CD38 ligation induces discrete cytokine mRNA expression in human cultured lymphocytes

Human CD38 is a surface glycoprotein expressed by different immuno-competent cells such as immature and activated lymphocytes, plasma cells and natural killer cells. It has recently been reported that the CD38 molecule exerts adenosine diphosphate ribosyl cyclase activity and is associated with distinct transmembrane signaling molecules. This study reports that ligation of CD38 by specific monoclonal antibodies (mAb) induces multiple cytokine mRNA expression in cultured peripheral blood mononuclear cells (PBMC). The mRNA for tumor necrosis factor-alpha, interleukin (IL)-1 beta, IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-12 were always detected, whereas interferon-gamma and IL-10 mRNA expression were seen in most, but not all PBMC cultures. Low levels of IL-2, IL-4 and IL-5 mRNA were also found. The key observation of this work is that CD38 ligation in PBMC induces a large spectrum of cytokines, many of which overlap with those induced via CD3 activation. The main differences between CD38 and CD3 activation are the low to undetectable levels of IL-2 mRNA, and the sustained IL-1 beta and IL-6 mRNA accumulation found in PBMC cultures following treatment with anti-CD38 mAb. Furthermore, PBMC proliferation was not found to be a prerequisite for CD38-mediated cytokine induction. Together, these results suggest that human CD38 activates a signaling pathway which leads to the induction of a discrete array of cytokines, and that this pathway only partially overlaps with that controlled by T cell receptor CD3.     

Ouabain effects on activated lymphocytes: augmentation of CD25 expression on TPA-stimulated cells and of CD69 on PHA-and TPA-stimulated cells

The objective of this study was to compare the baroreflex sensitivity (BRS) assessed by the new, non-invasive, spontaneous sequence method (BRS-sequence) with the Valsalva manoeuvrebased BRS. Fourteen healthy volunteers were studied in the supine position, during 60 degrees head-up tilt (HUT) and during -30 degrees head-down tilt (HDT). Blood pressure and R-R intervals were continuously and non-invasively recorded using a Finapres device. The BRS-sequence was assessed by analysing the slopes of spontaneously occurring sequences of three or more consecutive beats in which systolic blood pressure and R-R interval of the following beat increased or decreased in the same direction in a linear fashion; it was compared with data obtained during the Valsalva manoeuvre in each position. The time and frequency domain indices of R-R interval variability were also evaluated. The mean difference of BRS between the two non-invasive methods was 3.86 ms/mmHg with a standard deviation of 9.14 ms/mmHg. BRS was decreased during HUT and increased during HDT as assessed by both techniques. The changes in BRS were associated with vagal withdrawal and sympathetic activation during HUT and enhancement in the cardiac vagal tone and reduction in the sympathetic activity during HDT. We conclude that the BRS-sequence technique provides a reliable method to study the neural control of the circulation, although the body position in consecutive measurements needs to be standardized.     

Increased CD69 and human leukocyte antigen-DR expression on T lymphocytes in insulin-dependent diabetes mellitus of long standing

To better define prevailing activation of circulating T cell subsets in insulin-dependent diabetes mellitus (IDDM) of recent onset (DM; n = 31; median age +/- SD, 28 +/- 6.9 yr) and of long standing (DML; n = 27; age, 33 +/- 10.4 yr; median duration of disease, 105 months), CD4+ and CD8+ T cells were analyzed to determine their naive and memory subsets as well as their expression of human leukocyte antigen (HLA)-DR, interleukin-2 receptor alpha-chain (CD25), and CD69 by three-color flow cytometry. Twenty-six healthy subjects (HS; age, 32.0 +/- 8.2 yr) served as controls. No deviation was seen in either IDDM group compared to HS in CD25 expression on CD4+ or CD8+ cells or in their CD45RA+ or CD45RA- subsets. HLA-DR expression, however, was increased (P < 0.05) in total CD8+ cells and CD45RA+ cells, with CD45RA- CD8+ cells joining the prevailing pattern only in DML. Among CD4+ cells, increased expression of HLA-DR molecules was restricted to total and CD45RA- cells in DML. CD69 expression did not differ between IDDM and HS, but differed between DML (CD4+, CD8+, and CD45RA- CD4+) and DM only. In conclusion, our data demonstrate that HLA-DR expression in IDDM is restricted to memory cells (CD45RA-) among CD4+ cells in DML and is more markedly confined to naive (CD45RA+) than to memory CD8+ cells, whereas the early activation antigen CD69 is more readily expressed in DML than in DM. The observed activation of circulating T cells suggests an ongoing immune process in IDDM both at clinical manifestation and after long duration.     

CD73 expression and fyn-dependent signaling on murine lymphocytes

CD73 is a glycosyl phosphatidylinositol-anchored protein with both ecto-enzyme activity (ecto-5'-nucleotidase) and signal transducing capabilities for human T lymphocytes. We now report an analysis of the distribution and function of CD73 in murine lymphoid tissues made possible by the development of the first monoclonal antibodies (mAb) specific for murine CD73. Subsets of T and B lymphocytes are CD73+ and the level of expression increases with lymphocyte maturation in both species. Among B cells, CD73 is largely restricted to cells which have undergone isotype switching. The signal transmitting function of CD73 is also conserved, as splenic T cells treated with anti-CD73 mAb plus phorbol 12-myristate 13-acetate proliferate and secrete IL-2. Fyn-/- mice are unresponsive to CD73 ligation, however, demonstrating the requirement for this tyrosine kinase in CD73-mediated signal transduction. CD73 is down-regulated after mAb plus cross-linking, suggesting that expression may be controlled by interaction with a ligand. Only small numbers of thymocytes are CD73+, so CD73 receptor functions are unlikely to be important for developing T cells. However, immunohistochemical analysis reveals that reticular and vascular cells throughout the thymus and other lymphoid tissues are markedly CD73+. Therefore, CD73 might mediate lymphocyte-stromal cell interactions or condition the local microenvironment to facilitate lymphocyte development and/or function.     

Effect of 15-deoxyspergualine on antigen-specific lymphocyte activation measured by CD69 expression

The sarcosine oxidase from Bacillus sp. NS-129 has been crystallized by vapor diffusion using ammonium sulfate as precipitant. The tetragonal crystals have P4(1)2(1)2 (or P4(3)2(1)2) symmetry with a = b = 177.0 and c = 72.6 A. The crystals probably contain two sarcosine oxidase molecules per asymmetric unit and diffract to at least 2.7 A resolution.     

Donor cell induced CD69 expression and intracellular IL-2 and IL-4 production by peripheral blood lymphocytes isolated from kidney transplant recipients

Flow cytometry assays, which measure CD69 activation and intracellular cytokine production, have been used to measure peripheral blood lymphocyte (PBL) responses to in vitro antigen exposure. In the present study, we show that, in healthy individuals and immunosuppressed kidney transplant recipients, CD69 expression and intracellular cytokine production by peripheral blood T cells compare favorably to thymidine uptake as a measure of PBL response to alloantigen in mixed leukocyte culture (MLC). Heparinized whole blood from 23 healthy individuals was incubated for 24-48 h with 3rd party allogeneic monocytes; blood from twelve kidney transplant recipients was incubated with monocytes from their kidney donor and with monocytes from unrelated individuals. The percentage of T cells expressing surface CD69 or intracellular IL-2 or IL-4 was determined by 3-color flow cytometry. We identified 5 donor-specific response patterns in our kidney transplant group. One transplant recipient was hyporesponsive; his cells did not express CD69 or produce IL-2 in response to either donor or 3rd party allogeneic cells. All other transplant recipients expressed CD69 and IL-2 in response to 3rd party allogeneic cells. Two had no response to donor cells (donor-specific hyporesponsiveness), three had donor-specific anergy (CD69 expression without cytokine production in response to donor cells), five had a donor-specific Thl response (CD69 expression and IL-2 production in response to donor cells), and one had a donor-specific Th2 response (CD69 expression and IL-4 but not IL-2 production in response to donor cells). Rapid measures of donor-specific hyporesponsiveness such as CD69 activation antigen expression and intracellular cytokine production may prove valuable in monitoring lymphocyte function and aid in the long-term management of kidney transplant recipients.     

Gamma delta T lymphocytes in oriental cutaneous leishmaniasis: occurrence and variable delta gene expression

It has been suggested that T lymphocytes expressing gamma delta T-cell receptors could play an important role in defence against some intracellular infectious pathogens. The present study was undertaken to characterize the occurrence and variable delta gene expression of T lymphocytes expressing the gamma delta T-cell receptor in oriental cutaneous leishmaniasis. Eleven cases of oriental cutaneous leishmaniasis were investigated by immunohistological analysis using an alkaline phosphatase-anti-alkaline phosphatase (APAAP) technique. In three cases, we observed an increased percentage of gamma delta T cells (about 20% of CD3+ cells). In these cases gamma delta T cells generally expressed the V delta 2 segment, and only rarely the V delta 1 gene product. V delta 2+ cells were predominantly localized in the dermis, and were virtually absent in the epidermal compartment. The rare gamma delta T cells observed in the epidermis were almost exclusively V delta 1+. This study demonstrates that an increase of gamma delta T cells may be found in oriental cutaneous leishmaniasis, although it is not a constant feature of the disease. The finding of a preferential expansion of the V delta 2 subset suggests that this subpopulation of gamma delta T cells might be selectively involved in the recognition of Leishmania antigens. The distinct compartmentalization of gamma delta T-cell subpopulations indicates that these subsets may recognize distinct sets of antigens.     

Loss of CD28 expression on T lymphocytes: a marker of replicative senescence

A massive accumulation of neutrophils, mainly due to enhanced interleukin-8 (IL-8) levels, is believed to contribute to the deleterious effects of Pseudomonas aeruginosa lung infection, e.g., in cystic fibrosis (CF). Antibodies to phospholipase C, an exoenzyme of P. aeruginosa, are detected early and at high levels in CF patients. However, P. aeruginosa produces at least two types of phospholipase C (PLC), one haemolytic (PLC-H) and the other non-haemolytic (PLC-N), both with mol.wts of c. 77 kDa. Experiments were performed to evaluate the potential contribution of P. aeruginosa PLC to neutrophil accumulation during infection. Therefore, P. aeruginosa PLC-H and PLC-N were compared with regard to IL-8 generation from human monocytes. Purified PLC-H as well as culture supernates (mol.wt > 50 kDa) of a P. aeruginosa strain capable of producing both PLC-H and PLC-N, and mutant strains deficient in the production of one or other phospholipase, or both, were examined. Purified PLC-H (only at low concentrations up to 1 unit/4 x 10(5) monocytes), induced a dose-dependent increase in IL-8 release and IL-8-specific mRNA expression over that of unstimulated cells (at 4-, 12- and 24-h incubation times). Higher concentrations of PLC-H led to a decrease in IL-8 release and IL-8-specific mRNA expression. These findings were confirmed by the results obtained with the supernates of cultures of mutant strains of P. aeruginosa PAO1 that produced either a PLC-H or PLC-N or neither. Stimulation and inhibition of IL-8 release and mRNA expression were associated with a culture supernate fraction of mol. wt > 50 kDa and containing PLC-H. These results contribute to the understanding of the role of both P. aeruginosa PLC in IL-8 generation during their interaction with human monocytes.     

Gamma delta TCR-bearing intraepithelial lymphocytes regulate class II major histocompatibility complex molecule expression on the mouse small intestinal epithelium

OBJECTIVE: To examine the hypothesis that addition of therapeutic plasma exchange (TPEX) to an immunosuppressive drug regimen increases that regimen's efficacy to halt the progression of chronic progressive multiple sclerosis (CPMS). METHODS: The literature was searched for prospective controlled clinical trials evaluating the efficacy of TPEX in CPMS. Six studies were eligible for meta-analysis. Their results were combined, using Cochran's and Peto's methods. Three outcome measures were studied: 1) the change in Kurtzke's disability status scale (DSS) scores, 2) the relative odds of neurologic decline by 1 or more DSS grades, and 3) the relative odds of neurologic improvement by 1 or more DSS grades, in the treatment versus the comparison group of patients. Reported results of neurologic evaluations at 6, 12, 24, and 36 months of follow-up were analyzed separately. RESULTS: TPEX significantly (P < .05) reduced the proportion of patients who experienced neurologic decline (by 1 or more DSS grades) at 12 months of follow-up (relative odds of decline = 0.441, 95% confidence interval = 0.210-0.929). CONCLUSIONS: There is a need for further clinical research into the possibility of a beneficial effect of TPEX in patients with CPMS likely to experience neurologic decline over the ensuring 12 months. Targeting treatment to a particular subgroup of CPMS patients may be necessary for TPEX to prove effective.     

Interleukin-12 enhances CD26 expression and dipeptidyl peptidase IV function on human activated lymphocytes

Research of a cellular pathway activated by IL-12 which may result in new therapeutical approaches for IL-12, led us to find an intriguing relationship between IL-12 and CD26/DPPIV ectopeptidase on activated T cells. Both the percentage and median fluorescence intensity (MFI) of CD26+ cells in the PHA-stimulated PBMC or lymphoblasts increased when IL-12 (optimum dose, 2 ng/ml) was present. Maximum CD26 expression was observed on day-2 cultures of lymphoblasts, the presence of IL-12 receptor probably being necessary for this upregulation. In addition, CD26 upregulation correlated with enhanced DPPIV function. Enzyme affinity and secretion of the soluble form of DPPIV were not affected by IL-12. Kinetic behaviours of Ag expression and enzymatic activity support a different CD26 regulation pathway by IL-12. These data suggest that the correlation found in vivo between the CD26 expression and Th1-like immune responses is due to this IL-12-dependent upregulation.     

Coexpression of CD69 and HLADR activation markers on synovial fluid T lymphocytes of patients affected by rheumatoid arthritis: a three-colour cytometric analysis

Haploid and diploid strains of Aspergillus nidulans have been repeatedly treated with the strong mutagen 6-N-hydroxylaminopurine (HAP) which causes only base substitutions. An enormous amount of variability may be rapidly accumulated in haploid or diploid strains of A. nidulans. In particular, in the diploids the analysis of the results shows that after 12 cycles of treatment the conidia differ from each other for about ten recessive lethals and therefore probably for several hundreds of mutations. The viability of the heterozygous multiply mutant diploids is not appreciably different from that of untreated controls. In the diploid strains the accumulated variability was very high. The treatment of a haploid strain during vegetative growth also caused a strong accumulation of mutations, even though deleterious, because they can be maintained in the heterokaryotic condition.     

Expression of CD2 on porcine B lymphocytes

BACKGROUND/AIMS: Deposition of paramagnetic substances in basal ganglia, resulting in increased signals in T1-weighted magnetic resonance images (bright basal ganglia), is frequently seen in liver cirrhosis. The present study describes the prevalence of bright basal ganglia and its clinical significance in patients with long-standing portal vein thrombosis in the absence of liver cirrhosis. METHODS: Six patients with angiographically proven complete portal vein thrombosis and cavernomatous transformation without signs of acute or chronic liver disease were studied by magnetic resonance imaging of the brain, neuropsychiatric evaluation, psychometric tests, electroencephalography, and determination of arterial ammonia levels and of serum manganese concentrations from peripheral venous blood. RESULTS: Five out of six patients demonstrated increased signal intensity in the basal ganglia. Overt portal-systemic encephalopathy was not noted prior to or at the time of evaluation. Normal EEG results were recorded in all patients. Only one of the six patients had pathological results in at least two out of four psychometric tests. This latter patient had had a large right-sided brain infarction. Arterial ammonia concentrations were normal in four of the six patients; one patient with increased ammonia levels had concomitant renal insufficiency with azotemia. The other four patients had no relevant concomitant diseases. Serum manganese levels were non-significantly increased compared with a control group (p=0.06), but they were significantly correlated to basal ganglia signal intensity (R=0.88; p=0.02). CONCLUSIONS: Our results demonstrate that bright basal ganglia primarily represent shunt-induced alterations. They are not directly associated with disturbed liver function nor with portal-systemic encephalopathy.     

Expression of activation antigen, CD69, on human local eosinophils

The activation antigen, CD69, has been shown to be expressed on activated lymphocytes. CD69 has also been shown to be associated with the signal transduction process. Recently, we demonstrated the expression of CD69 on lung eosinophils obtained from the bronchoalveolar lavage fluid of patients with eosinophilic pneumonia. In this study, we performed flowcytometric analysis of CD69 expression on eosinophils obtained from the bronchoalveolar lavage fluid, pleural effusion or peripheral blood of various patients. Whatever the disease, local eosinophils expressed significant levels of CD69, whereas most peripheral blood eosinophils did not express CD69. These findings suggest that CD69 is a marker for local eosinophils and that local eosinophils may differ functionally from peripheral blood eosinophils.     

Downregulation of T cell receptor expression by CD8(+) lymphocytes in kidney allografts

Allospecific CD8(+) T lymphocytes are an important component of the cellular response in allograft rejection. These cells recognize and engage MHC class I antigens, leading to allospecific cytolytic responses and graft rejection. In mouse kidney allografts that survive to 3 wk after transplantation, we noted that the majority of CD8(+) cells do not express surface alpha/beta T cell receptor alpha/beta(TCR), gamma/deltaTCR, or CD3. However, these CD8(+)TCR- cells did express surface markers characteristic of T cells, including Thy1.2, CD2, and CD5. In addition, the CD8(+)TCR- cells expressed mRNA for TCR Vbeta gene families, and nearly half stained positive for cytoplasmic Vbeta8 protein, suggesting that they are T cells that have downregulated alpha/betaTCR protein expression from their cell surfaces. When these surface TCR- cells were isolated from kidney allografts by flow cytometry and cultured in the presence of either allogeneic or syngeneic stimulators, nearly 100% of cells reacquired normal levels of alpha/betaTCR expression with disproportionate usage of Vbeta8 chains. After recovery of their surface TCR expression, the CD8(+)TCR- population demonstrated strong alloreactivity in culture. These results suggest that the substantial number of CD8(+)TCR- cells found in long-term surviving mouse kidney allografts are alpha/beta-T cells that have downregulated their cell surface expression of TCR. While in other systems this phenotype may identify cells that have engaged antigen, our results indicate that loss of TCR expression by CD8(+) kidney graft-infiltrating cells may not depend on antigen engagement and that elements in the microenvironment of the kidney graft play a key role in this process. Factors that modulate expression of TCR by graft-infiltrating lymphocytes may have an important role in regulating rejection responses.     

Activation of CD8+ T lymphocytes through the T cell receptor turns on CD4 gene expression: implications for HIV pathogenesis

T cell activation through the T cell receptor is necessary to achieve a specific and effective immune response. We report here that stimulation of CD8+ T cells through the T cell receptor complex leads to de novo expression of the CD4 antigen on the cell surface that results in susceptibility of CD8+ T cells to HIV infection. In addition, activation of peripheral blood mononuclear cells from HIV-infected individuals results in the appearance of double-positive CD4+/CD8+ T cells, which become infected by endogenous HIV. HIV DNA sequences could be detected in uncultured and sorted mature CD3+CD8+ T cells from HIV+ individuals. These results suggest a new mechanism by which HIV could attack the immune system and may help to explain the CD8+ T cell defects in AIDS patients.     

Rapid expression of the CD69 antigen on T cells and natural killer cells upon antigenic stimulation of peripheral blood mononuclear cell suspensions

The CD69 antigen has been identified as the earliest activation marker on the surface of cytokine- or mitogen-activated lymphocytes. The expression of this molecule may be a useful early marker of antigen- or allergen-specific activation of lymphocytes in vitro. We evaluated the expression of the CD69 and CD25 antigens on antigen- or allergen-stimulated lymphocytes and the proliferative responses as detected by thymidine incorporation. Peripheral blood mononuclear cells (PBMC) of allergic patients sensitized to Dermatophagoides pteronyssinus, bovine casein, or nickel sulfate were cultured in the absence or presence of clinically relevant allergens, tetanus toxoid, or recombinant interleukin (IL)-2. The respective binding of CD69 or CD25 antibodies to PBMC and thymidine incorporation were measured. An early expression of CD69, but not of CD25, antigen was detectable after 24-72 h of stimulation on up to 80% of natural killer (NK) cells and up to 10% of CD4+ T cells in PBMC cultures. Anti-IL-2 antibodies inhibited these increases of CD69 on NK cells and T cells by up to 60%. After 6 days of antigenic stimulation, the rates of both CD25+ and CD69+ lymphocytes were higher. Seventy-four percent of the CD25+ PBMC but only 55% of the CD69+ cells were CD3+ T lymphocytes at this time. No qualitative differences were detectable in allergen- or tetanus-toxoid-stimulated PBMC from allergic patients. The high expression of CD69 on NK cells in antigen-stimulated cultures suggests that these cells are easily activated by cytokines from antigen-stimulated T cells. CD69+ NK cells may serve as early-indicator cells in cultures with antigen- or allergen-stimulated mononuclear cells.     

Ligation of CD28 receptor by B7 induces formation of D-3 phosphoinositides in T lymphocytes independently of T cell receptor/CD3 activation

The co-stimulatory role of B7/CD28 interactions is important in promoting T cell activation. Very little is known about the intracellular events that follow CD28 engagement although recent evidence has implicated coupling of CD28 to a protein tyrosine kinase signal transduction pathway. In this study we have investigated the putative role of D-3 phosphoinositides as mediators of CD28 receptor signaling, since phosphoinositide (PI) 3-kinase, the enzyme responsible for D-3 phosphoinositide formation, is a known substrate for protein tyrosine kinases associated with certain T cell surface receptors such as CD4 and interleukin-2 receptor. The lipid products of PI 3-kinase activity have been suggested to play a role in mitogenic signaling and growth regulation in other cells. Chinese hamster ovary cells (CHO) previously transfected with B7 cDNA, induced time-dependent elevation above basal levels of phosphatidylinositol(3,4)-bisphosphate (PtdIns(3,4)P2) and PtdIns(3,4,5)P3, while parental CHO cells that did not express B7 had no effect on these lipids. Moreover, the elevation of these same lipids by CD3 ligation was potentiated in an additive manner by CHO-B7+ but not by CHO-B7- cells. CHO-B7+ and CHO-B7- cells did not activate phospholipase C as evidenced by their inability to modulate basal or CD3-induced changes in the levels of phosphatidic acid or D-4 and D-5 phosphoinositides. These data imply that PI 3-kinase but not phospholipase C, may be an important signal transduction molecule with respect to CD28-mediated co-stimulation and T cell activation following ligation by B7.     

Granulated CD56 lymphocytes of decidual tissue

The reproduction is a complex mechanism wich is subtly immunoregulated since the semiallogenic fetus is not rejected by the mother. At the feto-maternal tissue interface, where fundamental mechanisms of tolerance occur, a large and unusual lymphocytes population takes place. These cells recruited during the earlier stages of pregnancy are very granulated and express brightly the N-CAM molecule (or CD56) with numerous adhesion and activation molecules. Nevertheless, they seem fonctionnally inactivated. They phenotypically belong to the peripheral blood minor fraction of NK cells which are CD56++CD16-CD3-. Although their function in the establishment of the gestation is not clearly known, because of their phenotypical heterogeneity, they may have two distinct functions: cytotoxicity and suppression. Their cytokine pattern, preferentially of the Th1 type, inform us about their potential functions and their interactions with other cells populations in this tissue, allowing us to understand the main mechanism in the establishment of the pregnancy.     

p38 MAPK is required for CD40-induced gene expression and proliferation in B lymphocytes

We have investigated the activation of the p38 MAPK pathway in response to CD40 engagement in multiple B cell lines and in human tonsillar B cells to define the role of p38 MAPK in proliferation, NF-kappaB activation and gene expression. Cross-linking CD40 rapidly stimulates both p38 MAPK and its downstream effector, MAPKAPK-2. Inhibition of p38 MAPK activity in vivo with the specific cell-permeable inhibitor, SB203580, under conditions that completely prevented MAPKAPK-2 activation, strongly perturbed CD40-induced tonsillar B cell proliferation while potentiating the B cell receptor (BCR)-driven proliferative response. SB203580 also significantly reduced expression of a reporter gene driven by a minimal promoter containing four NF-kappaB elements, indicating a requirement for the p38 MAPK pathway in CD40-induced NF-kappaB activation. However, CD40-mediated NF-kappaB binding was not affected by SB203580, suggesting that NF-kappaB may not be a direct target for the CD40-induced p38 MAPK pathway. In addition, SB203580 selectively reduced CD40-induced CD54/ICAM-1 expression, whereas CD40-dependent expression of CD40 and CD95/Fas and four newly defined CD40-responsive genes cIAP2, TRAF1, TRAF4/CART and DR3 were unaffected. Our observations show that the p38 MAPK pathway is required for CD40-induced proliferation and that CD40 induces gene expression via both p38 MAPK-dependent and -independent pathways.