Ways of realizing apoptosis of human lymphocytes induced by UV-light and reactive oxygen species

Changes of DNA structural condition, the level of membrane Fas-receptor expression, caspase-3 functional activity, concentrations of Ca2+, p53 and cytochrome c proteins of human lymphocytes in dynamics of apoptosis development induced by UV-light (240-390 nm) at doses 151, 1510, 3020 J/m2 and reactive oxygen species (superoxide anion-radical, hydroxyl radicals, hydrogen peroxide, singlet oxygen) have been studied. UV-light and reactive oxygen species have been established to induce fragmentation of lymphocyte DNA after 20 h incubation of the modified cells. It has been shown, that the increase in the expression level of membrane death Fas-receptors is observed during 1-5 h after exposure oflymphocytes to UV-light and ROS compared with intact cells. Also revealed is augmentation of lymphocyte caspase-3 functional activity 4 h after generation of singlet oxygen, hydroxyl radical and hydrogen peroxide addition, as well as 8 and 24 and 6 and 8 h after UV-irradiation of the cells at doses 151 and 1510 J/m2, correspondingly. Using DNA-comet method made it possible to tape that DNA damages (single-strand breaks) appear 15-20 min after lymphocyte UV-irradiation at doses 1510 and 3020 J/m and addition of hydrogen peroxide in concentration 10(-6) mol/l (C1 type comet) and reach their maximum 6 h after modification of the cells (C2 and C3 type comets). It has been observed, that 6 h after exposure oflymphocytes to hydrogen peroxide and UV-light at doses 1510 and 3020 J/m2, the p53 level of investigated cells raises. It has also been shown that the higher level of calcium in lymphocyte cytosol in conditions of UV-light exposure (1510 J/m2) and exogenous generation of reactive oxygen species is caused by Ca2+ exit from intracellular depots as a result of activating the components of the phosphoinositide mechanism for transferring information into a cell. Ideas about correlation between alterations of the calcium level and initiation of programmed cellular destruction of human lymphocytes after exposure to UV-irradiation and ROS is proposed. The authors come to the conclusion about the leading role of receptor-mediated (Fas-dependent) caspase- and p53-dependent ways of realizing apoptosis oflymphocytes induced by UV-light at doses 151 and 1510 J/m2 and active oxygen metabolites. The pattern of the possible intracellular events leading to apoptotic destruction of lymphocytes after their UV-irradiation is offered.     

Organization of the human T-cell receptor genes

T lymphocytes recognize antigens through their membrane bound T-cell receptors. Whereas the conventional T-cell receptors are heterodimers of alpha and beta chains, expressed at the surface of CD3+ CD4+ and CD3+ CD8+ T lymphocytes, the gamma delta T-cell receptors are found at the surface of a subset of T-lymphocytes of phenotype CD3+ CD4- CD8-. The synthesis of the T-cell receptor chains results from the junction (or rearrangement) of DNA segments: Variable (V) gene and joining (J) segment for the alpha and gamma chains, V gene, D (diversity) and J segments for the beta and delta chains. In this review, we summarize the recent findings on the genomic organization of the alpha, beta, gamma and delta T-cell receptor loci in human.     

Nonequivalent effects of PKC activation by PMA on murine CD4 and CD8 cell-surface expression

The membrane glycoproteins CD4 (L3T4) and CD8 (Lyt2) are expressed on distinct populations of mature murine T lymphocytes, and are thought to be receptors for monomorphic determinants expressed on MHC class II and class I molecules, respectively. Although they differ in their ligand specificity, it has been presumed that CD4 and CD8 perform equivalent functions in the T cells that bear them. Since activation of protein kinase C (PKC) is known to cause rapid down-regulation of various receptors, including the T cell receptor complex (TcR complex), we treated cells with phorbol 12-myristate 13-acetate (PMA), a PKC activator, to determine whether cell-surface expression of CD4 and CD8 would be similarly affected by this intracellular mediator. Brief or relatively prolonged treatment with PMA induced mature murine T cells to reduce their surface expression of the TcR complex and of CD4, but not of CD8. Similarly, PMA rapidly induced transfected L cells to down-regulate surface CD4 expression, but had no effect on surface CD8 expression. Most significantly, PMA treatment induced CD4+CD8+ immature thymocytes to rapidly reduce their surface CD4 expression, but, again, it had no immediate effect on the surface expression of CD8. These results indicate that CD4 and TcR complex cell-surface expression are both sensitive to PKC activation by brief treatment with PMA, whereas CD8 expression is not, and suggest that CD4 and CD8 surface expression levels are regulated by distinct intracellular mechanisms.     

Effects of ultraviolet irradiation on structural and functional condition of T- and B-lymphocytes of human blood

The influence of ultraviolet (UV) radiation on structural and functional condition of membranes T- and B-lymphocytes of human blood is investigated. It is shown, that intergral flow of UV-light (240-390 nm) in doses 151-1359 J/m2 leads to the increase of the expression of some HLA-antigen (DR1, DR2, DR5, DR7) and Fc-receptors on membranes of B-lymphocytes. The level of HLA-antigen was increased by 20-500% from the initial state after exposure to different doses. The growth of the expression of Fc-receprots was registered in 80% of the donors in the range from 20 up to 540%. The minimum dose of UV-light (151 J/m2) caused the maximum effect. The reduction of the level of Fc-receptors by 10-90% relatively to the native condition was registered for T-cells in 80% of the donors. Groups of the donors were revealed, T-lymphocytes of which differed by the sensitivity to UV-radiation. The effect of "strengthening--weakening", resulting to the reduction of the initially high parameters and to increase in the initially low ones was found.     

免疫性炎症与前列腺体积及雄激素受体表达的关系

目的:探讨免疫性炎症与前列腺体积及雄激素受体表达的关系。方法:回顾性的分析了105例手术获得的前列腺标本。使用免疫组化的方法研究前列腺组织中CD4、CD8和雄激素受体表达情况。如果CD4或CD8阳性则被定义为免疫性炎症,并进一步探讨了免疫性炎症与前列腺体积、雄激素受体表达之间的关系。结果:在前列腺增生组织中,CD4、CD8和雄激素受体表达的阳性率分别为20(19.0%),21(20.0%)和48(45.7%)。在免疫性炎症组,前列腺体积为67.0±26.3ml,而在非免疫性炎症组,为54.0±24.2ml,有显著性差别。免疫性炎症组雄激素受体表达的阳性率为65.6%,而非免疫性炎症组雄激素受体表达的阳性率为37.0%(X~2=7.35,P<0.05)。结论:免疫性炎症与前列腺体积、雄激素受体表达明显相关。免疫性炎症可能导致前列腺增生进展,因此,抗炎治疗可能是治疗前列腺增生的一个新的靶点。     

Regulation of CD4 and CD8 surface expression on human thymocyte subpopulations by triggering through CD2 and the CD3-T cell receptor

Human thymocytes bearing the CD4 and/or CD8 antigens can be fractionated into cells with an immature and more mature phenotype based on their quantitative expression of the CD3 Ag (J. Immunol. 138:3108; J. Immunol. 139:1065). We show that the expression of CD4 and CD8 on thymocyte subpopulations with low CD3 (CD3L) and high CD3 (CD3H) is regulated by activation through the CD2 molecule and perturbation of the CD3-T cell receptor complex (CD3-Ti). Similar to its previously reported effects on peripheral T cells, PMA was able to induce the down-regulation of surface CD4, but not CD8, on thymocyte subpopulations. PMA could induce CD4 and CD8 phosphorylation in both CD3L and CD3H fractions. These results suggest that if changes in phosphorylation represent the mechanism by which CD4 and CD8 are able to transmit signals, this mechanism is operative in both CD3L and CD3H subpopulations. Treatment with anti-T11(2) and anti-T11(3) antibodies (CD2 activation pathway) resulted in partial down-regulation of CD4 but not CD8 surface expression on both CD3L and CD3H thymocytes. Similar treatment had no detectable effect on peripheral T cells. The down-regulation of surface CD4 induced by activation via CD2 could be inhibited by treatment of thymocytes with anti-CD3 antibodies. Treatment of thymocytes with anti-CD3 alone or following CD2 activation induced the selective down-regulation of surface CD8 within 15 minutes. These results suggest that CD2 and CD3-Ti triggering may regulate CD4 and CD8 surface expression on thymocytes. Furthermore, these results suggest that "cross-talk" between the CD2 and CD3-Ti pathway of activation may involve CD4 and CD8 molecules.     

Transgenic mice to study T-cell receptor gene regulation and repertoire formation

Transgenic mice have been obtained with genes coding for an alpha beta T-cell receptor that recognizes the male-specific antigen H-Y in association with the Db class I major histocompatibility complex molecule. Most if not all of the T-cells express the beta chain encoded by the transgene and show allelic exclusion of endogenous beta genes. In contrast, the expression of the alpha transgene does not completely block rearrangement and formation of functional endogenous alpha genes. In H-2b transgenic female mice the transgenic T-cell receptor is functionally expressed on at least 30% of CD8+ peripheral T-lymphocytes as indicated by their ability to lyse male target cells. Also in transgenic H-2b male mice a large proportion of peripheral T-cells appear to express the transgenic receptor. However, these cells do not react with male target cells because they show only low level or no expression of CD8 cell interaction molecules. Tolerance is established in the male transgenic thymus through deletion of CD4+CD8+ immature thymocytes.     

Lateral membrane protein associations of CD4 in lymphoid cells detected by cross-linking and mass spectrometry

Interactions of membrane proteins are important in various aspects of cell function. However, weak membrane protein-protein interactions are difficult to study using techniques such as co-immunoprecipitations. CD4 is a cell surface protein involved in T cell activation and the binding of the human immunodeficiency virus to HIV target cells. Here we report the use of cross-linking followed by affinity purification of CD4 in combination with mass spectrometry for identification of proteins that are in the proximity of CD4. Besides the components of the CD4 receptor complex, CD4 and lck, we have identified by tandem mass spectrometry 17 tryptic peptides from transferrin receptor CD71, three peptides from protein phosphatase CD45, and one peptide from 4F2 lymphocyte activation antigen CD98. The efficiency of the cross-linking did not correlate with the level of cell surface expression of the detected molecules, excluding a possible bias of the cross-linking toward the most abundant cell surface molecules. Whereas the association of CD4 with CD45 has been reported, the associations with CD71 and CD98 have not been previously described. We used small-scale immunoprecipitation after cross-linking in combination with fluorescence resonance energy transfer (FRET) measurements to investigate the association between CD4 and CD71. Our data show that CD71 self-associates on the cell surface, that a small fraction of CD4 can be detected by copurifying it with CD71 after cross-linking, and that the level of association between CD4 and CD71 significantly increases after phorbol 12-myristate 13-acetate-induced endocytosis of CD4. This suggests that a small fraction of CD4 associates with clusters of CD71. As both molecules undergo endocytic recycling, the association and cross-linking result from their clustering in the same pit and/or vesicle. The CD4-CD98 association probably results from nonspecific cross-linking.     

Peripheral pool of CD8+ T-cells contains lymphocytes with antigen-specific receptors that recognize syngeneic MHC class II molecules

The capacity of T-lymphocytes to recognize "nonself" and tolerating "self" is formed as a result of positive and negative selection in the thymus. While obtaining and testing specificity of T-hybridomas, we demonstrated that the major part of peripheral pool of CD8+ T-lymphocytes carried receptors specific to "self" MHC class II molecules. Such an unexpected specificity of receptors has been found in some T-cell hybridomas produced by fusion of activated peripheral CD8+ T-lymphocytes with a tumor partner transfected by the coreceptor CD4 gene. The reactivity to "self" is not an experimental artifact due to an increased avidity of interaction of the hybridoma cells with antigen-presenting cells. Also, it is not an expression of reactivity of T-cells to superantigens, products of endogenous viruses of mouse breast cancer. The formation of a pool of such T-cells involves both cells with double receptor specificity and cells coexpressing two alpha-chains of T-cell receptor. Their appearance in the periphery can be due to the capacity of thymocytes differentiating in the direction of CD4+ cells to avoid negative selection via change of expression of coreceptor CD4 to CD8.     

Quantitative changes in characteristics of immunity in longlivers

In 93 persons aged 90 to 103 years old the subpopulation compositon of lymphocytes and the level of thymic serum activity (TSA) was studied. In the presence of polymorbidity the low level of TSA was observed in 81% of examinees, with the quantitative deficiency in the relative and absolute number of CD3+CD8+ T-lymphocytes (62% and 78% respectively) and the excess in the number CD3+CD4-CD8- T-lymphocytes (42% and 57% respectively). Among CD3+CD4-CD8- T-lymphocytes cells with phenotypes CD3+/-CD4-CD8-CD16- and CD3+CD4-CD16+ were determined. In prolonged imunomonitoring the reciprocacity of changes in the amount of CD3+/-CD4-CD8-CD16- T-lymphocytes was established in relation to the level of TCA and the number of CD3+CD8+ T-lymphocytes. The reversibility of these changes is indicative of the potential of reactivity of the immune system in longlivers. The positive result of laser therapy in persons with quantitative deficiency of CD3+CD8+ T-lymphocytes and the presence of cells with phenotype CD3+/-CD4-CD8-CD16- was associated with the restoration of the size of CD3+CD8+ subpopulation and a decrease in an amount of CD3+/-CD4-CD8-CD16-. The unfavorable prognostic sign was, seemingly, the quantitative deficiency of CD3+CD8+ T-lymphocytes with the low level of TCA in the absence of CD3+/-CD4-CD8-CD16- T-lymphocytes.     

TCRbeta transmembrane tyrosines are required for pre-TCR function.

The pre-TCR promotes thymocyte development in the alphabeta lineage. Productive rearrangement of the TCRbeta locus triggers the assembly of the pre-TCR, which includes the pTalpha chain and CD3 epsilongammadeltazeta subunits. This complex receptor signals the up-regulation of CD4 and CD8 expression, thymocyte proliferation/survival, and the cessation of TCRbeta rearrangements (allelic exclusion). In this study, we investigate the function of two conserved tyrosine residues located in the TCRbeta chain transmembrane region of the pre-TCR. We show that replacement of both tyrosines with alanine and expression of the mutant receptor in RAG-1(null) thymocytes prevents surface expression and abolishes pre-TCR function relative to wild-type receptor. Replacement of both tyrosines with phenylalanines (YF double mutant) generates a complex phenotype in which thymocyte survival and proliferation are severely disrupted, differentiation is moderately disrupted, and allelic exclusion is unaffected. We further show that the YF double mutant receptor is expressed on the cell surface and associates with pTalpha and CD3epsilon at the same level as does wild-type TCRbeta, while association of the YF double mutant with CD3zeta is slightly reduced relative to wild type. These data demonstrate that pre-TCR signaling pathways leading to proliferation and survival, differentiation, and allelic exclusion are differently sensitive to subtle mutation-induced alterations in pre-TCR structure.     

Experimental inflammatory bowel disease--role of T cells.

BACKGROUND: Our experiments were aimed to test: 1. which lymphocyte subpopulations participate in mouse colitis, produced by intrarectal (i.r.) deposition of trinitrobenzene sulphonic acid (TNBSA, TNP hapten); 2. the expression of cell adhesion molecules on lymphocytes draining the site of reaction; 3. the influence of mouse haplotype on the development of colitis. METHODS: CBA/J, BALB/c and C57BI/6 inbred and outbred Swiss Webster strains were used. Mesentheric lymph node (MLN) cells of immunized animals, unseparated or separated into CD4+, CD8+ or gammadelta+ and alphabeta+ T cell subpopulations or depleted of B lymphocytes, were transferred into recipients which were challenged i.r. with TNBSA. Inflammatory reaction in the colon was confirmed macro- and microscopically and by myeloperoxidase (MPO) level. MLN lymphocyte surface markers were tested cytofluorimetrically using appropriate antibodies. RESULTS: Sensitization with TNP results in chronic colitis (hapten dose-dependent colon weight gain and cellular infiltrate, significant increase of MPO level) only in CBA/J and BALB/c strains and can be adoptively transferred in a cell-dose dependent manner into syngeneic recipients by T alphabeta+ cells of both CD4+ and CD8+ subpopulations. T gammadelta+ cells were ineffective and B lymphocytes do not participate in the passive transfer reaction. In MLN the number of T lymphocytes positive for cell adhesion molecules particularly LPAM-1 (V-CAM1) and LPAM-2 increases significantly. CONCLUSIONS: Both CD4+ and CD8+ lymphocytes participate in the development of TNP-induced colitis. High MPO level may suggests that both Th1 and Th2 cells are involved. Colitis is accompanied by a significant accumulation in MLN of T lymphocytes positive for several cell surface adhesion molecules characteristic for memory T cells. Significant differences in susceptibility to develop colitis were found between different strains of mice.     

Phosphorylation of CD4 and CD8 molecules following T cell triggering

CD4 and CD8 molecules have been implicated in the regulation of T cell activation. In the present study, CD4 and CD8 were modified by increased phosphorylation when T cell clones or T cells were either exposed to phorbol-12-myristate- 13-acetate or were triggered via the CD3-T cell receptor complex. Activation of T cells through the CD2 sheep erythrocyte binding protein, using anti-T11(2) and -T11(3) antibodies, also resulted in CD4 and CD8 phosphorylation. These findings suggest that signals derived from two different receptor pathways can converge and result in similar molecular modifications of CD4 and CD8. Furthermore, phorbol myristate acetate treatment or activation via the CD2 pathway induced phosphorylation of the CD4 and CD8 molecules of thymocytes, suggesting that these molecules may be functional in thymus. Together, our findings indicate that CD4 and CD8 phosphorylation is a consequence of T cell triggering, and suggest that CD4 and CD8 phosphorylation may represent a molecular signaling mechanism among the CD3-T cell receptor complex, CD2, CD4, and CD8.     

Reduced expression of distinct T-cell CD molecules by collagenase/DNase treatment

DNase/collagenase treatments are widely used to obtain single-cell suspensions of tumour cells and tumour-infiltrating T lymphocytes (TIL) from solid tumours. Since the functional integrity of such cells has been questioned, we have studied whether treatments with commonly used preparations of these enzymes could affect the expression of lymphocyte surface molecules and lymphocyte proliferative responsiveness. With peripheral-blood-derived T cells as a model, flow-cytometric analysis revealed strongly reduced expression of distinct CD molecules for each enzyme, notably CD2, CD4, CD8 and CD44 for DNase, and CD4, CD14, CD16, and CD56 for collagenase. The effects were found to be due to protease contaminations present in all but the purest enzyme preparations tested. Addition of serum or trypsin inhibitor abolished the effects. Since serum-free media are widely used to expand tumour-infiltrating T cells for clinical therapeutic use, data from early phenotypic analyses can be strongly misleading. Even after an 18-h rest period following the enzyme treatments, re-expression of the affected membrane markers was still far from complete. On the other hand, despite strongly reduced expression of CD2 molecules on the lymphocyte membrane, anti-CD2-induced proliferation was not affected, showing the redundancy of this signal molecule. Since other important T cell activation molecules (TCR, CD3, CD28) were not affected by enzymatic treatment, the use of expensive, highly purified collagenase/DNase preparations does not seem to be mandatory in clinical studies with expanded TIL.     

Role of chorionic gonadotropin in the differentiation of thymocytes

The effects of chorionic gonadotropin, a basic hormone of pregnancy, on the differentiation of the human thymocytes were studied. The hormone does not affect the phenotype as determined by expression of the membrane molecules CD3, CD4, CD8 and functional activity of intrathymic pre-T-lymphocytes, but stimulates production of autocrine growth factors by these cells. Cultivation of cortical thymocytes in the presence of chorionic gonadotropin induces their phenotypic maturation with predominant development of CD4+ CD8- cells. In addition, at a high physiological dose (100 MU/ml) the hormone induces functional maturation of the cortical thymocytes. Thus, the data obtained allow us to consider this hormone as a factor regulating antigen-independent differentiation of T-lymphocytes during pregnancy and determining development of the immune system in embryogenesis.     

Enhancement of human immunodeficiency virus type 1 envelope-mediated fusion by a CD4-gp120 complex-specific monoclonal antibody.

The entry of human immunodeficiency virus type 1 (HIV-1) into cells is initiated by binding of the viral glycoprotein gp120-gp41 to its cellular receptor CD4. The gp120-CD4 complex formed at the cell surface undergoes conformational changes that may allow its association with an additional membrane component(s) and the eventual formation of the fusion complex. These conformational rearrangements are accompanied by immunological changes manifested by altered reactivity with monoclonal antibodies specific for the individual components and presentation of new epitopes unique to the postbinding complex. In order to analyze the structure and function of the gp120-CD4 complex, monoclonal antibodies were generated from splenocytes of BALB/c mice immunized with soluble CD4-gp120 (IIIB) molecules (J. M. Gershoni, G. Denisova, D. Raviv, N. I. Smorodinsky, and D. Buyaner, FASEB J. 7:1185-1187 1993). One of those monoclonal antibodies, CG10, was found to be strictly complex specific. Here we demonstrate that this monoclonal antibody can significantly enhance the fusion of CD4+ cells with effector cells expressing multiple HIV-1 envelopes. Both T-cell-line-tropic and macrophage-tropic envelope-mediated cell fusion were enhanced, albeit at different optimal doses. Furthermore, infection of HeLa CD4+ (MAGI) cells by HIV-1 LAI, ELI1, and ELI2 strains was increased two- to fourfold in the presence of CG10 monoclonal antibodies, suggesting an effect on viral entry. These findings indicate the existence of a novel, conserved CD4-gp120 intermediate structure that plays an important role in HIV-1 cell fusion.     

Elimination of CD8+ thymocytes in transgenic mice expressing an anti-Lyt2.2 immunoglobulin heavy chain gene.

Individual T cell populations are characterized by specific surface proteins, namely by the T cell receptor complex (TCR) and by two accessory molecules, CD8 (Lyt2) and CD4 (L3T4). CD8 and CD4 are required for T cell interactions with class I or class II major histocompatibility complex molecules. In the thymus, immature CD8(-4)-TCR- cells differentiate, possibly via a short stage of CD8+4- thymocytes, into CD8+4+ TCR+ T cells and mature further into the main T cell populations, the CD8+4- TCR+ cytotoxic T lymphocytes and the CD4+8- TCR+ T helper cells. In order to analyse the differentiation steps involving CD8, we generated transgenic mice expressing mu heavy chain genes from an anti-Lyt2.2 hybridoma. Transgenic lines expressing either the complete (mu sm) or only the secreted mu protein (mu s) suffer from a severe depletion of their CD8+4+ thymocytes affecting also the mature CD8+4- and CD4+8- populations. The depletion is correlated to the expression of transgenic mu-chain proteins within thymocytes. This intrathymocyte expression of the mu chain prevents CD8-4- thymocytes from further differentiation, most probably via intracellular interactions between mu heavy chain and CD8 proteins. These results show that CD8 plays an important role during thymocyte maturation.