Perforin, granzyme B, and FasL expression by peripheral blood T lymphocytes in emphysema

Background

It is generally accepted that emphysematous lungs are characterized by an increase in the numbers of neutrophils, macrophages, and CD8⁺ T lymphocytes, the lasts having increased cytotoxic activity. Because systemic inflammation is also a component of emphysema, we hypothesize that peripheral CD8⁺ T lymphocytes of emphysematous smokers who show evidence of systemic inflammation will have higher expression of cytotoxic molecules.

Methods

We assessed parameters of systemic inflammation in normal individuals (smokers or non-smokers) and in emphysematous subjects with an active smoking history by measuring serum interleukine-6, C-reactive protein, and tumor necrosis factor. Expression of perforin, granzyme B, and FasL protein by CD8⁺ T lymphocytes, CD4⁺ T lymphocytes, and natural killer cells were assessed by flow cytometry while perforin, granzyme B, and FasL mRNA expression were measured on purified systemic CD8⁺ T lymphocytes by real-time PCR.

Results

Emphysematous smokers had higher levels of serum interleukine-6 than normal subjects. Even with the presence of systemic inflammation in emphysematous smokers, the percentage of peripheral CD8⁺ T lymphocytes, CD4⁺ T lymphocytes, and NK cells expressing perforin and granzyme B protein was not different between the three groups.

Conclusion

Despite evidence of systemic inflammation, peripheral T lymphocytes of emphysematous smokers did not show higher levels of cytotoxic markers, suggesting that increase of activated T lymphocytes in the emphysematous lung may be due to either activation in the lung or specific peripheral recruitment.     

Cell specificity of granzyme gene expression

Granzymes are serine proteases present in secretory granules of cytolytic T lymphocyte lines. We have studied the expression of the granzyme family (granzyme A, B, C, D, E, F, and G) in different lymphoid cell populations and cell lines as well as in nonlymphoid cells and tissues. Our data show that with few exceptions expression of granzyme genes is restricted to T cells and their thymic precursors. In mature T cells granzymes are expressed only upon activation. The same is true for thymocytes, with the exception of grazyme A that is expressed also in non-stimulated cells. In T cells and thymocytes the distribution of mRNAs coding for different granzymes depends on the subpopulation tested and the activation protocol. Highly cytolytic PEL express granzymes A and B but none of the other granzymes.     

The granzyme B-Serpinb9 axis controls the fate of lymphocytes after lysosomal stress

Cytotoxic lymphocytes (CLs) contain lysosome-related organelles (LROs) that perform the normal degradative functions of the lysosome, in addition to storage and release of powerful cytotoxins employed to kill virally infected or abnormal cells. Among these cytotoxins is granzyme B (GrB), a protease that has also been implicated in activation (restimulation)-induced cell death of natural killer (NK) and T cells, but the underlying mechanism and its regulation are unclear. Here we show that restimulation of previously activated human or mouse lymphocytes induces lysosomal membrane permeabilisation (LMP), followed by GrB release from LROs into the CL cytosol. The model lysosomal stressors sphingosine and Leu-Leu-methyl-ester, and CLs from gene-targeted mice were used to show that LMP releases GrB in both a time- and concentration-dependent manner, and that the liberated GrB is responsible for cell death. The endogenous GrB inhibitor Serpinb9 (Sb9) protects CLs against LMP-induced death but is decreasingly effective as the extent of LMP increases. We also used these model stressors to show that GrB is the major effector of LMP-mediated death in T cells, but that in NK cells additional effectors are released, making GrB redundant. We found that limited LMP and GrB release occurs constitutively in proliferating lymphocytes and in NK cells engaged with targets in vitro. In Ectromelia virus-infected lymph nodes, working NK cells lacking Sb9 are more susceptible to GrB-mediated death. Taken together, these data show that a basal level of LMP occurs in proliferating and activated lymphocytes, and is increased on restimulation. LMP releases GrB from LROs into the lymphocyte cytoplasm and its ensuing interaction with Sb9 dictates whether or not the cell survives. The GrB-Sb9 nexus may therefore represent an additional mechanism of limiting lymphocyte lifespan and populations.     

A1/Bfl-1 expression is restricted to TCR engagement in T lymphocytes

We analyzed regulation of the prosurvival Bcl-2 homologue A1, following T-cell receptor (TCR) or cytokine receptor engagement. Activation of CD4(+) or CD8(+) T cells by antigenic peptides induced an early but transient IL-2-independent expression of A1 and Bcl-xl mRNA and proteins, whereas expression of Bcl-2 was delayed and required IL-2. Cytokines such as IL-2, IL-4, IL-7 or IL-15 prevented apoptosis of activated T cells that effect being associated with the maintenance of Bcl-2, but not of A1 expression. However, restimulation of activated or posteffector T cells with antigenic peptide strongly upregulated A1 mRNA and maintained A1 protein expression. IL-4, IL-7 or IL-15 also prevented cell death of naive T cells. In those cells, cytokines upregulated Bcl-2, but not A1 expression. Therefore, in naive, activated and posteffector T cells, expression of A1 is dependent on TCR but not on cytokine receptor engagement, indicating that A1 is differently regulated from Bcl-xl and Bcl-2.     

The expression and potential function of cellular prion protein in human lymphocytes

We examined expression of the normal cellular prion protein (PrP(C)) in human peripheral blood mononuclear cells (PBMC) and in transfected neuroblastoma cells with a panel of six monoclonal antibodies (Mabs). While all six of the Mabs reacted strongly with the neuroblastoma cells, only four of the Mabs reacted with PrP(C) expressed by human PBMC. PrP(C) is expressed at high levels in human T cells, B cells, monocytes, and dendritic cells, but not in red blood cells. Immunoblotting studies revealed that the PrP(C) glycoforms and the composition of the N-linked glycans on PrP(C) in human PBMC are different from those of the brain or the neuroblastoma cells. In human PBMC and the neuroblastoma cell lines the N-terminal portion of the PrP(C) is hypersensitive to proteolytic digestion, suggesting that the N-terminus of the PrP(C) on the surface of a living cell lacks secondary structure. We found that the level of PrP(C) expressed on the surface of human T lymphocytes was up-regulated as a consequence of cellular activation. Accordingly, memory T cells express more PrP(C) than na?ve T cells. In addition, the proliferation of human T lymphocytes stimulated with an anti-CD3 Mab was inhibited by anti-PrP(C) Mabs. Collectively, these results suggest that PrP(C) can participate in signal transduction in human T lymphocytes.     

Stimulation of S-adenosylmethionine synthetase in human lymphocytes by streptococcal M protein

The effects of the specific antigen M5 protein of group A streptococci on AdoMet synthetase activity and AdoMet levels in peripheral blood (PB) lymphocytes were studied and were compared with the effects of the nonspecific polyclonal T cell mitogen PHA. M5 protein stimulated AdoMet synthetase activity, whereas PHA had a biphasic effect with an early inhibitory effect and a later stimulatory effect on AdoMet synthetase activity. S-Carbamyl-L-cysteine (SCC), an inhibitor of human lymphocyte AdoMet synthetase, reduced AdoMet levels and inhibited the blastogenic response of PB lymphocytes to both M5 protein and PHA. Inhibition of the response to M5 protein was stronger than that to PHA. However, the inhibitory effects of SCC were totally reversible by washing the cells. It is our hypothesis that such differences in the biochemical events triggered by specific antigen as opposed to a polyclonal mitogen may determine the direction of the functional differentiation of T lymphocytes.     

A mechanism of restricted human immunodeficiency virus type 1 expression in human glial cells.

We characterized in detail the life cycle of human immunodeficiency virus type 1 (HIV-1) in human glioma H4/CD4 cells which stably express transfected CD4 DNA (B. Volsky, K. Sakai, M. Reddy, and D. J. Volsky, Virology 186:303-308, 1992). Infection of cloned H4/CD4 cells with the N1T strain of cell-free HIV-1 (HIV-1/N1T) was rapid and highly productive as measured by the initial expression of viral DNA, RNA, and protein, but all viral products declined to low levels by 14 days after infection. Chronically infected, virus-producing H4/CD4 cells could be obtained by cell cloning, indicating that HIV-1 DNA can integrate and remain expressed in these cells. The HIV-1 produced in H4/CD4 cells was noninfectious to glial cells, but it could be transmitted with low efficiency to CEM cells. Examination of viral protein composition by immunoprecipitation with AIDS serum or anti-gp120 antibody revealed that HIV-1/N1T-infected H4/CD4 cells produced all major viral proteins including gp160, but not gp120. Deglycosylation experiments with three different glycosidases determined that the absence of gp120 was not due to aberrant glycosylation of gp160, indicating a defect in gp160 proteolytic processing. Similar results were obtained in acutely and chronically infected H4/CD4 cells. To determine the generality of this HIV-1 replication phenotype in H4/CD4 cells, nine different viral clones were tested for replication in H4/CD4 cells by transfection. Eight were transiently productive like N1T, but one clone, NL4-3, established a long-lived productive infection in H4/CD4 cells, produced infectious progeny virus, and produced both gp160 and gp120. We conclude that for most HIV-1 strains tested, HIV-1 infection of H4/CD4 is restricted to a single cycle because of the defective processing of gp160, resulting in the absence of gp120 on progeny virus.     

M241 (CD1) expression on B lymphocytes

The human thymus leukemia-like antigens (CD1a-c) consist of three similar glycoproteins found on subpopulations of normal thymocytes, T cell acute leukemias, and cutaneous dendritic cells. The CD1c antigen recognized by the M241 monoclonal antibody was detected on the circulating mononuclear cells of three children with severe combined immunodeficiency disease (SCID). Two-color immunofluorescence analysis demonstrated that M241 expression (43 to 95%) was limited to cells expressing the B cell-restricted antigens B4 (CD19), B1 (CD20), and surface immunoglobulin. To confirm M241 expression on normal cells of the B lineage rather than aberrant expression limited to SCID B cells, its expression was demonstrated serologically and biochemically on purified B cells from spleen, tonsil, and peripheral blood. Parallel analyses with monoclonal antibodies NA1/34 and 4A76 demonstrated that the CD1a and CD1b molecules were negative on all B cells that were studied. It has been hypothesized that the CD1 molecules represent the human counterpart of the murine thymus leukemia antigens due to their similar size, limited tissue distribution, and association with beta 2-microglobulin. This study suggests that a subset of CD1 antigens detected by M241 (CD1c) may represent a human analog of a murine Qa antigen due to its extended distribution on normal peripheral B cells.     

Structural features and restricted expression of a human alpha-tubulin gene.

The nucleotide sequence of a human alpha-tubulin gene (b alpha 1) is described. This gene is extensively homologous to a rat alpha-tubulin gene in its coding regions, 3'-untranslated region and, indeed, in segments of its largest intron. However, with the exception of three short conserved blocks of homology, the 5' flanking regions of the rat and human genes are unrelated. Hence, these genes each encoding an identical protein are transcribed under the influence of divergent promoters. Blot analyses using RNA from a variety of transformed cells derived from different tissues indicate that expression of the human alpha-tubulin gene is restricted to cells of neurological origin. Among neurological cell types b alpha 1 expression is further restricted to adherent cells that are morphologically differentiated. The data presented suggest that the b alpha 1 gene encodes a prominent neuronal and glial alpha-tubulin and that b alpha 1 expression is a function of the differentiated state of these cells.     

Functional analysis of granzyme M and its role in immunity to infection

Cytotoxic lymphocytes express a large family of granule serine proteases, including one member, granzyme (Grz)M, with a unique protease activity, restricted expression, and distinct gene locus. Although a number of Grzs, including GrzM, have been shown to mediate target cell apoptosis in the presence of perforin, the biological activity of Grz has been restricted to control of a number of viral pathogens, including two natural mouse pathogens, ectromelia, and murine CMV (MCMV). In this article, we describe the first reported gene targeting of GrzM in mice. GrzM-deficient mice display normal NK cell/T cell development and homeostasis and intact NK cell-mediated cytotoxicity of tumor targets as measured by membrane damage and DNA fragmentation. GrzM-deficient mice demonstrated increased susceptibility to MCMV infection typified by the presence of more viral inclusions and transiently higher viral burden in the visceral organs of GrzM-deficient mice compared with wild-type (WT) mice. The cytotoxicity of NK cells from MCMV-infected GrzM-deficient mice remained unchanged and, like WT control mice, GrzM-deficient mice eventually effectively cleared MCMV infection from the visceral organs. In contrast, GrzM-deficient mice were as resistant as WT control mice to mouse pox ectromelia infection, as well as challenge with a number of NK cell-sensitive tumors. These data confirm a role for GrzM in the host response to MCMV infection, but suggest that GrzM is not critical for NK cell-mediated cytotoxicity.     

Activated mouse B cells lack expression of granzyme B

Recently, it has been reported that human B cells express and secrete the cytotoxic protease granzyme B (GrB) after stimulation with IL-21 and BCR cross-linking. To date, there are few clues on the function of GrB in B cell biology. As experimental transgenic murine systems should provide insights into these issues, we assayed for GrB in C57BL/6 B cells using an extensive array of physiologically relevant stimuli but were unable to detect either GrB expression or its proteolytic activity, even when Ag-specific transgenic BCRs were engaged. Similar results were also obtained with B cells from DBA/2, CBA, or BALB/c mice. In vivo, infection with either influenza virus or murine γ-herpesvirus induced the expected expression of GrB in CTLs, but not in B cell populations. We also investigated a possible role of GrB on the humoral immune response to the model Ag 4-hydroxy-3-nitrophenylacetyl-keyhole limpet hemocyanin, but GrB-deficient mice produced normal amounts of Ab with typical affinity maturation and a heightened secondary response, demonstrating conclusively the redundancy of GrB for Ab responses. Our results highlight the complex evolutionary differences that have shaped the immune systems of mice and humans. The physiological consequences of GrB expression in human B cells remain unclear, and the current study suggests that experimental mouse models will not be helpful in addressing this issue.     

A p50 surface antigen restricted to human urinary bladder carcinomas and B lymphocytes

Summary We have previously described the derivation of a monoclonal antibody, S2C6, to a novel 50 Kdalton antigen associated with human urinary bladder carcinoma. No reactions were obtained with carcinomas of unrelated origin or with normal urothelial cells. However, the antibody also reacted with a similar antigen on some cell lines of B lymphocyte origin. Using large panels of target cells we have now shown that this reactivity was entirely restricted to cells of the B lineage within the haematopoietic system. As opposed to its apparent restriction to malignant cells of the urothelium, the S2C6 antigen was expressed by normal B lymphocytes as well as by many malignant B cells (chronic lymphocytic leukaemia, hairy cell leukaemia and immunocytoma). Pre-B cells derived from acute lymphocytic leukaemia and plasma cells from multiple myeloma lacked the antigen. Expression was significantly enhanced on cultured B cells from Burkitt lymphomas and on Epstein-Barr virus-transformed lymphoblastoid cell lines including those of the pre-B phenotype derived from fetal bone marrow. As judged from the molecular size and the distribution pattern displayed by the S2C6 antigen it appears to be distinct from other B cell antigens previously described. A possible relation of the S2C6 antigen to a receptor for B cell growth factors is discussed.     

Exercise increases prolactin-receptor expression on human lymphocytes.

Plasma prolactin has been shown to increase during stress; the immune system is responsive to prolactin and affected by stress. Therefore, this study was undertaken to investigate the effects of acute graded, maximal treadmill exercise on prolactin-receptor expression by lymphocytes. Eight healthy men underwent one exercise and one nonexercise session. Blood was sampled immediately before and after the exercise. On the day of the nonexercise session, two resting blood samples were obtained at the same times as the exercise session samples to act as baseline data. Plasma prolactin concentrations were significantly elevated in response to exercise and correlated positively with total prolactin-receptor expression per B lymphocyte. An increase in total prolactin-receptor expression per B lymphocyte in response to exercise also was observed. In addition, exercise significantly increased the total number of circulating B lymphocytes expressing prolactin receptor as well as the total number of circulating B lymphocytes. These data support the idea that exercise may enhance the interaction between immune target cells and prolactin, a stress hormone capable of enhancing immune function.     

Regulation of the expression of lactate dehydrogenase isozymes in human lymphocytes

Mitogen activation of human peripheral lymphocytes leads to a switch in the isozymes of LDH; resting cells contain low activities of only the B₄ and B₃A forms, whereas activated cells contain high activities of the A₄ and A₃B forms. B₄ LDH is not altered in activated cells. In this study we show that the appearance of the A subunits occurs concomitantly with a several fold increase in the steady state levels of LDH-A mRNA. Responses in LDH-A mRNA are observed within 12 hrs of activation, and are, thus, associated with the G0/G1 transition or with early G1 (Marjanovicet al. Exp. Cell Res. (1991) 193: 425–431). Maximal expression of LDH-A mRNA requires both phorbol ester and concanavalin A, implying a complex regulatory pathway involving cascade systems activated through both the antigen receptor (TR) and protein kinase C.     

Kinetics of activation antigen expression by in vitro-stimulated human T lymphocytes

In this study a panel of monoclonal antibodies was used to investigate the kinetics of the appearance of activation-linked surface determinants as well as cytoplasmic and nuclear determinants in human T cells following lectin stimulation. Well known activation markers, such as Ia/DR, transferrin receptor, IL-2 receptor, T10, and gp24, were compared and investigated together with the T13 structure, recently found in this laboratory. T13, not demonstrable on resting T cells, could be seen within 24 hr after lectin stimulation. Kinetics of the appearance were similar to IL-2 receptor and transferrin receptor expression. Ia/DR synthesis was investigated separately for each polypeptide and the cytoplasmic invariant gamma-chain expression could be demonstrated for the first time with a gamma-chain-specific monoclonal antibody VIC-Y1. Moreover, gamma-chain synthesis seems to precede alpha- and beta-chain occurrence in human T cells. In addition, data from quantitative studies on antigenic densities are presented.     

Uridine enhances expression of the fragile X chromosome in human lymphocytes

Summary We have studied the effect of uridine on the expression of fragile X (fra[X]) in lymphocyte cultures established in the folate and thymidine deficient medium TC199. The results indicate that uridine enhances the expression of fra(X) and gives a higher mitotic rate. The excess of uridine during DNA synthesis might further promote the previously suggested cycle of misincorporation and removal of deoxyuridine monophosphate when the pool of deoxythymidine triphosphate is continuously depleted.