Effects of CD40 binding on ovarian carcinoma cell growth and cytokine production in vitro.

The poor prognosis associated with ovarian carcinoma (OVCA) is linked to the high incidence of local recurrence. There is a pressing need to identify factors that can play a role in OVCA growth and spread. Here, we focused on CD40, a member of the tumour necrosis factor (TNF) receptor superfamily with important functions in immune response. The expression of CD40 has been reported on various types of carcinoma cells, but its biological role is still poorly understood. The aim of the present study was to investigate the expression and function of the CD40 in OVCA cell lines. Detectable CD40 levels ranging from low to very high were found on the cell surface of several OVCA cell lines by flow cytometry analysis. Co-culture with a murine cell line transfected with CD40 ligand (CD40L) inhibited cell growth and up-regulated the secretion of proinflammatory cytokines interleukin (IL)-6, IL-8 and TNF-alpha in high-level CD40-expressing OVCA cell lines. Similarly, an increase of IL-6 and IL-8 release could be obtained by adding a soluble form of CD40L to the OVCA cultures. These results suggest that CD40-CD40L interaction is an important pathway affecting growth regulation and cytokine production in OVCA.

Interleukin-6 and vascular endothelial growth factor release by renal cell carcinoma cells impedes lymphocyte-dendritic cell cross-talk.

Anti-tumour T cell response requires antigen presentation via efficient immunological synapse between antigen presenting cells, e.g. dendritic cells (DC), and specific T cells in an adapted Th1 cytokine context. Nine renal cell carcinoma (RCC) primary culture cells were used as sources of tumour antigens which were loaded on DC (DC-Tu) for autologous T cell activation assays. Cytotoxic activity of lymphocytes stimulated with DC-Tu was evaluated against autologous tumour cells. Assays were performed with 75 grays irradiated tumour cells (Tu irr) and with hydrogen peroxide +/- heat shock (Tu H(2)O(2) +/- HS) treated cells. DC-Tu irr failed to enhance cytotoxic activity of autologous lymphocytes in seven of 13 assays. In all these defective assays, irradiated tumour cells displayed high interleukin (IL)-6 and vascular endothelial growth factor (VEGF) release. Conversely, when tumour cells released low IL-6 levels (n = 4), DC-Tu irr efficiently enhanced CTL activity. When assays were performed with the same RCC cells treated with H(2)O(2) + HS, DC-Tu stimulation resulted in improved CTL activity. H(2)O(2) + HS treatment induced post-apoptotic cell necrosis of tumour cells, totally abrogated their cytokine release [IL-6, VEGF, transforming growth factor (TGF)-beta1] and induced HSP70 expression. Taken together, data show that reduction in IL-6 and VEGF release in the environment of the tumour concomitantly to tumour cell HSP expression favours induction of a stronger anti-tumour CTL response.

Doxorubicin induces Fas-mediated apoptosis in human thyroid carcinoma cells.

Doxorubicin remains the most extensively used drug in the chemotherapy of thyroid cancer. However, drug resistance often limits the efficacy of chemotherapy in clinical practice. Several anticancer drugs exert their cytotoxic effect by triggering Fas-mediated apoptosis in some cell types. However, no investigations have been conducted to determine whether doxorubicin causes apoptosis in thyroid carcinomas. In the present study, we assessed the cytotoxic and apoptotic effects of doxorubicin on two thyroid cancer cell lines (FTC 238 and FTC 133). Cytotoxic effects of doxorubicin were evaluated by a 3-(4,5 dimethylthiazol-2yl) 2-5 diphenyltetrazolium bromide (MTT) assay. Apoptosis was quantified by fluorescein isothiocyanate-conjugated annexin V/flow cytometric analysis and by DNA fragmentation. Fas expression was measured by flow cytometric analysis. After a 24-hour incubation, doxorubicin induces a dose-dependent cytotoxicity in the two cell lines. Treatment with doxorubicin (0.5 and 1 microM) for 24 hours induced cell apoptosis and upregulated Fas expression. A significant correlation was found between the fluorescence intensity values obtained with annexin V staining and those observed for Fas expression (r = 0.996; p < 0.001 or r = 0.957; 0.02 < p < 0.05 for FTC 238 or FTC 133 cells, respectively). In conclusion, doxorubicin exerts its cytotoxic effects, at least partly, through Fas-mediated apoptosis in thyroid cancer cells. These results may have clinical implications for thyroid cancer therapy.

Design of a flow cytometric assay for the determination of natural killer and cytotoxic T-lymphocyte activity in human and in different animal species.

BACKGROUND: The most common assay used to detect natural killer (NK) and cytotoxic T-lymphocyte (CTL) activity is the (51)Cr release assay. The numerous disadvantages of this method led us to evaluate cytotoxicity functions by flow cytometry. We described a flow cytometric assay to assess NK and CTL activity from different species. METHODS: This assay is based on a dual fluorescent staining of target cells. The dye, DIOC18((3)) (3, 3'-dioctadecyloxacarbocyanine perchlorate), is used to stain the membrane of different target cells. Propidium iodide (PI) is used to label dead target and effector cells. This labeling allows a clear discrimination between both cell populations. RESULTS: A good correlation was observed between the percentage of target lysis and the effector-to-target cell (E/T) ratios with human and porcine peripheral blood mononuclear cells (PBMC) as effector cells. The flow cytometric assay was shown to be as sensitive and as reliable as the (51)Cr release performed with human cells. The assay was also applied successfully to measure NK cell activity in other animal species (pig, rabbit, hen, and mouse) and to measure murine CTL activity against the influenza virus. CONCLUSIONS: We provide evidence that the flow cytometric assay using DIOC18((3)) is highly reproducible and is suitable to measure different types of cell cytotoxicity.

Peripheral blood and intrathyroidal T cell clones from patients with thyroid autoimmune diseases.

For a better understanding of the pathogenesis of thyroid autoimmune diseases, we have studied morphological and functional properties of T clones from peripheral blood lymphocytes (PBL) and from intrathyroidal lymphocytes (ITL) obtained from 3 patients with Graves' disease or 1 Hashimoto's thyroiditis. Investigations were carried out on clones cultured alone or cocultured with autologous thyrocytes. Clonage efficiency ranged from 30% to 33% for PBL and 10% to 36% for ITL. A predominance of CD4-positive clones was observed whatever the origin of the lymphocytes or the autoimmune pathology. Gamma interferon (IFN-gamma) was detected in the majority (17/19) of the clones tested. Intracytoplasmic interleukin (IL-4) was secreted in 7/19 clones and both cytokines were produced in 5/19 clones. In coculture a proliferative response and tumour necrosis factor (TNF-alpha) production were observed with 6 clones (4 from Graves thyrocytes and 2 from thyroiditis). No cytotoxic clone was derived from Graves or thyroiditis tissues. These data demonstrate that the large majority of T clones are principally CD4-T cells; all the clones secreted TNF-alpha and a large majority produced IFN-gamma. Only a few clones produced IL-4 alone or associated with IFN-gamma. Six T clones induced proliferative response and of TNF-alpha secretion in coculture. Further investigations must be performed on these antigen-reactive T clones to analyse their role in the pathogenesis of the human thyroid autoimmune diseases.

Effect of PSC 833 on the efficacy of doxorubicin in vitro in a medullary thyroid carcinoma cell line.

In medullary carcinoma of the thyroid (MTC), multidrug resistance (MDR) remains the major obstacle to effective chemotherapy. In this work MDR was investigated in TT cells, a human MTC cell line. We studied the effect of an efficient MDR agent (SDZ PSC 833) on doxorubicin (DOX)-induced cytotoxicity in TT cells cultured in monolayers. The toxicity was evaluated with four tests: MTT test, lacticode-hydrogenase and glutathione assays, and neutral red uptake. PSC 833 (3 microM) partially reversed the resistance to DOX in vitro after a 48-hour coincubation, followed by a 24 hour-post incubation. Under these conditions, PSC 833 was not toxic at the concentration used. Our results suggest that PSC 833 has the potential to reverse the MDR phenotype in MTC cells.

TCR repertoire and cytokine profiles of cerebrospinal fluid- and peripheral blood-derived T lymphocytes from patients with multiple sclerosis.

The cerebrospinal fluid (CSF) represents an important source of T lymphocytes that could be involved in the inflammatory response occurring in the central nervous system in multiple sclerosis (MS). In order to investigate whether the Vbeta gene usage of CSF T lymphocytes is restricted, we analyzed the TCR Vbeta expression in twelve CSF expanded by in vitro culture compared to the paired in vitro-stimulated peripheral blood T lymphocytes. The overexpression of one or two Vbeta genes was demonstrated in ten CSF, but the type of Vbeta over expressed varied from one patient to another. For one patient, the Vbeta repertoire was also investigated by single cell cloning. High frequency of BV6S7-expressing T cell clones was observed in the CSF while no BV6S7 clone was derived from the peripheral blood T lymphocytes suggesting that these cells could be involved in the immunopathological process in the central nervous system (CNS). The cytokine patterns of the T cell clones derived from the CSF- and peripheral blood-T lymphocytes of this patient were determined. The CSF T cell clones produced higher levels of cytokines than the peripheral blood T cell clones. The high frequency of IL-4-producing-T cell clones observed in CSF demonstrate that T cells which could downregulate the inflammatory process are present in the CNS.

Expression of membrane and soluble intercellular adhesion molecule-1 in Graves' disease.

We have investigated the in vitro expression of membrane and soluble intercellular adhesion molecule-1 (ICAM-1) by human thyroid cells from 20 patients with Graves' disease and 5 normal subjects. Membrane ICAM-1 was not detected by flow cytometry analysis in non-cultured thyrocytes from either normal or Graves' disease tissues. It appeared on thyroid cells after a 24-h culture in monolayers and showed a regular dose-dependent increase. The same results were obtained with soluble ICAM-1 (sICAM-1) in culture media from cells cultured in monolayers, vesicles or follicles. No change was obtained with different concentrations of fetal calf serum added to the media. Coculture of Graves' disease thyrocytes with autologous peripheral blood lymphocytes (PBL) or intrathyroidal lymphocytes (ITL) enhanced the expression of both membrane and sICAM-1 whatever the culture model. When normal thyrocytes were cocultured with PBL, sICAM-1 increased but with ITL sICAM-1 remained unchanged. High concentrations of gamma interferon induced an increase of both membrane and sICAM-1 in the three culture models. However the increases were greater with vesicles and follicles. Only sICAM-1 levels were raised with 0.1, 1 and 10 microM retinoic acid. These results suggest that ICAM-1 appears in culture, possibly due to mechanical effects such as adherence to plates and cell-to-cell contacts. Moreover, its expression is modulated by several factors such as cytokines or retinoic acid. Further investigations are needed to establish whether ICAM-1 is really involved in the pathogenesis of Graves' disease.

Longitudinal study of HIV-specific cytotoxic lymphocytes in HIV type 1-infected patients: relative balance between host immune response and the spread of HIV type 1 infection.

To evaluate the contribution of a specific cytotoxic response in the control of HIV infection in relation to clinical status, we performed serial analysis of anti-Env and anti-Gag cytotoxic activity in 13 infected individuals over a 6- to 10-year period, using cryopreserved peripheral blood mononuclear cells (PBMCs). Autologous EBV-transformed B cell lines infected in vitro with recombinant vaccinia viruses expressing HIV-1 env and gag genes were used as targets. Without any stimulation of the effector cells, we were able to show an anti-HIV cytotoxic activity in the PBMCs of 12 of 13 HIV-1-infected patients, consistent with chronic immune activation in HIV infection. Different patterns of HIV-specific cytotoxic activity were observed, and the extent of this cytotoxic response varied between the clinically defined groups of individuals. No direct relationship was observed with the number of CD4 and CD8 lymphocytes during the observation period. However, patients who remained asymptomatic had a more vigorous cytotoxic response than patients with clinical deterioration during the observation period, and a significant difference was observed for HIV Gag-specific CTL activity. From these data, we suggest that the HIV-specific cytotoxic response has a protective role in the course of HIV infection.

Action of peripheral or intrathyroidal lymphocytes on autologous thyrocytes cultured in follicles in collagen gel.

We have studied the action of peripheral blood lymphocytes (PBLs) and intrathyroidal lymphocytes (ITLs) on the biochemical and hormonal metabolism of autologous thyrocytes cultured in follicles in a collagen gel. The production of tumour necrosis factor alpha (TNF-alpha) in culture was also measured. Thyroid tissues and lymphocytes were obtained from ten patients with Graves' disease and from five control subjects. Lymphocyte-induced cytotoxicity was evaluated in autologous thyrocytes cultured in a collagen gel by several tests; neutral red uptake, lactate dehydrogenase activity and glutathione level. Hormonal metabolism was assessed by evaluating tri-iodothyronine (T3) and total cAMP production under TSH stimulation. TNF-alpha levels were measured in supernatants after 5 days of coculture. PBLs altered biochemical metabolism, T3 synthesis and cAMP production in autologous thyroid follicles. These inhibitions were greater than those obtained with ITLs. No difference was seen between cells obtained from patients with Graves' disease and those from normal subjects. TNF-alpha levels secreted by PBLs were higher than those secreted by ITLs. The concentrations of this cytokine decreased in coculture. Significant correlations were observed between the decrease in biochemical and hormonal parameters and TNF-alpha levels. Exogenous TNF-alpha and high doses of interferon gamma inhibited follicle metabolism, especially hormone secretion. In conclusion, thyrocytes cultured in follicles provide a more sensitive model than monolayer cultures for analysis of lymphocyte-induced interactions. Lymphocytes gradually inhibit the biochemical and hormonal metabolism of autologous thyroid follicles depending on the isolation method. These alterations may be particularly attributed to TNF-alpha secreted by lymphocytes. The cytokine-induced inhibition of thyroid hormonal function apparently involves the adenylate cyclase system.

Polyamine deprivation prevents the development of tumour-induced immune suppression.

Mice grafted with the 3LL (Lewis lung) carcinoma exhibit immune suppression: spleen cells showed decreased spontaneous interleukin 2 (IL-2) production and T-CD4+ and T-CD8+ lymphocyte populations; in addition the polyamine content in the spleen was increased. By treating the mice with a polyamine-deficient diet containing neomycin, metronidazole and inhibitors of ornithine decarboxylase and polyamine oxydase, tumour growth was reduced and the immune abnormalities were reversed. The spleen cells overproduced IL-2 by reducing exogenous sources of polyamines, but total blockade of all major polyamine sources was necessary to obtain an optimal effect both on IL-2 production and on spleen polyamine content. Irrespective of whether polyamine deprivation was started at an early or at an advanced stage of tumour growth, T-lymphocyte populations were restored to normal values, demonstrating that polyamine deprivation not only prevents tumour-induced immune suppression, but reverses established immunological disorders. In contrast to what was observed regarding IL-2 production by spleen cells and natural killer (NK) cell activity, the polyamine oxidase (PAO) inhibitor did not enhance the number of T lymphocytes. These findings are consistent with a direct effect of the polyamines on immune effector cell metabolism. They suggest an important role of the gastrointestinal polyamines and of PAO activity in the regulation of IL-2 production.

Effect of lymphocytes on hormonal secretion by autologous thyrocytes cultured in monolayers.

We studied the lymphocyte-induced alterations in hormonal metabolism and the production of tumour necrosis factor alpha (TNF-alpha) during coculture of thyrocytes and autologous lymphocytes from 20 patients with Graves' disease and from five normal subjects. Thyroglobulin (Tg) mRNA was assessed by slot-blot analysis under TSH stimulation. Tg, tri-iodothyronine (T3) and cAMP secretion in the presence of TSH were measured by RIA after 3 or 5 days of coculture. TNF-alpha levels produced after 5 days incubation were also assayed in lymphocyte culture and coculture media. Lymphocytes isolated from peripheral blood (PBLs) altered the production of Tg, T3 and cAMP in autologous thyrocytes. Intrathyroidal lymphocytes (ITLs) decreased Tg and cAMP secretion but had no effect on T3 secretion. The reductions in Tg and cAMP levels obtained with mechanically isolated ITLs (M-ITLs) were generally higher than those obtained with ITLs isolated by dispase (D-ITLs). No difference was seen between Graves' disease and normal cocultures. PBLs secreted large concentrations of TNF-alpha, larger than those obtained with M-ITLs whereas D-ITLs produced low amounts of this cytokine. In coculture, TNF-alpha levels were lower than those observed in lymphocyte culture. Significant correlations were obtained between TNF-alpha levels and the decrease in Tg, T3 and cAMP concentrations. The percentage of T lymphocytes was higher in PBLs and D-ITLs than in M-ITLs. B lymphocytes levels were higher in ITLs, especially M-ITLs, than in PBLs. TNF-alpha production by B lymphocytes was maximal in M-ITLs. In conclusion, lymphocytes induced a decrease in hormonal thyroid metabolism when cocultured with autologous thyrocytes. These perturbations may be attributed, at least partly, to TNF-alpha secreted by lymphocytes. TNF-alpha interacts via the adenylate cyclase pathway of TSH signal transduction.

Immunomodulatory activity of beta-casein permeate medium fermented by lactic acid bacteria.

During fermentation, lactic acid bacteria may be able to release components that possess immunomodulatory activity. This activity was investigated in several culture supernatants arising from lactic acid bacteria cultured in a medium composed primarily of UF permeate of bovine milk; beta-CN was added as the sole protein source. Only a Lactobacillus helveticus supernatant allowed the modulation (both suppression and enhancement) of lymphocyte proliferation in vitro on human peripheral blood lymphocytes, but L. helveticus did not modulate the cytotoxic activity of natural killer cells or of lymphokine-activated killer cells. The addition of different quantities of culture supernatant to cultures of human mononuclear cells, stimulated by the mitogen concanavalin A, significantly increased the production of interferon-gamma and decreased the production of interleukin-2 and the expression of the alpha-chain of the interleukin-2 receptor (p55), all of which appear to be correlated with the decrease in lymphocyte proliferation. Our results suggest that the culture supernatant activity might be related to interaction with monocyte-macrophage and T helper cells, especially Th1-like cells.

Expression of the alpha-subunit of CR4 (CD11c) by peripheral blood and crevicular fluid neutrophils.

This work determined the levels of expression of CD11c by neutrophils (PMNs) collected from subjects with various periodontal conditions. The percentages of CD11c-positive crevicular fluid PMNs were significantly lower than those of peripheral blood PMNs, but the levels of CD11c expression were similar in PB-PMNs and CF-PMNs (P<0.001). On the other hand, no significant difference could be found between the groups, either for the percentages of CD11c-positive cells or for the CD11c expression levels.

Distinct pattern of IL-2 and IFN-gamma gene expression in CD4 and CD8 T cells: cytofluorometric analysis at a single cell level using non-radioactive probes.

IL-2 and IFN-gamma gene expression was analyzed using an original method for in situ hybridization (ISH) with non-isotopic probes and flow cytometric analysis (FC). This method permits rapid detection of mRNA at a single cell level among in vitro activated human peripheral blood mononuclear cells (PBMC) and purified CD4 and CD8 T cell subsets. After stimulation with PMA and ionomycin (PMA+Io), cells were fixed at different times and hybridized with digoxigenin (DIG)-labelled RNA antisense or sense probes specific for IL-2 and IFN-gamma. The level of cytokine gene expression in individual cells was visualized using FITC-conjugated anti-DIG antibodies and the fluorescent signal was analyzed by flow cytometry. Specific hybridization with IL-2 and IFN-gamma antisense probes was detected among activated PBMC within lymphoid cells identified by their light scattering properties. Kinetic analysis of the frequency of mRNA producing cells exhibited a biphasic pattern with an early peak at 6-8 hrs. when percentages of IL-2 and IFN-gamma expressing cells reached 35 +/- 7% and 18 +/- 4%, respectively. Similar data were obtained by enzymatic detection on cell smears using AP-conjugated anti-DIG. Combination of ISH with FC was applied to the comparison of the pattern of cytokine gene expression between CD4 and CD8 T cell subsets isolated by negative selection using immunobeads and magnetic separation. IL-2 was expressed by activated CD8 T cells (25-35%), but CD4 T cells were the major producers of IL-2 as assessed by the high frequency of mRNA expressing cells (60%) and the large amount of mRNA per cell relative to the mean fluorescence intensity. In contrast, IFN-gamma mRNA was preferentially expressed by CD8 T cells (27-37%) and a minority of CD4 T cells (17-23%). Despite quantitative differences, kinetic analysis of IL-2 gene expression in CD4 and CD8 T cells showed similar profiles with an early peak at 6-8 hrs. Upregulation of IL-2 gene expression in CD4 T cells by CD28 co-stimulation increases the amount of IL-2 mRNA per cell as visualized by mean fluorescence intensity. In addition the effect of CD28 co-stimulation on IL-2 mRNA stabilisation was demonstrated by the maintenance of a high frequency of IL-2 expressing CD4 T cells and an elevated level of mRNA per cell for prolonged period after PMA+Io stimulation. By contrast CD28 co-stimulation had no obvious effect on IFN-gamma expression.

The prognostic value of plasma viremia in HIV-infected patients under AZT treatment: a two-year follow-up study.

To determine the prognostic value of plasma viremia in long-term zidovudine (AZT)-treated HIV-infected patients, HIV-1 plasma viremia (PV) was quantified in 28 HIV-infected patients before and during AZT long-term treatment; the follow-up also included p24 antigenemia and CD4 cell counts. The variations of these markers during the follow-up period, the correlation with the clinical outcome (progressors versus nonprogressors), and the discrepancies between PV and surrogate markers were then analyzed. A significant and stable decrease in PV titer was observed in only nonprogressors (Friedman test, p < 0.005). At the end of follow-up, 11 (73%) of the 15 non-progressors were PV responders (patients who remained or became PV- long-term), whereas all the 13 progressors were PV nonresponders (patients who remained or became PV+). These results indicated a strong correlation between PV and clinical outcome (Fischer's exact test, p < 0.0001). The persistence, increase, or reappearance of viral replication appeared to be an important predictor of poor clinical outcome in HIV-infected patients under AZT treatment. This finding could provide a rational basis to help the clinician's decision in the clinical treatment of HIV-infected patients.

Relevance of 10 Caucasian HLA haplotypes in searches for unrelated bone marrow donors for 100 patients from a single center.

Unrelated donor searches for 100 Caucasian patients were referred to France Greffe de Moëlle Registry (FGM) from September 1987 (24,600 donors) to December 1993 (71,500 donors, 61% DR typed). After DR typing of HLA-A,B matched donors, unsuccessful searches were extended to other European Registries for 36 patients. Twenty two patients had a donor (FGM: 19, other Registries: 3) selected on: (1) HLA-A,B and DRB,DQB1 split identity; and (2) unidirectional relative response < 5% in MLR performed twice. Estimated probability of finding a compatible donor at 9 months in FGM was 12% (s.e. +/- 4%) and 25% at 2 years (s.e. +/- 6%). This probability was stringently dependent on a phenoidentity to one very common HLA-A,B,DR or B,DR haplotype (25% at 9 months when present, representing 19 of 19 patients with a compatible donor). Without this phenoidentity, the probability was zero per cent (P = 0.0001) in FGM searches and < 4% (n = 1) in extended searches. The MLR test was shown to be insensitive for screening for DPB1 mismatches. Clinical status influenced the probability of finding a compatible donor at one year ranging from 9% +/- 9% for ALL to 23% +/- 8% for CML (NS). Disregarding DPB1 mismatches is the most efficient way of increasing search efficiency.

Ex vivo studies of polymorphonuclear neutrophils from patients with early-onset periodontitis (III). CR3 and LFA-1 expression by peripheral blood and gingival crevicular polymorphonuclear neutrophils.

In this study, we assessed the LFA-1 (CD18/CD11a) and CR3 (CD18/CD11b) expression on peripheral polymorphonuclear leukocytes (PB-PMN) and crevicular fluid polymorphonuclear leukocytes (CF-PMN), by subjects with a healthy periodontium (n = 7), gingivitis (n = 8), early-onset periodontitis (n = 17) and adult periodontitis (n = 8). Using flow cytometry analysis, the %s of CD18, CD11a and CD11b positive cells and the absolute numbers of fluorescent molecules were determined. No significant difference could be found among the 4 groups, for these 2 kinds of parameters, in PB-PMN or CF-PMN. However, a great difference could be noted between the results obtained from PB-PMN and those obtained from CF-PMN. The %s of positive CF-PMN were significantly lower than those of PB-PMN for the 3 sub-units (p < 0.001). The levels of CD18 and CD11b expressed by CF-PMN were higher than those expressed by PB-PMN and the difference was significant for CD11b (p < 0.001). On the contrary, the level of CD11a expressed on CF-PMN was significantly lower than that expressed by PB-PMN (p < 0.001). Hence, our current results show that early-onset periodontitis PMN can be quite normal and this fact is not surprising insofar as, in our study, these cells were perfectly functional and all the subjects were in good health. We concluded that the analysis of the leukocyte adhesion receptors expression on PB-PMN does not appear useful for helping to establish a differential diagnosis between the different forms of periodontitis.(ABSTRACT TRUNCATED AT 250 WORDS)

Interleukin 12 induces the differentiation of major histocompatibility complex class I-primed cytotoxic T-lymphocyte precursors into allospecific cytotoxic effectors.

The production of interleukin 12 (IL-12) following allogeneic stimulation and its involvement in the differentiation of allospecific cytotoxic T lymphocytes (CTLs) have been investigated. Supernatants of mixed lymphocyte cultures had detectable levels of IL-12 p40 which were completely abrogated after depletion of responder cells from monocytes. While addition to the culture of anti-IL-12 neutralizing antibodies partially inhibited the allogeneic proliferative response and the subsequent CTL activity, addition of IL-12 stimulated both responses, suggesting that endogenously produced IL-12 plays a role in the development of alloreactivity. Furthermore, using primary mixed cultures of lymphocytes from major histocompatibility complex-recombinant siblings identical for class II antigens and displaying class I disparity, we demonstrated that addition of recombinant IL-12 at the sensitizing phase of the primary mixed lymphocyte culture induced CTL activity. Under these stimulation conditions, addition of recombinant IL-12 also triggered cell proliferation, indicating that IL-12 provides both growth and differentiation signals. The mechanism underlying this process does not appear to require IL-2, since IL-12-mediated CTL generation was not abrogated by anti-IL-2 alpha-chain antibodies. IL-12 increased granzyme B and perforin mRNA accumulation in major histocompatibility complex class I-primed lymphocytes, suggesting that this cytokine activates these two genes in CTL precursors. We conclude that IL-12 can stimulate the generation of alloreactive CTLs. We suggest that IL-12 may play a role in helper cell-independent CTL generation.