response to concanavalin A, which was also macrophage dependent, showed no H-2 restriction and could be replaced by a macrophage-conditioned medium. Nonadherent spleen cells, extensively devoid of macrophages, could substitute for macrophages but at a hundredfold excess. Cells isolated in the fluorescence-activated cell sorter with anti-Ig reagents also showed an antigen-presenting function. We raise the question whether our results imply an antigen-presenting function of B cells.     

Analysis of splenic lymphocytes with high electrophoretic mobility in adult and aged nude mice

Electrophoretic mobility (EPM) and surface markers of splenic lymphocytes in adult (8 weeks old) and aged (over 1 year old) nude mice were investigated. Splenic lymphocytes in nude mice showed a bimodal pattern consisting of low mobility lymphocytes (LML) corresponding to B cells and high mobility lymphocytes (HML). The HML of nude mice showed the following immunological characteristics: (1) surface Ig- cells; (2) asialo GM1+ cells; (3) an increase in natural killer (NK) activity after depletion of B cells; (4) abrogation of the HML peak and NK activity after treatment with anti-asialo GM1 and complement. These findings suggested that HML in nude mice were NK cells. The mobility of NK cells was slightly lower than that of T cells in normal mice, although their histograms greatly overlapped each other. In the spleen cells of nude mice, there was a significant increase in the numbers of Thy-1+ cells and a decrease in the intensity of asialo GM1 antigen as a function of age. The surface markers of HML were Thy-1+- asialo GM1++ in adult nude mice, but were Thy-1+ asialo GM1+ in aged nude mice. However, although HML in aged nude mice became Thy-1+, these had almost the same EPM as those in adult nude mice.     

Rotavirus induces proliferative response and augments non-specific cytotoxic activity of lymphocytes in humans.

In vitro cell-mediated immune responses to rotavirus in humans were studied. Peripheral blood mononuclear cells (PBMC) of healthy adults proliferated in response to stimulation with the infectious and u.v.-inactivated Wa strain of human rotavirus, showing a maximum response on day 7 of culture; however, cord blood lymphocytes failed to respond to rotavirus. A cross-reactive proliferative response of PBMC detected by stimulation with the NCDV strain of bovine rotavirus suggests the existence of epitopes common to both human and bovine rotaviruses, which are recognized by human T lymphocytes. The phenotype of the majority of activated lymphocytes was CD3+4+8-, indicating that the cells mainly activated were helper T cells. Culture supernatants of PBMC stimulated with rotavirus contained interleukin-2 (IL-2) and interferon-gamma (IFN-gamma). In addition, PBMC stimulated with rotavirus demonstrated significantly enhanced cytotoxic activity against natural killer (NK) sensitive K562 cells as well as an NK-resistant Epstein-Barr virus-immortalized lymphoblastoid cell line (LCL). Treatment of PBMC with anti-CD16 or NKH1A monoclonal antibody, both of which react with most NK cells and lymphokine-activated killer cells and complement markedly reduced the cytotoxic activity against K562 and LCL. These results suggest that stimulation of human PBMC with rotavirus results in the production of lymphokines, such as IL-2 and IFN-gamma, by rotavirus-reactive helper T cells and that these lymphokines augment NK activity and generate other forms of non-specific cytotoxic human lymphocyte activity. These cell-mediated immune responses observed in the present in vitro study might play an important role in protection and recovery from rotavirus infection.     

Cell-mediated immune phenomena induced by lymphokines from splenic lymphocytes of mice with chronic staphylococcal infection.

Splenic lymphocytes from normal mice and from mice displaying delayed hypersensitivity to Staphylococcus aureus were cultured in the presence or absence of specific staphylococcal antigens. The cell-free supernatant fluids from these lymphocyte cultures were assessed for their ability to alter the functional capacities of normal macrophages. It was found that supernatants from staphylococcus-immune cells cultured in vitro with antigen possessed migration inhibitory factor activity and also were capable of stimulating the incorporation of [14C]glucosamine into macrophage membrane glycoproteins. In addition, the lymphokine-containing supernatants were capable of inducing activation of normal macrophages so that they inhibited the multiplication of intracellular Listeria monocytogenes. Although it was not possible to snow any significant enhancement of intracellular killing of S. aureus by the activated macrophages, evidence is presented that suggests that cell-mediated immune responses to S. aureus may significantly enhance pahgocytosis of staphylococci and, thereby, may provide for their rapid clearance from extracellular fluids.     

The participation of thymus-derived and of bone marrow-derived lymphocytes of sensitized mice, in the proliferative response to specific antigen, in vitro

Spleen cells of mice primed with sheep red cells (SRC) responded with increased thymidine uptake when stimulated with SRC in vitro. Depletion of bone marrow-derived (B) cells by filtering the spleen cells through columns of plastic beads coated with anti-mouse immunoglobulin serum resulted in a drastic loss of the proliferative response. A similar loss occurred on depleting the thymus-derived (T) cells by treatment with anti-Θ serum and complement. Addition of peritoneal exudate cells failed to restore the reactivity of the purified T cells to SRC. The proliferative response could be restored to a level exceeding the sum of the individual responses, by mixing the purified T with the purified B cells. A synergistic interaction between T and B cells is suggested.     

Reactivity of mouse splenic B lymphocytes to adrenalin during the immune response

Bulletin of Experimental Biology and Medicine -     

Antigen-presenting capacity of guinea pig B lymphocytes in T cell proliferative response in vitro

The capacity of normal (unprimed) B cells and keyhole-limpet-hemocyanin(KLH)-primed B cells to present the antigen KLH to KLH-primed T cells in the guinea pig was examined with in vitro assay of lymphocyte proliferative response. Antigen-pulsed B-lymphocyte population, extensively devoid of macrophages (M phi), induced the proliferative response of KLH-primed T lymphocytes, although less effectively than the peritoneal M phi. The antigen presentation was antigen-specific. There was no substantial difference between the magnitude of T-cell response induced by KLH-primed B-cell population and that stimulated by unprimed, normal B-cell population. The antigen-presenting capacity of the B-cell population was abrogated by pretreatment with anti-guinea pig immunoglobulin (Ig) or anti-I-region-associated (Ia) antigen antiserum and complement, whereas it was not affected with anti-guinea pig M phi antiserum and complement. From studies using strain 2 and strain 13 guinea pigs, histocompatibility requirement between antigen-pulsed B cells and antigen-reactive T cells was suggested: B lymphocytes, as well as M phi, did elicit proliferative response to specific antigen, KLH or purified protein derivative of tuberculin (PPD), in syngeneic, but not in allogeneic, T cells. These results suggest that the Ia-positive, surface-Ig-bearing guinea-pig B lymphocytes - not only KLH-primed B cells but also unprimed B cells - are capable of presenting antigen to primed T cells in a major histocompatibility complex(MHC)-restricted fashion.