Some electrical properties of the surfaces of murine B- and T-lymphocytes and thymocytes. II. Effects of neuraminidase and ribonuclease.

The electrical properties of the peripheries of murine thymocytes, B-lymphocytes and T-lymphocytes were studied by measurement of electrophoretic mobilities and electron microscopic quantitation of adsorbed, positively charged CIH particles. The effects of neuraminidase and/or ribonuclease treatment upon these parameters were examined. Neuraminidase-susceptible groups accounted for 17%, 13% and 21% of the net surface negativity of T-lymphocytes, B-lymphocytes and thymocytes, respectively, and 28%, 63% and 78% respectively of particle binding. The calculated numbers of charges at the cellular electrokinetic surface per observed CIH particle were similar in control T-cells and thymocytes and higher than in B-lymphocytes. In neuraminidase-treated T- and B-lymphocytes the calculated charges per CIH particle were much lower than in thymocytes. These results may well indicate heterogeneities in the distribution of groups susceptible to neuraminidase, and also in the distribution and/or chemical nature of anionic groups susceptible to neither neuraminidase nor ribonuclease at the peripheries of different murine lymphocyte populations; however, at present we cannot discriminate between these and other possibilities.     

Some electrical properties of murine thymocytes, and B- and T-lymphocytes.

The electrical properties of the peripheries of mouse thymocytes, B-lymphocytes and T-lymphocytes have been examined in the fresh and glutaraldehyde-fixed states by electrophoresis, and in the fixed state by electron microscopic observation of the densities of binding of positively charged, colloidal iron hydroxide (CIH) particles. On the one hand, the electrophoretic mobilities of the T-lymphocytes were higher than those of either the B-lymphocytes or the thymocytes, in both the fresh and fixed states. On the other hand, the densities of CIH particles binding to the B-lymphocytes were higher than those on either the T-lymphocytes or the thymocytes. It is suggested that the disproportions between the electrokinetic and electron microscopic observations in the three classes of cells, are due to different (average) numbers of anionic sites, in the clusters of these sites marked by each CIH particle.     

The pool of free purine and pyrimidine nucleosides and bases in the thymocytes, and splenic T- and B-lymphocytes of C3HA mice during the growth of solid hepatoma 22a

Using reverse phase ion pair high performance liquid chromatography, the levels of free adenosine, inosine, adenine, xanthine, hypoxanthine, guanine and deoxycytidine in thymocytes and splenic T- and B-lymphocytes of C3HA mice, were studied under normal conditions and at different times (5 hrs, 1, 2, 3, 4, 5, 8 and 20 days) after transplantation of solid hepatoma 22a. The adenosine and inosine levels in thymus and spleen lymphocytes were 5 to 10 times as low as that of purine bases. Inosine was totally absent in T-and B-lymphocytes. The absolute content of adenine and guanine in thymus and spleen lymphocytes was higher compared to purine bases. It was shown that in all cases studied the decrease in hypoxanthine, xanthine and guanine levels in T- and B-lymphocytes during maximal tumour growth, i.e., on the 5th and 8th post-inoculation days as well as at the terminal period (20th day), was correlated with the decrease in the adenosine deaminase and functional activities of these cells. The level of free adenine in thymocytes and spleen T-lymphocytes during tumour growth showed a 2-4-fold increase in comparison with normal values. A dramatic decrease of intracellular concentration of deoxycytidine was observed in thymocytes and spleen T- and B-lymphocytes beginning with the 5th hour and over the whole subsequent period. The key role of the deoxycytidine decline during tumour growth as a possible cause of simultaneous impairment of DNA synthesis and purine deoxyribonucleoside phosphorylation in lymphocytes is discussed.     

Changes in the activity of granulocytopoiesis during increasing and reducing the reactions of the lysosomal apparatus of circulating neutrophils in hypovolemic hypotension

An interrelation between the intensity of release of the lysosomal content of circulating neutrophils and the activation degree of granulocytopoiesis and neutrophilia was revealed by means of pharmacological influence on the lysosomal membrane stability in case of hypovolemic hypotension in rabbits. The activation of granulocytopoiesis and neutrophilia increased while intensifying the release of lysosomal factors from the circulating neutrophils and sharply decreased while restricting it. The intensity of hypotension decreased while intensifying the reaction of the lysosomal apparatus of circulating neutrophils and increased while depressing the reaction.     

Relationship between T- and B-lymphocyte cooperation and their syngeneity]

Cooperation efficiency of (CBA x C57BL/6) F1 thymocytes and CBA bone marrow cells in immune response to SRBC was compared with the syngenic combination of the same cells. Selectivity of interaction of the T- and B-lymphocytes of different origin was studied in incomplete cyclophosphamines (CBA x C57BL/6) leads to CBA chimerae, where donors were primed with SRBC and the recipients were either intact or tolerant to the given antigen. F1 T-cells proved to interact with the CAB-B-cells 10-15 times less effectively than with the syngenic B-cells. It is suggested that similarity between the antigenic structure of the cell membrane of the T- and B-lymphocytes, aiding their physical contact, increased the action efficiency of the T-mediator on the B-cell.     

Change in the cooperation of T- and B-lymphocytes during the immune response of ram erythrocytes in the presence of liver injury by carbon tetrachloride

Changes in cooperation of T- and B-lymphocytes induced by the immune response to the ram erythrocytes under conditions of liver injury by CCl4 in donors of cells or recipients have been studied on CBA line mice in the adaptive transfer system. It is stated that application of CCl4 induces changes in functional properties of T- and B-lymphocytes and process of their cooperation. The pattern of these changes is determined by periods passed after application of the hepatotropic poison, e. i. by the degree of the liver injury and by the stage of the pathological process in it. Application of CCl4 exerts more pronounced inhibiting effect on B-lymphocytes than on T-lymphocytes.     

Measles Immune Suppression: Lessons from the Macaque Model

Measles remains a significant childhood disease, and is associated with a transient immune suppression. Paradoxically, measles virus (MV) infection also induces robust MV-specific immune responses. Current hypotheses for the mechanism underlying measles immune suppression focus on functional impairment of lymphocytes or antigen-presenting cells, caused by infection with or exposure to MV. We have generated stable recombinant MVs that express enhanced green fluorescent protein, and remain virulent in non-human primates. By performing a comprehensive study of virological, immunological, hematological and histopathological observations made in animals euthanized at different time points after MV infection, we developed a model explaining measles immune suppression which fits with the “measles paradox”. Here we show that MV preferentially infects CD45RA⁻ memory T-lymphocytes and follicular B-lymphocytes, resulting in high infection levels in these populations. After the peak of viremia MV-infected lymphocytes were cleared within days, followed by immune activation and lymph node enlargement. During this period tuberculin-specific T-lymphocyte responses disappeared, whilst strong MV-specific T-lymphocyte responses emerged. Histopathological analysis of lymphoid tissues showed lymphocyte depletion in the B- and T-cell areas in the absence of apoptotic cells, paralleled by infiltration of T-lymphocytes into B-cell follicles and reappearance of proliferating cells. Our findings indicate an immune-mediated clearance of MV-infected CD45RA⁻ memory T-lymphocytes and follicular B-lymphocytes, which causes temporary immunological amnesia. The rapid oligoclonal expansion of MV-specific lymphocytes and bystander cells masks this depletion, explaining the short duration of measles lymphopenia yet long duration of immune suppression.     

Thymocyte apoptosis induced by T cell activation is mediated by glucocorticoids in vivo

Glucocorticoids, administered in pharmacological doses, potently modulate immune system function and are a mainstay therapy for many common human diseases. Physiologic production of glucocorticoids may play a role in optimization of the immune repertoire both centrally and peripherally. Possible effects include alteration of lymphocyte development and down-regulation of cytokine responses, but essential roles remain unclear. To determine the part that endogenous glucocorticoids play in thymocyte development, we used fetal liver from mice lacking the glucocorticoid receptor GRko for immunological reconstitution of lethally irradiated wild-type (WT) mice. We find normal numbers and subset distribution of GRko thymocytes. GRko thymocytes also exhibit similar sensitivity to apoptosis induced by activating anti-CD3epsilon Ab as WT thymocytes in vitro. Surprisingly, GRko thymocytes are significantly more resistant than WT thymocytes to anti-CD3epsilon-mediated thymocyte apoptosis in vivo. Consistent with this finding, in vivo TCR complex activation induces sustained high levels of glucocorticoids that correlate strongly with thymocyte apoptosis in WT mice. We find that while direct engagement of the TCR complex may cause death of a subset of thymocytes, glucocorticoids are required for deletion of the majority of thymocytes. Thus, TCR stimulation by Ab administration may more accurately reflect polyclonal T cell activation than negative selection in vivo.     

Effect of unilateral nephrectomy in mice on the level of the humoral immune response to T-independent antigen

The influence of unilateral nephrectomy on the degree of humoral immune response to T-independent (polyvinylpyrrolidone, PVP) and T-dependent (sheep red blood cells, SRBC) antigens was studied. The increase in the number in antibody-forming cells (AFC) and nonspecific immunoglobulin-forming cells (nIFC) was investigated by means of the adaptive transfer model. The lethally irradiated recipients were injected with the antigen and also the spleen cells of operated and intact donors. PVP did not induce significant alterations of antibody genesis in mice receiving spleen cells of unilaterally nephrectomized animals comparing with recipients of intact spleen cells. At the same time, the kidney operation induced the increase in the number of AFC and nIFC when the SRBC were used. Hence the activation of humoral immune response induced by kidney operation was related not to the direct activation of B-lymphocytes but to T-cells. The possible causes of this activation were analyzed. Spleen cells of operated animals enhance both specific and antigen-dependent nonspecific immune response.     

Tetraspanins in the humoral immune response

The tetraspanins represent a large superfamily of four-transmembrane proteins that are expressed on all nucleated cells. Tetraspanins play a prominent role in the organization of the plasma membrane by co-ordinating the spatial localization of transmembrane proteins and signalling molecules into 'tetraspanin microdomains'. In immune cells, tetraspanins interact with key leucocyte receptors [including MHC molecules, integrins, CD4/CD8 and the BCR (B-cell receptor) complex] and as such can modulate leucocyte receptor activation and downstream signalling pathways. There is now ample evidence that tetraspanins on B-lymphocytes are important in controlling antibody production. The tetraspanin CD81 interacts with the BCR complex and is critical for CD19 expression and IgG production, whereas the tetraspanin CD37 inhibits IgA production and is important for IgG production. By contrast, the tetraspanins CD9, Tssc6 and CD151 appear dispensable for humoral immune responses. Thus individual tetraspanin family members have specific functions in B-cell biology, which is evidenced by recent studies in tetraspanin-deficient mice and humans. The present review focuses on tetraspanins expressed by B-lymphocytes and discusses novel insights into the function of tetraspanins in the humoral immune response.     

Role of CTLA-4 in the activation of single- and double-positive thymocytes

CTLA-4, a homologue of CD28, is a negative regulator of T cell activation in the periphery and is transiently expressed on the cell surface after T cell activation. However, the role of CTLA-4 in T cell activation in the thymus is not clear. This investigation was initiated to determine the role of CTLA-4 in the activation of CD4(+)CD8(+) double-positive (DP) and CD4(+)CD8(-) and CD4(-)CD8(+) single-positive (SP) thymocytes using fetal thymic organ cultures (FTOC) of MHC class II-restricted, OVA(323-339)-restricted TCR transgenic mice (DO11.10). We found that treatment of the FTOC with anti-CTLA-4-blocking Ab during activation with OVA(323-339) increased the proportion and number of DP thymocytes, but decreased the proportion and number of SP thymocytes compared with OVA(323-339)-stimulated FTOC without anti-CTLA-4 Ab treatment. In addition, anti-CTLA-4 Ab treatment inhibited OVA(323-339)-induced expression of the early activation marker, CD69, in DP thymocytes, but increased CD69 in SP thymocytes. Similarly, CTLA-4 blockage decreased phosphorylation of ERK in DP thymocytes by Ag-specific TCR engagement, but increased phosphorylation of ERK in SP thymocytes. CTLA-4 blockage inhibited deletion of DP thymocytes treated with a high dose of OVA(323-339), whereas CTLA-4 blockage did not inhibit deletion of DP thymocytes treated with a low dose of OVA(323-339). We conclude that CTLA-4 positively regulates the activation of DP thymocytes, resulting in their deletion, whereas blocking CTLA-4 suppresses the activation of DP thymocytes, leading to inhibition of DP thymocyte deletion. In contrast, CTLA-4 negatively regulates the activation of SP thymocytes.     

Role of Epac proteins in mechanisms of cAMP-dependent immunoregulation

This review presents observations on the role of Epac proteins (exchange protein directly activated by cAMP) in immunoregulation mechanisms. Signaling pathways that involve Epac proteins and their domain organization and functions are considered. The role of Epac1 protein expressed in the immune system cells is especially emphasized. Molecular mechanisms of the cAMP-dependent signal via Epac1 are analyzed in monocytes/macrophages, T-cells, and B-lymphocytes. The role of Epac1 is shown in the regulation of adhesion, leukocyte chemotaxis, as well as in phagocytosis and bacterial killing. The molecular cascade initiated by Epac1 is examined under conditions of antigen activation of T-cells and immature B-lymphocytes.     

Role of immune RNA in the interaction of T- AND B-lymphocytes]

"Immune" RNA preparations were obtained from the total population and also from the T- and B-lymphocytes of the spleens of the QBA line. Intact bone marrow cells or splenic cells activated with antigen served as target cells for the "immune" RNA. Investigations were carried out in the system of syngenic transfer. To study the target cells in the activated population of the spleen elimination of T-or B-lymphocytes was realized immediately after the incubation of the suspension of the splenic cells with the RNA preparations with the aid of anti-theta-or anti-B-antilymphocytic sera. T-lymphocytes served as the source of the biologically active RNA in the total preparation. B-lymphocytes of the spleen and the bone marrow served as target cells for the RNA of the cells of thymus origin. However, to detect the inducing action of the RNA simultaneous presence in the population of T- and B-lymphocytes is necessary.     

The induction of the migration of bone marrow stem elements to the periphery by the cytokines of cortisone-resistant thymocytes]

We present evidence about the functional activity of factors produced by cortisol-resistant thymocytes. Experiments in vivo have shown that the administration of the supernatant prepared from cortisol-resistant thymocytes leads to a strong stimulation of endogenous colony formation, significantly prolongs survival of sublethally irradiated mice, increases the rate of restoration of the number of thymocytes in the thymus after sublethal irradiation, and contributes to the recovery of the humoral immune response of nude mice to T-dependent antigens. The results obtained suggest that along with other cytokines, cortisol-resistant thymocytes spontaneously produce a chemotactic factor inducing migration of stem cells from the bone marrow to the periphery.     

Mechanisms of action of synthetic polyelectrolytes and polyampholytes on the immune response]

We investigated the effect of polyacrilic acid (PAA) on the immune response in mice of various strains on sheep red blood cells and also the influence of poly-2-methyl-5-vinyl-pyridine (PMVY), PAA and their statistical copolymers on antibody-forming cells (AFC) production in cultures of T- and B-lymphocytes in vivo. PAA was seen to increase accumulation of AFC in the spleen of mice depending on their genotypes. PMVP and PAA were found to intensify the cooperating interaction of T- and B-lymphocytes, whereas their copolymers exert quite an opposite effect. The injection of copolymers to the recipients of cooperating T- and B-lymphocytes practically results in the complete elimination of the cooperation effect between T- and B-lymphocytes in the immune response to sheep erythrocytes without cytostatic action of cell proliferation.     

Reactive oxygen species released from granulocytes stimulate 5-lipoxygenase activity in a B-lymphocytic cell line.

B-lymphocytes express 5-lipoxygenase (5-LO) protein but cellular leukotriene production is suppressed by selenium-dependent peroxidases. Thus it was of interest to check whether reactive oxygen species (ROS) which are released under inflammatory conditions can stimulate B-lymphocyte 5-LO and counteract peroxidase-mediated suppression of cellular 5-LO activity. It was found that 5-LO in the Epstein-Barr virus-transformed B-lymphocytic cell line BL41-E95-A is activated by addition of hydrogen peroxide or xanthine/xanthine oxidase and after increasing the oxidative state of the cell by azodicarboxylic acid bis(dimethylamide). Generation of endogenous ROS from mitochondria by antimycin A also lead to a threefold upregulation of 5-LO activity in B-cells. There was almost no detectable endogenous superoxide formation in BL41-E95-A cells after stimulation with 4beta-phorbol 12-myristate 13-acetate. Co-incubation experiments with BL41-E95-A cells and granulocytes demonstrated that granulocyte-derived ROS can activate B-lymphocyte 5-LO. Addition of superoxide dismutase and/or catalase to the B-lymphocyte/granulocyte co-incubations and to B-lymphocyte homogenates revealed that the 5-LO activation is due to the superoxide-derived release of hydroperoxides or hydrogen peroxide from granulocytes. The data suggest that ROS formation plays an important role in the regulation of cellular 5-LO activity in B-lymphocytes. As leukotrienes affect B-cell functions like cell proliferation, activation and maturation, this finding provides a new link between the formation of ROS and the regulation of immune responses.     

Dynamics of immune status indices of liquidators in the remote period after the accident at the Chernobyl NPP

In the report there are present the results of monitoring of the state of immune status in the remote period after the accident at the Chernobyl Nuclear Power Plant among three groups of liquidators, which were from Moscow and the Moscow district, the Northwestern region of Leningrad district and from Krasnoyarsk territory. During the monitoring of the liquidators from different regions there was found phenotype of immune status with regional characteristics. In the Moscow district the clinical displays of immune insufficiency prevail and in the North western region the modifications of immune status prevail among liquidators. In all the groups we could see quantitative and immunoregulatory disbalance with the increase of parameters of cellular activity such as CD95+, HLA-DR+, T-activated lymphocytes, fluctuation cytotoxic cells (CD8+, CD16+), persevering disimmunoglobulinemia with differently directed modification serum of immunoglobulins at decrease B-lymphocytes. By the end of the twenty-year period after the accident we can see a similar phenotype of immune status in all the groups with increasing of the marker of late activation (HLA-DR+) and apoptotic activity (CD95+) and it is potentially dangerous regarding cancerogenesis. According to the basis of long-term monitoring there has been discovered the immune characteristic of proliferative syndrome for the first time. The monitoring of 2004 has revealed a huge similarity immune status at non-malignant growth at significant decrease CD25+ in the Northwestern region and some differences in common IgE.     

The activation of the alternative complement pathway by fetal calf serum and mouse thymocytes.

Mouse thymocytes activated the alternative complement pathway of mouse serum in the presence of heated fetal calf serum. The activation required C3 from the fetal calf serum but was independent of antibody either in the murine or bovine serum. No other murine cells tested, including erythrocytes, peripheral blood lymphocytes, lymph node cells, spleen cells, and various cultured cell lines, activated the alternative complement pathway as effectively as thymocytes. In addition, sera from species other than cows could not substitute for fetal calf serum. The C3 deposited on thymocytes was in the form of both C3b (immune adherence positive) and C3bi (conglutinable). We propose that the basis of activation in this system is the specific protection of bovine C3b on mouse thymocyte surface.     

Mobilization of intracellular calcium from the sarcoplasmic reticulum of B-lymphocytes using cyclic guanosine 3'5'-monophosphate (cGMP)

Cyclic guanosine-3'5' monophosphate (cGMP) can mobilize intracellular calcium from the microsomal fraction of B-lymphocytes of the mouse spleen as a result of activation of cGMP-dependent microsomal proteinkinases. The existence of such a mechanism makes B-lymphocytes independent of extracellular calcium in response to agents whose effect on B-lymphocytes is mediated by calcium mechanisms.     

5C6-F4, a novel 100,000-dalton rat lymphocyte activation antigen defined by monoclonal antibody

Con A-activated rat thymocytes were used to immunize mice, and immune spleen cells were fused with NS/1 myeloma cells. One clone, designated 5C6-F4, reacted strongly with Con A-activated rat thymocytes and some LPS-activated rat spleen cells but not with normal thymocytes, spleen cells, or bone marrow cells of rat origin. The 5C6-F4 did not react with Con A-activated thymocytes of mouse origin. Immunoprecipitation of 5C6-F4 antigen from surface-iodinated Con A-activated rat thymocytes or LPS-activated rat spleen cells revealed its m.w. to be approximately 100,000. The kinetic studies of the expression of 5C6-F4 antigen revealed that 5C6-F4 antigen was detectable at 6 hr after Con A stimulation of rat spleen cells, whereas IL 2 receptor (IL 2R) was detectable at 12 hr. The appearance of 5C6-F4 antigen and IL 2R precede the onset of DNA synthesis of Con A-activated spleen cells. Thus, 5C6-F4 antigen is classified as early activation antigen. The 5C6-F4 inhibits the lymphocyte proliferation induced by mitogen and the IL 2-driven rat T cell proliferation. Sequential immunoprecipitation study as well as binding inhibition study indicated that the 5C6-F4 antigen is distinct from IL 2R molecule. The 5C6-F4 antigen appears to be a novel rat lymphocyte activation antigen that exhibits immunoregulatory function and also may serve as a useful marker of T cell activation.