Stimulation Of Peripheral Blood Research Articles

Increased peripheral blood T‐cell apoptosis and decreased Bcl‐2 in chronic obstructive pulmonary disease

Chronic obstructive pulmonary disease (COPD) is an inflammatory airway disease, usually associated with cigarette smoking. Stimulated peripheral blood T cells from patients with COPD have an increased propensity to undergo apoptosis. The mitochondrial apoptotic pathway is regulated by pro-apoptotic proteins (including p53 and Bax) as well as anti-apoptotic proteins (e.g. Bcl-2) and cytokines (IL-2, IL-4 and IL-7). We hypothesized that alterations in expression of these apoptosis-related proteins, cytokines and cytokine receptors may be important in determining the susceptibility of T cells to undergoing apoptosis in COPD. We further hypothesized that inhaled corticosteroids (GCS) contribute to the increased rates of T-cell apoptosis observed in COPD. The process of apoptosis (assessed by Annexin V and ssDNA staining), as well as Bcl-2, Bax, p53, IL-2, IL-4 and receptors IL-7R, IL-4R and IL-2Rgamma were investigated in PHA-stimulated peripheral blood-derived T cells, using flow cytometry. Fifteen patients with COPD receiving inhaled GCS (four of who received additional prednisolone), eight patients with COPD receiving symptom control medication, and 16 control subjects were studied. T cells (CD4(+) and CD8(+)) from GCS-treated COPD patients showed an increased propensity to undergo apoptosis, associated with significantly decreased Bcl-2 and IL-7 receptor expression. No significant differences were observed for the COPD patients who were receiving symptom control medication. These findings may suggest a negative peripheral effect of inhaled GCS on the immune system in COPD, although the clinical significance of these effects remains uncertain.

In vitro Effects of Beet Root Juice on Stimulated and Unstimulated Peripheral Blood Mononuclear Cells

Intake of fruits and vegetables rich in antioxidants is suggested to reduce the incidence of cancer and coronary heart disease in humans. Exceptional antioxidant activity of beet root extracts has been reported. Likewise in animal models, e.g., extracts of red beetroot Beta vulgaris var. rubra revealed significant tumor inhibitory effects. Red beetroot concentrate is universally permitted as a food ingredient. In this study, effects of a commercially available beetroot juice on freshly isolated human peripheral blood mononuclear cells stimulated with the mitogens phytohaemagglutinin and concanavalin A were investigated in vitro. Tryptophan degradation and neopterin formation were monitored in culture supernatants to determine effects of test substances on immunobiochemical pathways which both are induced by the pro-inflammatory cytokine interferon-γ. Compared to unstimulated cells, the mitogens induced significant formation of neopterin and degradation of tryptophan which is reflected by increasing concentrations of kynurenine together with diminished tryptophan levels in supernatants. Addition of beetroot extracts significantly suppressed these mitogen-induced changes, e.g. the rate of neopterin production as well as tryptophan degradation was dose-dependently suppressed. Our data show that beetroot extract is able to counteract pro-inflammatory cascades in peripheral blood mononuclear cells. Because inflammation is strongly involved in the development and progression of several clinical conditions including coronary heart disease and cancer, beneficial effect of beetroot extract may relate to this anti-inflammatory capacity.

Rapid identification of preformed alloreactive T cells for use in a clinical setting.

In clinical practice, HLA matching is generally applied to minimize the incidence of graft rejection after transplantation. Recently, graft rejection has been directly associated with the presence of preformed alloreactive T cells before transplantation. Despite this knowledge, assays to rapidly quantify preformed alloreactivity are not available for use in a clinical setting. In this study, such an assay was developed and evaluated in a large cohort to correlate alloreactive T-cell reactivity with HLA matching. Stimulator peripheral blood mononuclear cells were prestained with CD45-fluorescein isothiocyanate antibody and mixed with responder peripheral blood mononuclear cells. Activation-induced cytokine secretion was blocked using brefeldin A. After 6 hr, functionally active alloreactive responder CD4 and CD8 T cells were quantified among fluorescein isothiocyanate-negative cells by their expression of interferon-gamma on flow cytometry. Directly alloreactive CD4 and CD8 T cells among both stimulators and responders were easily distinguished after 6 hr of stimulation without being affected by bystander activation. Among 128 paired combinations, 23.4% of individuals had alloreactive CD8 T cells, 15.7% had alloreactive CD4 T cells, and 12.5% had alloreactivity in both T-cell subpopulations. Alloreactive T cells decreased from circulation within a few days after transplantation. In line with well-known clinical observations that associate HLA matching with graft outcome, the number of HLA-A and -B mismatches correlated with alloreactive CD8 T-cell frequencies in the whole study population, whereas it did not predict alloreactivity on an individual basis. Alloreactive T cells may rapidly be quantified after 6 hr of stimulation. Thus, the flow cytometric approach may be applied in a clinical setting to facilitate the individualization of immunosuppressive therapy and studies on the identification of patients who are at increased risk to develop graft rejection.

MTX-block during S-phase in BrdU-treated cells depends on species and medium used.

Stimulated peripheral blood cells from humans (Homo sapiens), cattle (Bos taurus), river buffaloes (Bubalus bubalis), and sheep (Ovis aries) were cultured in three different mediums, also differing in their respective quantities of folic acid. MTX (0.5 microgram/ml) and BrdU (20 micrograms/ml) were simultaneously added to cell cultures 24 h before harvest and removed 18 h later. The MTX-block in late S-phase was checked by counting the number of cells which replicate once (G + C bands in both chromatids, M1-cells) or twice (one chromatid stained = SCE, M2-cells) in the presence of BrdU. Compared with the control (with BrdU and without MTX), the cellular responses varied within and between species in relation to the medium used.

Stimulation of peripheral blood and intestinal mucosa cells by synthetic CpG oligodeoxynucleotides

The breakdown of tolerance to autologous bacterial flora has been implicated as a major factor contributing to the initiation and perpetuation of chronic inflammation in inflammatory bowel diseases (IBD). To test whether bacterial DNA is at the origin of inflammation in IBD, we have examined the response of lamina propria (LPMC) or peripheral mononuclear cells (PBMC) and purified T cells from IBD patients and control patients to stimulations with a set of oligodeoxynucleotides (ODNs) characterized by the presence or absence of cytosine-guanosine dinucleotides (CpG) and/or 3′ poly-guanosine (poly-G) extension. Furthermore we have evaluated the costimulatory activities of these ODNs on T cells activated via CD2 or CD3 pathway. We demonstrated that CpG ODNs induce higher proliferation of LPMC from inflammatory intestinal mucosa compared to healthy mucosa. We confirmed that CpG ODNs do not directly costimulate peripheral blood T cells activated by CD3 pathway. Finally, we revealed that CpG or non-CpG ODNs with 3′ poly-G extension inhibit completely CD2 activation of purified PB or LP T-cells whereas only CpG ODNs without poly-G extension enhance proliferation and IFN-γ production of PB T cells stimulated by CD2 pathway only in presence of NK and NK T cells. Our data suggest that NK T cells may be the primary target of ODNs and play a crucial role in indirect T-cell activation by ODN.

Simultaneous detection of 15 human cytokines in a single sample of stimulated peripheral blood mononuclear cells.

Cytokines secreted by cells of the immune system can alter the behavior and properties of immune or other cells. At a site of inflammation, sets of cytokines interact with immune cells, and their combined effect is often more important than the function of one isolated component. Conventional techniques, such as enzyme-linked immunosorbent assays, generally require large quantities of cells to characterize a complete cytokine profile of activated lymphocytes. The Bio-Plex system from Bio-Rad Laboratories combines the principle of a sandwich immunoassay with the Luminex fluorescent-bead-based technology. We developed a multiplex cytokine assay to detect different cytokines simultaneously in culture supernatant of human peripheral blood mononuclear cells stimulated with antigen and with mitogen. Fifteen human cytokines (interleukin 1alpha [IL-1alpha], IL-1beta, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-13, IL-15, IL-17, IL-18, gamma interferon, and tumor necrosis factor alpha) were validated with a panel of healthy individuals, rheumatoid arthritis patients, and juvenile idiopathic arthritis patients. Comparing the multiplex assay with a regular enzyme-linked immunosorbent assay technique with this donor panel resulted in correlation coefficients for all cytokines ranging from 0.75 to 0.99. Intra-assay variance proved to be less then 10%, whereas interassay variability ranged between 10 and 22%. This multiplex system proved to be a powerful tool in the quantitation of cytokines. It will provide a more complete picture in differences between activated lymphocyte cytokine profiles from healthy individuals and those from patients with chronic inflammatory diseases.

Enhanced activity of human IL-10 after nitration in reducing human IL-1 production by stimulated peripheral blood mononuclear cells.

Nitric oxide and superoxide form the unstable compound, peroxynitrite, which can nitrate proteins and compromise function of proinflammatory cytokines at sites of inflammation. Reduced function of proinflammatory proteins such as IL-8, macrophage inflammatory protein-1alpha, and eotaxin suggest an anti-inflammatory effect of nitration. The effects of nitration on anti-inflammatory cytokines such as IL-10 are unknown. We hypothesized that peroxynitrite would modify the function of anti-inflammatory cytokines like IL-10. To test this hypothesis, the capacity of recombinant human IL-10 to inhibit production of human IL-1beta (IL-1) from LPS-stimulated human PBMC was evaluated. Human IL-10 was nitrated by incubation with peroxynitrite or by incubation with 3-morpholinosydnonimine, a peroxynitrite generator, for 2 h and then incubated with LPS-stimulated PBMC for 6 h, and IL-1 was measured in the culture supernatant fluids. Human IL-1 production was significantly lower in the peroxynitrite- or 3-morpholinosydnonimine-nitrated IL-10 group than in the IL-10 controls (p < 0.05, all comparisons). This finding demonstrates that although peroxynitrite inhibits proinflammatory cytokines, it may augment anti-inflammatory cytokines and further point to an important role for peroxynitrite in the regulation of inflammation.

Plasmapheresis Modulates TH1/TH2 Imbalance in Patients with Systemic Lupus Erythematosus According to Measurement of Intracytoplasmic Cytokines

To examine the possible effect of plasmapheresis on the ratio of Th1/Th2 type cytokine-secreting cells we recruited eight patients with active systemic lupus erythematosus into the present study. They all failed to respond to conventional therapy. A sensitive multiparametric flow cytometric analysis was used for the detection of intracellular IL-4, IL-10 and IFN n . Stimulated peripheral blood cells were analysed by this procedure. Plasmapheresis was performed every second day for three occasions, using a continuous flow type blood cell separator, and a total of 100 u ml/body weight kg plasma was removed. Patients received 1 u mg/kg/day methylprednisolone during this period. As a result of the procedure, the rate of IFN n positive Th cells increased, while the rate of IL-4 and IL-10 expressing CD4 positive cells decreased. Together with these observations the concentration of anti-ds-DNA antibodies decreased after plasmapheresis. A decrease in disease activity index (SLE-DAI) indicated the clinical effectiveness of the therapy.

Intracellular cytokine profile in T-cell subsets of multiple sclerosis patients: different features in primary progressive disease.

To evaluate the expression of cytokines in both CD4+ and CD8+ T cells derived from peripheral blood of untreated multiple sclerosis (MS) patients with either relapsing-remitting (RR), secondary progressive (SP) or primary progressive (PP) MS and healthy controls (HC). MS is an immune-mediated disease and cytokines hove been hypothesized to contribute significantly to disease progression. Compared to the relapse-onset (RR, SP) form of the disease, PPMS patients have different clinical, immunological and pathological features. Surprisingly, the ability of their circulating T cells to produce immunoregulatory cytokines has not been extensively studied so far. Seventy-two MS patients (24 RR, 26 SP, 22 PP) and 34 HC were studied. Stimulated peripheral blood derived CD4+ and CD8+ T MS patients express significantly more CD4+ and CD8+ T cells were analyzed for IFN-gamma, IL-2, TNF-alpha, IL-4, IL-10 and IL-13 production. cells producing IFN-gamma compared to HC. Compared to the other forms of the disease, PPMS patients display a significant decrease in CD4+ T cells producing IL-2, IL-13 and TNF-alpha and a significant increase in CD8+ T cells producing IL-4 and IL-10. The data presented here demonstrate that patients with PPMS express less pro- and more anti-inflammatory cytokine producing T cells compared to the relapse-onset form of the disease, confirming the view on PPMS as a distinct disease entity.

Measurement of cytokine secretion, intracellular protein expression, and mRNA in resting and stimulated peripheral blood mononuclear cells.

Quantitation of cytokine production is a valuable adjunct to standard immunologic assays in defining several pathologic processes. Nevertheless, there is little agreement about which tissues should be assayed, which type of assay should be performed, and which stimulation protocol should be used. As these types of assays enter the clinical arena, there is need for standardization. There is also a need to maximize the amount of information which may be derived from a single sample. We compared secreted interleukin 4 (IL-4), IL-2, IL-6, tumor necrosis factor alpha (TNF-alpha), and gamma interferon proteins as measured by enzyme-linked immunosorbent assay with intracellular cytokine production (IL-2 and gamma interferon) as detected by flow cytometry and quantitative competitive PCR for IL-2, IL-4, TNF-alpha, and gamma interferon mRNA and cDNA. Results from unstimulated cells and cells stimulated with phorbol myristate acetate, phytohemagglutinin, and phorbol myristate acetate plus phytohemagglutin were compared. All three methodologies detected significant stimulation of cytokine production. The combination of phytohemagglutinin and phorbol myristate acetate was overall the most-potent stimulus.

Effects of High-Dose Inhaled Fluticasone Propionate via Spacer on Cell-Mediated Immunity in Healthy Volunteers

Systemic corticosteroids are known to alter cell-mediated immunity (CMI). However, the effects of inhaled steroids on CMI are unclear. We therefore sought to assess CMI following high-dose inhaled steroids in healthy subjects. Ten healthy nonasthmatic subjects self-administered fluticasone propionate (FP), 440 microg bid, with a spacer device. CMI was assessed by delayed hypersensitivity skin testing to multiple antigens and in vitro by phytohemagglutinin (PHA) stimulation of peripheral blood T lymphocytes. Percentages of CD3(+), CD4(+), and CD3(+)CD8(+) cells expressing CD69(+) were determined by three-color flow cytometry. Studies were conducted before and after 4 weeks of FP treatment. After 4 weeks of FP treatment, two of nine subjects became anergic, whereas six of nine subjects had reduced skin responses (one subject was excluded). Mean total skin test score fell from 18.4+/-10.9 to 9.1 +/-7.2 mm (p = 0.02). There was a decline in tuberculin responses in all four subjects who were positive prior to FP treatment. Following FP treatment, the percentage of unstimulated (from control subjects receiving saline solution) CD3(+)CD4(+)CD69(+) cells declined from 14.8+/-4.2% to 8. 5+/-4.6% (p = 0.02) and the CD3(+)CD8(+)CD69(+) cells decreased from 29.7+/-12.7% to 17.1 +/-5.0% (p = 0.007). PHA stimulation produced significant increases in the percentage of CD3(+)CD4(+)CD69(+) cells before and after FP treatment (67.0+/-9.1%, p<0.02 before FP; 55.4+/-17.0%, p<0.02 after FP), and in the percentage of CD3(+)CD8(+)CD69(+) cells before and after treatment (79.7+/-9.3%, p<0.03 before FP; 71.2+/-11.4%, p = 0. 008 after FP). High doses of FP suppress the proportion of activated circulating T cells but do not affect the ability of T cells to respond to direct stimulation with PHA. However, depression of skin test responses to antigens following treatment with FP suggests an impairment of in vivo clinical manifestations of T-cell activation by a mechanism that requires further investigation.

Cytokine gene expression occurs more rapidly in stimulated peripheral blood mononuclear cells from human immunodeficiency virus-infected persons.

Evaluation of cytokine gene expression following in vitro stimulation is one means of examining the dysregulation of the immune system in human immunodeficiency virus (HIV) infection. We have assessed differences in the immune status of non-HIV-infected (HIV-) and HIV-infected (HIV+) individuals by evaluating the kinetics of the expression of cytokine genes. We compared detailed time courses of cytokine mRNA expression in HIV- and HIV+ peripheral blood mononuclear cells (PBMC) and found that there is a significant shift (P<0.01) for all cytokines examined (interleukin 2 [IL-2], IL-6, IL-10, gamma interferon, and tumor necrosis factor alpha [TNF-alpha]) to an earlier time of mean peak mRNA expression by HIV+ PBMC (between 4 and 8 h) compared to HIV- PBMC (8 h) in response to either phytohemagglutinin (PHA) or anti-CD3 stimulation. Additional studies showed that although PHA-stimulated HIV+ PBMC showed decreased median IL-2, IL-4, and TNF-alpha mRNA levels, they typically demonstrated more rapid kinetics (increased mean 4-h/24-h cytokine mRNA ratios), with significant differences for IL-4 (P<0.05) and TNF-alpha (P<0.005), compared to HIV- PBMC. The use of fresh or frozen cells gave comparable cytokine mRNA data; however, the secretion of some cytokine proteins (IL-2 receptor, IL-10, and TNF-alpha) appeared to be reduced in HIV+ PBMC that had been frozen and thawed. Our studies demonstrate that the kinetics of cytokine gene expression can reveal additional dysregulation of the immune system in HIV infection, suggesting that PBMC of HIV-infected persons exist in an activated state in vivo that permits them to express cytokine genes more rapidly than a normal PBMC.

Noninsulin-dependent diabetes mellitus occurs in mice ectopically expressing the human Axl tyrosine kinase receptor.

The axl tyrosine kinase receptor is aberrantly expressed on myeloid cells of many individuals afflicted with chronic myelogenous leukemia (CML) and other myeloid leukemias. Although previous studies demonstrated this kinase to have oncogenic potential, it is not known whether axl actively participates in the onset and/or progression of CML. We addressed this question by generating transgenic mice possessing constitutive ectopic expression of human axl throughout cells of the myeloid hematopoietic lineage through the use of the granulocyte colony-stimulating factor (GCSF) receptor promoter. The transgenics did not exhibit hematopoietic malignancies, but did exhibit phenotypic characteristics associated with noninsulin-dependent diabetes mellitus (NIDDM) including hyperglycemia and hyperinsulinemia, severe insulin resistance, progressive obesity, hepatic lipidosis, and pancreatic islet dysplasia. The obese-diabetes phenotype was similar to that observed in the agouti and melanocortin-4(-/-) mutants, however the axl transgenics were not hyperphagic. Axl transgenic animals expressed elevated serum tumor necrosis factor (TNF)-alpha levels that were further enhanced upon in vitro lipopolysaccharide (LPS) stimulation of peripheral blood. Administration of the axl ligand, gas6, to peripheral transgenic blood samples eliminated excessive TNF-alpha production in response to LPS stimulation. As a means to better understand axl-gas6 biology, transgenic animals were produced which systemically expressed the gas6-binding axl proteolytic cleavage product. A more severe NIDDM phenotype occurred in these mice. The observed phenotypes may be related to the axl receptor or proteolytic cleavage product competing with related axl family receptors for binding of the gas6 ligand. We conclude that axl expression in myeloid cells in itself does not lead to the onset or progression of leukemia and suggest that ectopic axl expression affects endogenous modulation of TNF-alpha production indirectly resulting in the NIDDM phenotype.

Lesions in Cattle Exposed to Mycobacteriumbovis -inoculated Calves

Nine1999 Harcourt Publishers Ltdcalves were housed for periods ranging from 24 to 117 days in close contact with cattle inoculated intranasally with Copyright Mycobacterium bovis. These “in-contact” calves were examined immunologically and bacteriologically during the period of exposure, and pathologically and immunocytochemically post mortem. Three became infected by day 14, as indicated by the detection of M. bovis in nasal mucus. In-vitro interferon-γ production and lymphocyte proliferation were detected after stimulation of peripheral blood with M. bovis antigens in the majority of in-contact animals by day 28; this provided support for the role of immunological mechanisms in pathogenesis. Tuberculous lesions were found in the submandibular and bronchomediastinal lymph nodes and in the lungs of the in-contact calves; in distribution and appearance the lesions resembled those observed in naturally occurring disease. The distribution of M. bovis antigen and the numbers of mycobacteria within pulmonary lesions are reported. 1999 W.B. Saunders and Company Ltd.

Early Rheumatoid Arthritis is Associated with Diminished Numbers of TH 1 Cells in Stimulated Peripheral Blood

Rheumatoid arthritis (RA) has been associated with an altered TH 1 /TH2 balance. Here we present first results with a technique to quantitate stimulated TH1 and TH2 subsets in whole blood by means of cytofluorimetric detection of intracytoplasmic TH1 and TH2 cytokines. A group of 10 patients suffering from initial stages of RA exhibited a significantly reduced percentage of TH1 cells (11.0 ± 2.0%) in the peripheral blood as compared to 13 healthy, age matched controls (28.8 ± 2.0%). The TH2 response, as determined by intracellular expression of IL-4, remained unchanged. The data may indicate a defect in the THO-TH 1 differentiation or/and a selective trapping of TH1 cells into the affected joints.

Expression and catalytic activities of protein tyrosine kinases (PTKs) Fyn and Lck in peripheral blood T cells from elderly humans stimulated through the T cell receptor (TCR)/CD3 complex

Optimal signal transduction through the T cell receptor (TCR)/CD3 complex requires the coordinated activities of protein tyrosine kinases (PTKs) Fyn and Lck in addition to protein tyrosine phosphatases (PTPases) such as CD45. Although T cells stimulated with anti-CD3 monoclonal antibodies (mAb) exhibit age-related reductions in tyrosine phosphorylations of cellular proteins, it is unknown if the reductions represent abnormalities in PTKs or PTPases. In the current studies, immune complex kinase assays showed that the stimulation of peripheral blood T (PBT) cells from young humans with cross-linked anti-CD3 ε mAb OKT3 induced increased Fyn catalytic activity while anti-CD3 stimulation failed to induce significant increases in Lck activation. By contrast, Fyn activation in anti-CD3 stimulated PBT cells from a substantial proportion of elderly humans was reduced compared to anti-CD3 stimulated PBT cells from young humans. Also, we failed to find any increase in anti-CD3 stimulation of Lck activity in PBT cells from elderly subjects that could compensate for the decline in Fyn activity. However, no age-related alterations were detected in PBT cell expression of Fyn or Lck that might contribute to the changes in enzymatic activity. The results of other experiments demonstrated that the functional activities of PTPases in PBT cells from elderly subjects were equivalent to PBT cells from young subjects. These observations suggest that aberrant regulation of TCR/CD3 coupled PTKs may contribute to the age-related defects in signaling cascades and immune responsiveness of human T cells.

Interleukin‐4 and Interferon‐Gamma Production by Leishmania Stimulated Peripheral Blood Mononuclear Cells from Nonexposed Individuals

Interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) production by Leishmania reactive peripheral blood mononuclear cells (PBMC) from non-exposed individuals was investigated. IFN-gamma was measured in culture supernatants after antigen stimulation. For the measurement of IL-4, antigen stimulated cells were pulsed with PMA and ionomycin before IL-4 release was measured. L. donovani and L. major antigens induced IL-4 production (105-1748 pg/ml) in 13 and seven cultures, and IFN-gamma production (1.7 - > 66 IU/ml) in 14 and 11 of 20 cultures, respectively. IL-4 production rose steeply after 6 days of antigen stimulation suggesting a response due to antigen recognition. Both IL-4 and IFN-gamma production was abrogated by depletion of CD2+ or CD4+ but not CD8+ cells. CD2+ or CD4+ but not CD8+ enriched cultures produced cytokines as unseparated PBMC. Thus, in non-exposed individuals circulating Leishmania reactive CD4+ T cells could be demonstrated. The cells from different individuals showed different patterns of IFN-gamma and/or IL-4 production upon antigenic stimulation. In experimental leishmaniasis the early balance between IFN-gamma and IL-4 is important for the clinical outcome. Our findings call for studies of the importance of cytokine production by cross-reactive T cells for the outcome of L. donovani infections in humans and show that the method for IL-4 detection is useful for this purpose.

Involvement of CD31 in Lymphocyte-Mediated Immune Responses: Importance of the Membrane-Proximal Immunoglobulin Domain and Identification of an Inhibiting CD31 Peptide

CD31 (PECAM-1) is an immunoglobulin gene superfamily cell adhesion molecule found on vascular endothelium, platelets, and leukocytes. Lymphocyte expression of CD31 is most closely associated with the CD45RA+CD8+ naive T phenotype. CD31 has recently been shown to play a role in leukocyte egress to inflammatory sites. The mechanism of CD31 adhesion remains under investigation. Several investigators have reported evidence for a heterotypic ligand. We have previously shown that CD31 is phosphorylated with cell activation, which suggests a possible role for CD31 in cell activation events. We therefore studied the effects of CD31 antibodies on in vitro assays of lymphocyte activation. One CD31 antibody, LYP21, inhibited the mixed lymphocyte reaction (MLR) in a specific and dose-dependent fashion. An LYP21 epitope was localized to the sixth Ig domain of CD31. This peptide and a scrambled control peptide were synthesized and used to study effects of this epitope on lymphocyte activation. The CD31 peptide strongly inhibited the MLR. Because CD31 is expressed on both stimulator and responder populations, stimulator peripheral blood leukocytes and responder lymphocyte populations were separately incubated with CD31 peptide or control peptide and then washed before mixing. The CD31 peptide inhibited the MLR equally when either stimulator or responder cells were preincubated with the CD31 peptide. We further sorted responder cells into CD31-high and CD31-low populations and separately incubated these subsets with peptides. The CD31 peptide strongly inhibited MLRs, regardless of level of responder-cell CD31 expression. Examination of MLR reactions involving the CD31 peptide showed dispersed small aggregates of cells, rather than the single large aggregate observed in control MLRs. The CD31 peptide did not affect activation of lymphocytes by phorbol myristate acetate (PMA) and ionomycin. These results suggest that a surface CD31-ligand interaction may have a functional role in alloimmune lymphocyte activation and identify a functionally important domain of CD31.

The effect of dexamethasone, cyclosporine, and rapamycin on T-lymphocyte proliferation in vitro: Comparison of cells from patients with glucocorticoid-sensitive and glucocorticoid-resistant chronic asthma

Inhibition of T-lymphocyte activation may provide a useful approach to the treatment of chronic severe asthma. We compared rapamycin, a novel immunosuppressive drug, with cyclosporine and dexamethasone for its effects in inhibiting proliferation of T lymphocytes from patients with glucocorticoid-resistant and glucocorticoid-sensitive asthma. Phytohemagglutinin-stimulated peripheral blood T lymphocytes from 11 patients with clinically glucocorticoid-resistant and 8 patients with glucocorticoid-sensitive chronic asthma were tested for sensitivity to these drugs in a highly reproducible proliferation assay. All drugs inhibited proliferation in a dose-dependent manner (10 −6 to 10 −10 mol/L). T lymphocytes from the patients with glucocorticoid-resistant asthma were significantly less sensitive ( p < 0.01) to dexamethasone than those of patients with glucocorticoid-sensitive asthma over a wide concentration range. In contrast, cyclosporine and rapamycin inhibited cells from both patient groups to an equivalent extent. The presence of exogenous interleukin-2 abrogated the inhibitory effect of dexamethasone but not that of cyclosporine or rapamycin, suggesting that dexamethasone may act principally by inhibition of interleukin-2 production, whereas the latter drugs exert distinct or additional inhibitory effects. Stimulation of peripheral blood T lymphocytes with phytohemagglutinin for 24 hours before addition of the drugs abolished the inhibitory effect of dexamethasone and significantly reduced that of cyclosporine. The inhibitory effect of rapamycin was, however, unaltered. These data suggest that dexamethasone and cyclosporine exert their effects only at an early stage of T-lymphocyte activation, whereas rapamycin is able to inhibit lymphoblasts. The fact that the inhibitory mechanisms of these drugs are different might explain why cyclosporine and rapamycin are effective in inhibiting T lymphocytes from both patients with glucocorticoid-sensitive and those with glucocorticoid-resistant asthma. The data further suggest that cyclosporine and rapamycin may be effective for the therapy of glucocorticoid-resistant asthma.

Metaphase yields from staphylococcal enterotoxin A stimulated peripheral blood lymphocytes of unirradiated and irradiated aged rhesus monkeys.

The mitogen phytohemagglutinin (PHA) works well in both human and cynomolgus monkey (Macaca fascicularis) lymphocyte cultures to stimulate T cell proliferation. T cells from rhesus monkeys (Macaca mulatta) are less responsive than human cells, producing few metaphases when thousands are required, e.g. in biological dosimetry studies. We show that staphylococcal enterotoxin A (SEA), one of the most potent mitogens known, at a concentration of 0.5 microgram/ml stimulated peripheral lymphocytes to grow with a mitotic index (MI) averaging 0.13 metaphases/cell in old, irradiated rhesus macaques. This was significantly greater (p < 0.001) than that produced by PHA (MI < 0.01) in lymphocytes from the same animals. Whole blood was cultured for 96, 120 and 144 h for five irradiated individuals and for two controls. All cells cultured with SEA produced a high MI with a peak response at 120 h whereas the same cultures showed low MI for each PHA stimulated culture.