Ethanol inhibits mitogen-induced calcium mobilization in mouse splenocytes.

Ethanol inhibited the mitogen-induced initial increase in cytoplasmic free-calcium [Ca2+]i in mouse splenocytes. This effect was concentration-dependent, reversible, and observed at pharmacologically relevant concentrations (24-166mM). Other short-chain alcohols such as propanol, butanol, and pentanol also inhibited this mitogen-induced increase in [Ca2+]i. The potencies of these alcohols to produce this effect were highly correlated (r = 0.98, p less than 0.001) with their membrane/buffer partition coefficients. Analysis of mouse splenocyte subpopulations demonstrated that this effect was manifest in both B and T lymphocytes. Within T lymphocyte subpopulations, both CD4+ and CD8+ T cells were affected. These results suggest that the inhibition of [Ca2+]i increase may be an early event mediating ethanol-induced immunosuppression and that this may be a predisposing factor to infection and malignancies associated with alcoholism.     

Stress modulates calcium mobilization in immune cells

Both acute and chronic restraint stress modulated mitogen-induced increases in cytoplasmic free-calcium concentrations ([Ca2+]i) in mouse spleen cells. Dual-color analysis of lymphocyte subpopulations demonstrated that acute (2 hour) restraint stress suppressed mitogen-stimulated increases in [Ca2+]i in CD4+ T cells, but enhanced [Ca2+]i in CD8+ T cells. Chronic restraint stress (2 hours daily for up to 21 days) resulted in a significant suppression of mitogen-stimulated increases in [Ca2+]i in CD4+ T cells at 3 and 7 days, but not at 21 days. CD8+ T cells were unaffected by chronic stress. Chronic stress (for 7 days) had a modest suppressive effect on mitogen-induced Ca2+ responses in B cells. Within T lymphocyte subpopulations, both acute and chronic stress predominantly affected CD4+ T cells, which may induce a functional reversal of the CD4/CD8 ratios in vivo. Such a reversal could result in suppression of a variety of immune responses such as lymphocyte proliferation and antigen-specific antibody production. These findings indicate that the inhibitory effects of stress on calcium mobilization in lymphocytes may be an early event mediating stress-induced immunosuppression.     

Murine lymphocytes exhibit heterogeneity in their proliferative responsiveness to calcium ionophore.

The calcium ionophore, A23187, when used alone was found to induce proliferation of murine T cells, at concentrations of 0.5-1 mM. This response required the presence of syngeneic splenic adherant cells (SAC) as a source of accessory cells. Interestingly, only CD4+ T cells but not CD8+ T cells or B cells responded to the calcium ionophore by proliferation. The inability of CD8+ T cells or B cells to respond was not related to decreased elevation in the intracellular ionized calcium [Ca2+]i concentration induced by the ionophore, because activated CD4+ T, CD8+ T and B cells all exhibited similar elevation in [Ca2+]i. The inability of CD8+ T cells to respond to calcium ionophore was probably due to insufficient production of autocrine growth factors, such as IL-2, inasmuch as the addition of exogenous IL-2 could completely restore the CD8+ T cell responsiveness. Also, exogenous rIL-1 could partially restore purified T cell response to calcium ionophore, whereas, rIL-6 failed to do so. IL-2, but not IL-4, acted as an autocrine growth factor for T cells responding to the calcium ionophore in the presence of SAC, since, antibodies against IL-2 or IL-2 receptor (IL-2R) but not against IL-4, could inhibit the T cell proliferation. Furthermore, exogenous rIL-2 but not rIL-4 supported the proliferation of T cells to calcium ionophore in the absence of accessory cells. Our results suggest that murine lymphocytes exhibit heterogeneity in their proliferative responsiveness to calcium ionophore and that this may not depend on the early activation signal such as the elevation in [Ca2+]i) induced by the ionophore but may depend on subsequent signals which regulate endogenous growth factor production.     

Influence of aging on intracellular free calcium and proliferation of mouse T-cell subsets from various lymphoid organs.

The influence of aging on T-cell activation and proliferation was examined in lymphocytes derived from peripheral blood, spleen, and lymph nodes of WBB6F1 C57B1/6J x WB/Re) mice. Following activation with anti-CD3 monoclonal antibodies, the greatest age-related changes were seen in CD4+ cells derived from spleens of 27- to 30-month-old mice. These CD4+ lymphocytes showed reduced [Ca2+]i signaling and decreased proliferation in the presence of exogenous interleukin 2. CD8+ cells from spleens of old animals showed reduced [Ca2+]i but not altered proliferation. Both CD4+ and CD8+ cells derived from peripheral blood of old mice showed decreased peak [Ca2+]i, but no defect in cell proliferation. In contrast, age-related deficits in either [Ca2+]i or proliferation were not observed in CD4+ and CD8+ cells from lymph nodes. Additionally, the percentage of CD4+ cells was decreased in all lymphoid organs from old mice, while the percentage of CD8+ cells was similar in lymphoid organs of old and young mice. Old mice had a significant increase in expression of Pgp-1 in CD4+ cells from spleen and peripheral blood and CD8+ cells derived from lymph node. Our studies indicate that there are differential effects of aging in T lymphocytes derived from different lymphoid organs in mice. Among the cell sources and subsets examined, the age-related changes noted in CD4+ cells from mouse peripheral blood were the most similar to those previously observed in the corresponding peripheral blood lymphocyte subset in humans.     

Heterogeneity among T cells in intracellular free calcium responses after mitogen stimulation with PHA or anti-CD3. Simultaneous use of indo-1 and immunofluorescence with flow cytometry

Measurement of intracellular ionized calcium concentrations ([Ca2+]i) has been indispensable in elucidating the central role of [Ca2+]i as a trigger of cellular responses to activating stimuli. Such studies have employed the dye quin2, which has not been readily adapted to analysis of individual small cells. We show here that the calcium response of large numbers of single cells can be analyzed with the use of flow cytometry and the recently described dye, indo-1. Such analyses demonstrate for the first time the heterogeneous nature of the [Ca2+]i response to mitogenic stimuli within populations of peripheral blood lymphocytes (PBL). By simultaneous quantitation of one- or two-color surface immunofluorescence labels, some of this heterogeneity of [Ca2+]i response in PBL is shown to be related to cellular immunophenotype. Almost all T cells responded to anti-CD3 antibody; however, the response is greater among CD4+ than CD8+ cells, and within the CD4+ population the rate of response to stimulation by antibody to CD3 differed between subpopulations defined by expression of the common leukocyte marker p220. In contrast, not all T cells responded to phytohemagglutinin (PHA), even at very high doses. As with anti-CD3, after stimulation with PHA, CD4+ cells showed a larger proportion of responding cells than did CD8+ cells. In separate experiments, indo-1 was found not to impair reproductive viability of PBL, thereby providing the potential for analysis of functional activity after the separation of cells by sorting on the basis of the [Ca2+]i response to stimuli. Mixing experiments indicated that a response of a subpopulation representing as little as 0.3% of total cells could be readily detected. Thus, the flow cytometric assay with indo-1 is the first technique that allows the quantitative analysis of response differences of small subpopulations of cells and intercellular variation in [Ca2+]i.     

The effect of endotoxemia on concanavalin A induced alterations in cytoplasmic free calcium in rat spleen cells as determined with Fluo-3.

The effects of acute (3 h) and chronic (30 h) in vivo infusions of Escherichia coli endotoxin on the Ca2+ homeostasis of rat spleen cells was investigated. Conditions were established for obtaining reliable estimates of [Ca2+]i in these cells using the newly-developed Ca2+ indicator Fluo-3. The resting [Ca2+]i of splenocytes and T lymphocyte-enriched preparations were 119 +/- 35 and 102 +/- 31 nM, respectively. Treatment of the cells with concanavalin A (Con A) resulted in a rapid increase in [Ca2+]i. The magnitude of the increase was positively correlated with the concentration of Con A, whereas the time required to reach the maximum [Ca2+]i was inversely related to the amount of Con A. The peak [Ca2+]i was attained more rapidly in splenocytes (i.e. less than or equal to 30 s) than in the T cell-enriched fraction (i.e. 1.5-2.0 min). Both the resting [Ca2+]i and the Con A-induced increase in [Ca2+]i were similar to values previously reported for other lymphocyte cell types using different Ca2+ indicators, thereby supporting the values obtained with Fluo-3. Infusions of saline or endotoxin prior to the isolation of the cells did not result in significant alterations of either resting [Ca2+]i or the cells' response to Con A. Since chronic infusions of endotoxin have previously been shown to cause a reduction in blastogenic responsiveness of splenocytes to Con A, these data suggest that the endotoxin-induced lesion occurs distal to the mobilization of intracellular Ca2+.     

Patterns of costimulation of T cell clones by cross-linking CD3, CD4/CD8, and class I MHC molecules

The ability of mAb to class I MHC molecules, CD3, or CD4/CD8 to stimulate human T cell clones alone or in combination was examined. Cross-linking each of these surface Ag with appropriate mAb and goat anti-mouse Ig (GaMIg) resulted in a unique pattern of increase in intracellular free calcium ([Ca2+]i) and different degrees of functional activation. Cross-linking class I MHC molecules provided the most effective stimulus of IL-2 production and proliferation. Cross-linking more than one surface Ag induced a compound calcium signal with characteristics of each individual response. Cross-linking CD3 + HLA-A,B,C caused a rapid and prolonged increase in [Ca2+]i and synergistically increased IL-2 production and proliferation of all clones. Cross-linking CD3 + CD4/CD8 also generated a compound calcium signal and increased IL-2 production and DNA synthesis. Purposeful inclusion of CD3 was not required for costimulation as cross-linking HLA-A,B,C + CD4/CD8 also increased [Ca2+]i, IL-2 production, and proliferation. Cross-linking three surface Ag, CD3 + HLA-A,B,C + CD4/CD8, resulted in the greatest initial and sustained [Ca2+]i, IL-2 production, and DNA synthesis. Although there was a tendency for the various stimuli to increase both [Ca2+]i and functional responsiveness, neither the magnitude nor duration of the increased [Ca2+]i correlated with the amount of IL-2 produced or the ultimate proliferative response. To determine whether costimulation required that the various surface molecules were cross-linked together, experiments were carried out using isotype specific secondary antibodies. Augmentation of [Ca2+]i and costimulation of functional responses were noted when class I MHC molecules were cross-linked and CD3 was bound, but not cross-linked. Similarly, costimulation through CD3 and CD4/CD8 was observed when CD4/CD8 was cross-linked and the CD3 complex was engaged by an anti-CD3 mAb which was not further cross-linked. In contrast, costimulation by class I MHC molecules and CD4/CD8 was only observed when these molecules were cross-linked together. These data demonstrate that cross-linking class I MHC determinants or CD4/CD8 provides a direct signal to T cell clones that can be enhanced when CD3 is independently engaged. The results also indicate that T cell clones can be stimulated without engaging CD3 by the combination of signals delivered via class I MHC molecules and CD4/CD8, but only when these determinants were cross-linked together. These studies have demonstrated that these cell surface molecules differ in their capacity to deliver activation signals to T cell clones and also exhibit unique patterns of positive cooperativity in signaling potential.     

Intact antigen receptor-mediated generation of inositol phosphates and increased intracellular calcium in CD4 CD8 T lymphocytes from MRL lpr mice

The predominant T lymphocytes that accumulate in the peripheral lymphoid tissues of mice homozygous for the lpr gene bear the phenotype CD3+CD4-CD8-. By certain functional criteria these cells would appear to have impaired CD3-mediated signal transduction, in that they do not respond to alloantigen and produce little if any detectable IL-2 or other lymphokines. However, the signal pathway appears adequate for achieving other T cell functions, including induction of high affinity IL-2R, and thymic deletion. To clarify the basis of this seeming discrepancy, we examined transmembrane signal transduction in T cell subsets of lpr/lpr (lpr) and +/+ mice, as defined by increased [Ca2+]i and the generation of inositol phosphates (InsPs). Stimulation of lpr CD4-CD8- cells with anti-CD3 antibody produced prompt and sustained increases in the concentration of [C2+]i and in InsPs. Similar responses occurred in mature T cells from lpr and +/+ mice, except for the somewhat slower kinetics of their increased [Ca2+]i. In marked distinction to the anti-CD2-mediated response, Con A, even in high doses, could not stimulate any increase of [Ca2+]i in lpr CD4-CD8- cells, and only modest increases in InsPs. Mature T cells, whether of lpr or +/+ origin, yielded normal increased [Ca2+]i with Con A. The reason for the differences in signal transduction between anti-CD3 and Con A stimulation of lpr CD4-CD8- cells may relate to the absence of surface structures on these immature T cells that are required for activation by Con A but not by anti-CD3. The data demonstrate that the CD3 complex in lpr CD4-CD8- T cells can couple to phospholipase C to hydrolyze phosphoinositides. These activation properties of lpr CD4-CD8- T cells have interesting functional parallels to thymocytes at the time of thymic selection, as well as tolerance induction of mature T lymphocytes.     

Cholera toxin and its B subunit do not change cytosolic free calcium concentration

Using the fluorescent Ca2+ probe Quin-2 it has been reported that cholera toxin (CT) and its B subunit (B-CT) increase cytosolic free Ca2+ concentration ([Ca2+]i) in entherocytes, thymocytes and fibroblasts. In this work we show, however, that the fluorescence increases of Quin-2-loaded cells (rat thymocytes, mouse splenocytes, P-388 macrophages and 3T3 fibroblasts) observed upon addition of CT or B-CT are not caused by an increase in [Ca2+]i. The observed effect appears to be accounted for by EDTA-2Na admixtures (present as conservation agent in all CT and B-CT preparations) which 'unquenches' the fluorescence of Quin-2 acid leaked out from the cells into the extracellular medium and produces influorescent complexes with contaminating heavy metal ions. Thus the mitogenic effect of B-CT is not obviously connected with the cytosolic free Ca2+ increase but is probably due to ganglioside-mediated protein phosphorylation.     

A Na(+)-dependent Ca2+ exchanger generates the sustained increase in intracellular Ca2+ required for T cell activation.

Movement of extracellular Ca2+ is required for the sustained increase in [Ca2+]i necessary for T cell activation. However, the mechanisms mediating mitogen-stimulated Ca2+ movement into T cells have not been completely delineated. To explore the possibility that a Na(+)-dependent Ca2+ (Na+/Ca2+) exchanger might play a role in the mitogen-induced increases in [Ca2+]i required for T cell activation, the effects of inhibitors of this exchanger were examined. Inhibitors of Na+/Ca2+ exchange suppressed the sustained increase in [Ca2+]i stimulated by ligation of the CD3-TCR complex, but did not affect mobilization of intracellular Ca2+ stores. Consistent with the importance of this prolonged increase in [Ca2+]i in T cell activation, Na+/Ca2+ exchange inhibitors, but not inhibitors of the Na+/H+ antiporter, inhibited DNA synthesis stimulated by immobilized anti-CD3 mAb. Inhibition only occurred when the agents were present during the first hours after stimulation. These agents also inhibited IL-2 production, but not expression of the IL-2R or of an early activation Ag, 4F2. Inhibition of IL-2 production did not account for the inhibition of T cell proliferation as addition of exogenous IL-2 or phorbol ester (PDB) did not overcome the inhibition. In contrast, activation pathways that are not thought to require an increase in [Ca2+]i such as IL-1 + PDB or engagement of CD28 in the presence of PDB were less sensitive to the suppressive effects of inhibitors of Na+/Ca2+ exchange. Thus, proliferation induced by these stimuli was not suppressed by low concentrations of these inhibitors and IL-2 production induced by mAb to CD28 + PDB was not inhibited by any concentration of inhibitors of Na+/Ca2+ exchange. These results suggest that stimulation of a Ca2+ transporter with the same spectrum of inhibition as the Na+/Ca2+ exchanger in other tissues mediates the sustained increase in [Ca2+]i required for T cell activation after CD3 ligation.     

Reduced proliferation in T lymphocytes in aged humans is predominantly in the CD8+ subset, and is unrelated to defects in transmembrane signaling which are predominantly in the CD4+ subset

Peripheral blood lymphocytes (PBL) from elderly donors have a reduced proliferative response to phytohemagglutinin (PHA) and anti-CD3 monoclonal antibodies (mAb) compared to those from young donors. To examine whether this is due to intrinsic deficiencies in proliferative potential of T-cell subsets, we compared the growth of unsorted PBL vs sorted CD4+ or CD8+ CD11- cells after anti-CD3 mAb or PHA stimulation. Unsorted PBL of elderly donors (greater than 65 years) showed a significant decrease in proliferation compared to young donors (20-30 years) when stimulated with anti-CD3 mAb or PHA. Sorted CD4+ and CD8+ cells were grown in culture in the absence of accessory cells under optimized growth conditions (CD28 mAb, interleukin 2 and beta-mercaptoethanol present). CD4+ cells from elderly donors showed no reduced growth after anti-CD3 mAb stimulation and only slightly decreased growth after stimulation with PHA. CD8+ CD11- cells from elderly donors, however, showed a 20-30% reduction in the proportion of cells proliferating in response to the mitogens and up to 40% reduction in the rate of cell-cycle progression of the responding cells. We examined whether this reduced proliferation is related to decreased efficiency of signal transduction by comparing this to the mobilization of intracellular free calcium ([Ca2+]i) and calcium channel activity after stimulation with anti-CD3 mAb or PHA. [Ca2+]i was measured in CD4 and CD8 subsets of young and elderly donors using a flow cytometric assay with the dye indo-1. Compared to cells from young donors, CD4+ cells from elderly donors showed a [Ca2+]i response which was up to 26% lower after stimulation with CD3 and 10% lower after stimulation with PHA. This appeared to be related to decreased calcium channel activity in elderly donors, rather than mobilization of intracellular Ca2+ stores. CD8+ cells from elderly donors, however, had a slightly, but significantly, greater [Ca2+]i response to CD3 mAb and PHA than did cells from young donors. Since the age-dependent defect in proliferation is mainly in CD8+ cells, but the [Ca2+]i decline is predominantly in the CD4+ subset, these results suggest that the reduced proliferation of T cells from older donors is not related to decreased efficiency of transmembrane signal transduction.     

Effect of cytochalasins on cytosolic-free calcium concentration and phosphoinositide metabolism in leukocytes

Cytochalasins are routinely used to stimulate a variety of functions in eukaryotic cells even though their precise mode of action remains to be elucidated. In the present work we used the fluorescent Ca2+ indicator quin2 to study the effect of various cytochalasins, cytochalasins A, B, C, D, E (CA, CB, CC, CD, CE) and dihydrocytochalasin B (dhCB) on the intracellular Ca2+ concentration ([Ca2+]i) in various types of leukocytes, viz, neutrophils and lymphocytes. In human neutrophils, cytochalasins increase [Ca2+]i mainly by releasing Ca2+ from membrane-bound, intracellular stores. Thus, in order to readily appreciate the effect of cytochalasins on [Ca2+ )i, these cells must be loaded with low intracellular quin2 concentrations. On the other hand, in peripheral blood lymphocytes, splenocytes and thymocytes, the increase in [Ca2+]i is predominantly due to an increased Ca2+ influx from the extracellular medium. In addition, we found that in neutrophils these drugs prolong the increase in [Ca2+]i induced by chemotactic peptides, probably by increasing the cell permeability to Ca2+. Finally, in thymocytes, cytochalasins potentiate the production of inositol phosphates induced by the polyclonal mitogen concanavalin A (conA).     

N(omega)-nitro-L-arginine decreases resting cytosolic [Ca2+] and enhances heat stress-induced increase in cytosolic [Ca2+] in human colon carcinoma T84 cells

N(omega)-nitro-L-arginine (LNNA) inhibits the synthesis of heat shock proteins in animals and cultured cells exposed to heat stress. Heat shock protein synthesis is known to be Ca2+-dependent. In this study, we have characterized the effect of LNNA on [Ca2+]i before and after heat stress in human colon carcinoma T84 cells. In untreated cells incubated in the presence of external Ca2+, the resting [Ca2+]i was 201+/-3 nM. If these cells were exposed to 45 degrees C for 10 min, [Ca2+]i increased by 50+/-2%. Preincubation with LNNA (100 microM) without subsequent heating led to a decrease in [Ca2+]i in a LNNA concentration-dependent manner. Preincubation with LNNA followed by heating increased [Ca2+]i to levels 88+/-5% greater than cells heated without LNNA pretreatment. Incubating cells in medium without external Ca2+ (no heating, no LNNA treatment) lowered resting [Ca2+]i to 115+/-2 nM and greatly reduced the increase in [Ca2+]i observed if cells were heated in the presence of Ca2+, indicating that external Ca2+ plays an important role in the maintenance of [Ca2+]i in T84 cells. With external Ca2+ absent, LNNA pretreatment further reduced [Ca2+]i in unheated cells, and heating failed to enhance [Ca2+]i. We determined (with external Ca2+ present) that the heat-stress induced increase in [Ca2+]i in T84 cells was blocked by dichlorobenzamil, a Na+/Ca2+ exchanger inhibitor, suggesting that the exchanger mediates Ca2+ entry. The median inhibitory concentration (IC50) in cells not treated with LNNA was 0.970+/-0.028 microM. With LNNA pretreatment, the IC50 was 5.099+/-0.107 microM. Heat stress of T84 cells did not affect the binding affinity of the Na+/Ca2+ exchanger for external Ca2+, but it increased the maximal velocity of the exchanger. In unheated cells, preincubation with LNNA decreased the binding affinity of the exchanger for Ca2+, but after heat treatment, both the binding affinity and maximal velocity of the exchanger increased. Our data are consistent with the idea that LNNA affects the activity of the Na+/Ca2+ exchanger. We also determined there are intracellular Ca2+ pools in T84 cells sensitive to thapsigargin, monensin, and ionomycin. Treatment with TMB-8, a blocker of Ca2+ sequestration and mobilization, or ionomycin inhibited the LNNA-induced decrease in [Ca2+]i observed in the absence of external Ca2+, suggesting that LNNA promotes Ca2+ sequestration.     

The epidermal growth factor-induced calcium signal in A431 cells

Addition of epidermal growth factor (EGF) to human A431 cells causes a 2-4-fold increase in cytoplasmic free Ca2+ concentration ([Ca2+]i) as measured by quin-2 fluorescence. The EGF effect is rapid but transient: [Ca2+]i reaches a maximum within 30-60 s and then returns to its resting value (182 +/- 3 nM) over a 5-8-min period. The EGF-induced [Ca2+]i rise is completely dependent on extracellular Ca2+, is abolished by La3+ and Mn2+, and is not accompanied by changes in membrane potential (mean values of -64 mV). Serum also elicits a transient [Ca2+]i rise in A431 cells, but this response is not dependent on the presence of extracellular Ca2+. The tumor promoter 12-O-tetradecanoylphorbol 13-acetate completely inhibits the EGF- and serum-induced increases in [Ca2+]i without affecting basal [Ca2+]i levels. Our results, together with previous 45Ca2+ uptake data (Sawyer, S. T., and Cohen, S. (1981) Biochemistry 20, 6280-6286), suggest that while serum factors trigger the release of Ca2+ from internal stores, EGF acts by opening a voltage-independent Ca2+ channel in the plasma membrane. The data further suggest a role for protein kinase C in attenuating the Ca2+-mobilizing mechanisms of EGF and serum.     

Muscarinic receptor-mediated increase in cytoplasmic free Ca2+ in isolated bovine adrenal medullary cells. Effects of TMB-8 and phorbol ester TPA

The change in cytoplasmic free calcium, [Ca2+]i in isolated bovine adrenal medullary cells during stimulation by acetylcholine (ACh) in Ca2+-free incubation medium was measured using the fluorescent Ca2+ indicator quin2. ACh (1-100 microM) caused an increase in [Ca2+]i by mobilization of Ca2+ from the intracellular pool. Nicotine (10 microM) did not increase [Ca2+]i in the absence of extracellular Ca2+. Pretreatment of the cells with atropine (10 microM) completely inhibited ACh-induced increase in [Ca2+]i, whereas pretreatment with hexamethonium (100 microM) did not. The intracellular Ca2+ antagonist 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8), inhibited ACh-induced increase in [Ca2+]i. The activator of protein kinase C 12-O-tetradecanoylphorbol-13-acetate (TPA), but not its 'inactive' analog 4 alpha-phorbol-12,13-didecanoate (PDD), also inhibited ACh-induced increase in [Ca2+]i. These findings suggest that in bovine adrenal medullary cells, stimulation of muscarinic ACh receptor causes an increase in [Ca2+]i by mobilizing Ca2+ from the intracellular pool and that protein kinase C is involved in 'termination' or 'down regulation' of this response.     

Comparison of the significance of CD4+ and CD8+ T lymphocytes in the protection of mice against Encephalitozoon cuniculi infection

The role of T lymphocyte subpopulations in the protection against intraperitoneal (i.p.) and peroral Encephalitozoon cuniculi infections was compared in adoptive-transfer experiments using severe combined immunodeficient mice. Whereas CD8+ T cell-depleted, but not CD4+ T cell-depleted, BALB/c splenocytes failed to protect the mice against i.p. infection, only SCID mice reconstituted with both CD4+ T lymphocyte- and CD8+ T lymphocyte-depleted splenocytes succumbed to peroral infection. The results indicate that whereas CD8+ T cells are critical for the protection against an i.p. E. cuniculi infection, both CD4+ and CD8+ T lymphocyte subpopulations play a substantive protective role in a peroral infection, i.e., natural route of infection.     

Contribution of external and internal Ca2+ to changes in intracellular free Ca2+ produced by mitogens in Swiss 3T3 fibroblasts: the role of dihydropyridine sensitive Ca2+ channels

Changes in intracellular free Ca2+ concentration [( Ca2+]i) produced by growth factors and mitogens have been studied using aequorin-loaded Swiss 3T3 cells. Decreasing free Ca2+ in the external medium by using EGTA had no significant effect on the increase in [Ca2+]i produced by vasopressin, bradykinin, bombesin or prostaglandin E2, but reduced the increase in [Ca2+]i produced by platelet derived growth factor (PDGF) by 58%, by prostaglandin E1 44% and by prostaglandin F2 alpha 47%. The dihydropyridine Ca2+-channel antagonist nifedipine at 10 microM inhibited the [Ca2+]i response to PDGF by 41% in both the presence of and in the absence of external Ca2+. Methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl) pyridine-5-carboxylate (BAY K8644), a Ca2+-channel agonist, at 10 microM produced an increase in [Ca2+]i and decreased the [Ca2+]i response to PDGF by 39%. Nifedipine did not block 45Ca2+ uptake or release by inositol 1,4,5-trisphosphate in saponin-permeabilized Swiss 3T3 fibroblasts but BAY K8644 inhibited 45Ca2+ release by inositol 1,4,5-trisphosphate. The results suggest that the increase in [Ca2+]i caused by PDGF in Swiss 3T3 fibroblasts is due to the influx of external Ca2+ through dihydropyridine sensitive Ca2+ channels, as well as release of internal Ca2+.     

Antibody to a novel 95-kDa surface glycoprotein on human B cells induces calcium mobilization and B cell activation

These findings characterize a 95-kDa glycoprotein on the surface of B lymphocytes recognized by the mAb G28-8. This protein (designated Bgp95), previously classified as a CD39 molecule, is unique based on functional, cell distribution, and immunochemical criteria. Biochemical analyses revealed that Bgp95 is a 95-kDa glycoprotein with N-linked carbohydrate and is reduced to about 70-kDa after treatment with endoglycopeptidase F. In functional studies, stimulation by G28-8 mAb or its F(ab')2 fragments induced a G0 to G1 cell cycle transition and was synergistic with PMA, anti-mu, or anti-CDw40 in stimulating proliferation of resting B cells. G28-8 mAb also could induce increases of cytoplasmic free calcium concentration [Ca2+]i in a subpopulation of tonsillar or peripheral blood B cells. The G28-8 mAb alone induced a steady increase in [Ca2+]i detectable even 1 h after stimulation. Cross-linking the G28-8 mAb with a second mAb specific for murine kappa light chains induced a more rapid increase of [Ca2+]i which peaked at 10 to 20 min and then declined. At 1 h after stimulation, [Ca2+]i was higher in B cells stimulated with G28-8 alone than in B cells stimulated with G28-8 plus anti-kappa. The same conditions of cross-linking with the anti-kappa which increased the kinetics of the [Ca2+]i response decreased the proliferative response which otherwise followed co-incubation of the mAb with B cell growth factor or PMA. Thus, conditions leading to rapid but transient [Ca2+]i increase via Bgp95 may not be as effective at stimulating B cell proliferation as conditions favoring a slower prolonged [Ca2+]i response. Although the Bgp95 molecule is present on activated buoyant tonsillar B cells, mAb to Bgp95 did not trigger [Ca2+]i increases in these cells. These results suggest that the Bgp95 protein may function in early B cell activation and that its signal mechanisms are altered by the activation state of the cell.     

T cell receptor aggregation, but not dimerization, induces increased cytosolic calcium concentrations and reveals a lack of stable association between CD4 and the T cell receptor.

Exposure of T94, a CD4+ V beta 8-expressing murine Th cell clone, or immediately ex vivo CD4+ T cells to deaggregated, bivalent antibodies specific for either the TCR or CD3 failed to induce an increase in [Ca2+]i, or activation of phosphatidylinositol hydrolysis unless cross-linked with a secondary anti-Ig antibody. In contrast, we show that a combination of two mAb directed against different components of the TCR/CD3 complex (145.2C11, anti-CD3 epsilon and F23.1, anti-V beta 8) successfully induce second messenger formation, that is, without any requirement for a secondary antibody. This requirement for either a secondary antibody or two independent bivalent antibodies to activate second messenger production in T cells suggested that the signal transduction apparatus may be activated by multiple TCR/CD3 complexes being brought together on the T cell surface. This was supported by the observation that conditions inducing increased T cell [Ca2+]i through the TCR/CD3 complex also resulted in aggregation of the TCR/CD3 complex on the T cell surface. Conversely, binding of anti-TCR/CD3 antibodies to the T cell under conditions that did not induce increased [Ca2+]i also failed to induce surface TCR/CD3 redistribution. Cross-linking of the CD4 accessory molecule on T94 also resulted in increased [Ca2+]i, with kinetics similar to those observed after TCR/CD3 oligomerization. CD4 is involved in the recognition of invariant regions of MHC class II during Ag presentation and has been proposed to be associated with TCR/CD3 in the absence of Ag. Aggregation of TCR/CD3 and subsequent second messenger formation was achieved by combinations of mAb to distinct determinants within the complex due to the stable association of these determinants within the T cell membrane. We therefore assessed the functional association of CD4 with the TCR/CD3 complex by examining whether a combination of mAb directed against CD4 and CD3 or TCR induced second messenger formation. We found that anti-CD4 in combination with F23.1 or with 145.2C11 failed to induce increases in [Ca2+]i. Furthermore, mAb to CD4 failed to inhibit the increase in [Ca2+]i observed with the combination of 145.2C11 and F23.1. We therefore conclude that CD4 is not stably associated with TCR or CD3 in the absence of Ag/MHC class II composites.     

Role of the CD22 human B cell antigen in B cell triggering by anti-immunoglobulin

As B cells mature during ontogeny the CD22 human differentiation Ag is exported from the cytoplasm onto the membrane. Surface expression is lost in terminal differentiation and after activation. In tonsils, CD22 is expressed on the surface of 60 to 80% of the dense B cells. Some IgM+ dense cells, however, and buoyant in vivo activated B cells are CD22-. This differential expression of CD22 and the finding that an anti-CD22 mAb augmented anti-Ig induced B cell proliferation suggested that CD22 may play a role in B cell activation. In this study we have found that CD22+ but not CD22- B cells could be triggered by anti-IgM or anti-IgD to have increased free intracellular calcium ([Ca2+]i). The presence of CD22 rather than of IgD seems to determine the ability of B cells to respond to anti-Ig with a [Ca2+]i flux. Also the proliferative response to anti-Ig or anti-Ig + B cell growth factor was restricted to the CD22+ population. Anti-CD22 mAb, although not inducing [Ca2+]i on their own after binding to B cells, did augment [Ca2+]i fluxes by anti-Ig when cross-linked. Together these results suggest that CD22 may regulate triggering of B cells through surface Ig perhaps by acting as a "bridge" to transmit an early signal into the cytoplasm.