Enhancement of phytohemagglutinin-induced DNA synthesis of mouse thymocytes and T-lymphocytes by a neutral protease of polymorphonuclear leukocytes.

Mouse thymocytes were poorly triggered in vitro with phytohemagglutinin (PHA) to induce DNA synthesis and the response was markedly enhanced by culture supernatant (SUP) of polymorphonuclear leukocytes (PMN). The PMN factor was nondialysable, heat-labile and precipitated with 65% ammonium sulphate. Its molecular weight was approximately 19,000 on sephadex G-75. The sephadex fraction had a proteolytic activity on 3H-acetyl hemoglobin at neutral pH. The protease seemed to be a chymotrypsin-like enzyme on the basis of inhibition profile using various protease inhibitors. The thymocyte-helping activity in the protease fraction was absorbed by affinity columns of protease inhibitors. The PMN-protease also enhanced the DNA synthesis by antigen-stimulated lymph node cells and by PHA-stimulated T-lymphocytes, but not by LPS-stimulated B-lymphocytes. Only the lymphocytes already been stimulated with antigen or mitogen received preferentially the helping action by PMN-protease. The helping activity was effectively absorbed by PHA-stimulated thymocytes but not by nonstimulated thymocytes. These evidences seems to suggest that acceptor sites for the protease, newly developed on lymphocyte surface after stimulation, may play an important role in the enhanced DNA synthetic response.     

In vitro reactivity of human lymphocytes in chronic uraemia: analysis and interpretation

Altered cellular immunity complicating chronic uraemia includes lymphocytopenia, thymic atrophy, impaired allograft rejection and delayed hypersensitivity in skin tests, diminished appearances of lymphocytes on skin windows, and shortened in vitro survival of uraemic lymphocytes. Studies were undertaken to further characterize these lymphocyte defects.

Lymphocytes separated from peripheral blood of twenty-four uraemic patients were compared with those from fifty-one normal persons in their rates of RNA and DNA synthesis in both PHA-stimulated and unstimulated cultures, as determined by incorporation of radiolabelled precursors. Synthesis, expressed as absolute incorporation/10⁶ viable lymphocytes, was accelerated in the majority of both stimulated and unstimulated uraemic cultures. Serial studies over several months of twenty-five uraemic patients (fifteen maintained by haemodialysis, ten by renal allotransplantation), compared with nineteen normal controls, showed these differences to be consistent and persistent. While normal lymphocytes exhibited stability, uraemic cells fluctuated widely in their serial synthesis rates. In 25% of such cultures, unstimulated synthesis exceeded that induced by PHA. Increased synthesis without PHA, reflecting enhanced spontaneous blastogenesis, is compatible with decreased survival, numbers, and functional capacity of uraemic lymphocytes.

Reports by others of diminished PHA-responsiveness of uraemic lymphocytes are based upon whole-culture incorporation and/or expressed as ratios between stimulated and unstimulated cultures. Both shortened survival and accelerated spontaneous nucleic acid synthesis by uraemic lymphocytes cause such ratios to be misleadingly low. Such factors as cell numbers and viability at harvest, counting efficiency, and culture sterility are essential to avoid misinterpretations of such data based on incorporation of radiolabelled precursors to the nucleic acids.

     

ENHANCEMENT OF PHYTOHEMAGGLUTININ-INDUCED DNA SYNTHESIS OF MOUSE THYMOCYTES AND T-LYMPHOCYTES BY A NEUTRAL PROTEASE OF POLYMORPHONUCLEAR LEUKOCYTES

Mouse thymocytes were poorly triggered in vitro with phytohemagglutinin (PHA) to induce DNA synthesis and the response was markedly enhanced by culture supernatant (SUP) of polymorphonuclear leukocytes (PMN). The PMN factor was nondialysable, heat-labile and precipitated with 65% ammonium sulphate. Its molecular weight was approximately 19,000 on sephadex G-75. The sephadex fraction had a proteolytic activity on 3H-acetyl hemoglobin at neutral pH. The protease seemed to be a chymotrypsin-like enzyme on the basis of inhibition profile using various protease inhibitors. The thymocyte-helping activity in the protease fraction was absorbed by affinity columns of protease inhibitors. The PMN-protease also enhanced the DNA synthesis by antigen-stimulated lymph node cells and by PHA-stimulated T-lymphocytes, but not by LPS-stimulated B-lymphocytes. Only the lymphocytes already been stimulated with antigen or mitogen received preferentially the helping action by PMN-protease. The helping activity was effectively absorbed by PHA-stimulated thymocytes but not by nonstimulated thymocytes. These evidences seems to suggest that acceptor sites for the protease, newly developed on lymphocyte surface after stimulation, may play an important role in the enhanced DNA synthetic response. ACTA PATH. JAP. 27: 857-868, 1977.     

Increased expression of CR3 (C3bi receptor) on neutrophils in human inflammatory skin reactions

To help determine whether the neutrophils (PMN) found in skin inflammatory reactions are activated, we have compared the expression of the C3bi receptor (CR3) on such cells with that on autologous blood PMN in 10 pollen-sensitive subjects. Using skin chambers overlying denuded blister bases we collected PMN at 2 or 4 hr at sites of challenge with pollen antigen or buffer solution. These cells and PMN in autologous blood were incubated with monoclonal anti-CR3 antibody and the expression of CR3 was measured by indirect fluorescence and flow cytometry. Significantly more PMN were found at antigen than at buffer sites at 2 hr (7.02±0.45 × 10⁵ vs 0.71±0.25×10⁵) and at 4 hr (2.2±0.57×10⁶ vs 5.47×10⁵). The mean CR3 expressions on PMN at antigen and buffer sites were similar (117±7.4 vs 118±9.0); both were significantly greater than on blood PMN (17.6±1.5;P<0.005). PMN from both sites could be stimulated furtherin vitro with formyl-methionyl-leucyl-phenylalanine (FMLP) to express more CR3 to a level even greater than in FMLP-stimulated blood PMN (155±11 and 157±12, respectively, vs 108±7 in blood PMN). The incubation of blood PMN with the noncellular component of the chamber fluid led to a moderate (28–100%) increase in CR3 expression, but far less than the CR3 expression on the chamber fluid PMN themselves. Since surface CR3 is thought to be an activation marker important in PMN adhesion, these findings may be important in understanding the emigration of PMN in skin inflammatory reactions.     

Enhancement of in vitro immunoglobulin synthesis of human lymphocytes by lysosomal enzymes from polymorphonuclear leucocytes.

The effect of human peripheral blood polymorphonuclear leucocyte (PMN) extracts and PMN granule lysates on in vitro immunoglobulin (Ig) synthesis by autologous peripheral blood mononuclear cells was studied. The mononuclear cells were cultured for 3 days with or without autologous plasma. Newly synthesized Ig in the culture supernatants was measured using 14C-labelled amino acids by an immune coprecipitation method. Upon addition of a PMN extract to plasma-free cultures Ig synthesis was stimulated, the mean stimulation index (SI) of cultures from thirteen individuals, including nine normals, three patients with rheumatoid arthritis and one with psoriatic arthritis being 1-8 +/- 0-2 in comparison with control cultures (P less than 0-05). By contrast, in 10% fresh autologous plasma, PMN extracts yielded a mean SI of 0-9 +/- 0-1 indicating inactivation of the active extracts by plasma inhibitors. In experiments using PMN granule lysates containing high concentrations of beta-glucuronidase and cultured in RPMI 1640, the mean stimulation index was 3-2 +/- 0-7. Stimulation of Ig synthesis was also produced by trypsin. Stimulation of Ig synthesis was also produced by trypsin. Stimulating factors in PMN extracts were inhibited by Trasylol, a protease inhibitor. These results indicate that trypsin and proteolytic lysosomal enzymes in PMN increase Ig synthesis of human peripheral blood mononuclear cells. They suggest a possible new role of PMN in the potentiation of immunoglobulin synthesis.     

Mitogenicity of Mycoplasma fermentans for human lymphocytes.

The in vitro stimulation response of human lymphocytes to Mycoplasma fermentans was examined. M. fermentans stimulated DNA synthesis in blood lymphocytes from all of 20 healthy subjects examined. Only one of these subjects had complement-fixing antibodies to M. fermentans. Lymphocytes from 21 of 22 adenoids and from 1 spleen were also stimulated to DNA synthesis by M. fermentans. The organism induced DNA synthesis in both B and T lymphocytes from adenoids and spleen and preferentially in T lymphocytes from blood. M. fermentans was shown to activate adenoid lymphocytes to non-amtogem-specific antibody secretion demonstrable by a hemolytic plaque assay. It is concluded that M. fermentans can have a mitogenic effect on both B and T lymphocytes.     

Production of PAF-acether by synovial fluid neutrophils in rheumatoid arthritis

PAF-acether (PAF) is a pro-inflammatory phospholipid molecule potentially involved in the pathogenesis of arthritis. PAF and related metabolites have been isolated in the synovial fluid from patients with arthritis.The aim of this study was to determine PAF production by neutrophils isolated from synovial fluid and blood in patients with rheumatoid arthritis. Blood neutrophils from normal donors were also studied for their capacity to form PAF. Neutrophils were stimulated with the calcium ionophore A23187 (2µM) for 1 to 60min. PAF released in the medium and PAF associated to cells were measured.In synovial fluid neutrophils, PAF production began as soon as 1 min of stimulation (16.1 ± 6.3 pmol per 1 × 10⁶ cells) and reached a maximum at 20min: 29.2 ± 2.8 pmol per 1 × 10⁶ cells (mean ± SEM, n = 5). The amount of PAF released in the supernatant increased with the length of stimulation and was maximal after 30min (33.5%, percentage of released over total PAF, n = 5). After A23187 stimulation, similar amounts of PAF were produced by blood neutrophils from RA and control patients. However, neutrophils isolated from the joint had a lower capacity to produce PAF than blood neutrophils from the same patients.The present results demonstrate the synthesis and release of PAF by synovial fluid neutrophils. They suggest that neutrophils may be the source of PAF locally present in the joint. Newly synthetised PAF could participate in the amplification of the local inflammatory reaction.     

In vitro studies of lymphocytes from patients with plasma cell myeloma. I. Stimulation by mitogens and cytotoxic activities

Highly purified blood lymphocytes from patients with plasma cell myeloma were tested in different in vitro systems. The patients were untreated or had received a standardized 4-day treatment with melphalan and prednisolone every sixth week. They were tested 5 weeks after the last treatment to minimize the acute toxic effects of the drugs. Lymphocytes from healthy controls were included in each experiment.

Stimulation of lymphocytes was measured by incorporation of ¹⁴C-thymidine into DNA after activation with phytohaemagglutinin (PHA), concancavalin A (Con A) and pokeweed mitogen (PWM). Their cytotoxicity was tested against Chang cells (human cell line) and chicken red blood cells in presence of PHA or heat-inactivated rabbit antibodies to target cell antigens. Lysis was quantitated as release of radio-activity from target cells labelled with ⁵¹Cr-chromate.

Antibody-induced cytotoxicity of lymphocytes from untreated patients was normal or slightly elevated while that of treated patients was severely depressed. Also, lymphocytes from treated patients were significantly less stimulated to DNA synthesis by PWM than were control lymphocytes. PHA-induced cytotoxicity and stimulation of lymphocytes by Con A or PHA were normal in all groups.

These results suggest that treatment of myeloma patients with melphalan and cortisone selectively impairs lymphocytes which respond to PWM by DNA synthesis and which participate in antibody-mediated cytolysis.

     

Some properties of a phytohemagglutinin-induced lymphocyte mitogenic factor

The principles governing production and the effect on lymphocytes of the mitogenic factor (MF), secreted by human lymphocytes in vitro under the influence of phytohemagglutinin (PHA) and described previously by the author, were investigated. PHA was inactivated by anti-PHA antiserum and immunosorbents. Lymphocytes stimulated by PHA secrete MF in the G₁-period and at the beginning of the S-period of the cell cycle. During cultivation of the cells in a protein-free medium DNA and protein synthesis was sharply inhibited but MF production was 1.6 times higher than in media containing serum. The kinetics of the reaction of lymphocytes to MF is described: DNA synthesis (reflected in thymidine-H³ incorporation) began on the 5th–6th day and reached an maximum on the 6th–7th day.Belorussian Research Institute of Hematology and Blood Transfusion, Minsk. (Presented by Academician of the Academy of Medical Sciences of the USSR N. A. Fedorov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 80, No. 9, pp. 76–79, September, 1975.     

The role of macrophages in the activation of T lymphocytes by concanavalin A. III. Macrophage-independent activation

Lymphocytes from the mesenteric lymph nodes of Lewis rats were depleted of macrophages by absorption for 60 min at 37°C on 0.1-mm glass beads in the presence of 30% fetal calf serum. Lymphocytes which passed the column (total recovery 30-50%) contained less than 0.2% macrophages, as assessed by neutral red ingestion or nonspecific esterase staining. At cell densities which allowed optimal proliferation of concanavalin A (Con A)-stimulated, untreated lymphocytes (2 × 10⁶ cells/ml), macrophage-depleted lymphocytes were completely incapable of initiating DNA synthesis. The proliferative response to Con A was fully restored by adding back macrophages obtained from the peritoneal cavity. The help of macrophages was optimal at concentrations of 0.8 × 10⁵ cells/ml, which corresponds to about 4% of lymphocytes. Restitution of the mitotic response of macrophage-depleted lymphocytes was also achieved by raising cell densities to 5 × 10⁶/ml. At this cell density, addition of macrophages was without any effect. Early events in lymphocyte activation, such as the increase of the incorporation of ¹⁴C-oleate into membrane phospholipids after 4 h stimulation, was identical in the presence or absence of macrophages independent of the cell density ranging between 1 × 10⁶ and 10 × 10⁶ cells/ml. Similarly, the Con A-stimulated early increase in the incorporation of [³H]uridine into RNA was indistinguishable in macrophage-containing or -depleted lymphocytes. At low cell densities, only macrophage-containing lymphocytes proceeded to increase RNA synthesis, until S-phase, whereas in the absence of macrophages RNA synthesis approached background values after 40 h of Con A stimulation. Macrophage-depleted lymphocytes, too, proceeded with an increase in RNA synthesis, when stimulated at high cell densities. The data suggest that macrophages are not required for the initiation of lymphocyte activation. Their role in supporting mitosis may be the contribution of a second signal close to the G1/S boundary extending the size of the proliferating population.     

Mitogenic Responses to Lipopolysaccharide by B Lymphocytes from Patients with Systemic Lupus Erythematosus

Peripheral blood lymphocytes from 43 patients with systemic lupus erythcmatosus (SLE) and from age- and sex-matched normal controls were cultured with lipopolysaccharide (LPS) to examine the response to the polyclonal B-cell activator. Lymphocytes from active SLE patients incorporated 4840±471 (mean ± SE) cpm in response to LPS, whereas lymphocytes from inactive SLE patients incorporated 6906 ± 897 cpm. In contrast, lymphocytes from normal individuals incorporated 7452 ± 1126 cpm. Ig synthesis of lymphocytes from active SLE in response to LPS stimulation was also less than that of normal individuals. The helper T-cell function of active SLE, as examined by co-culturing irradiated SLE lymphocytes with unirradiated normal lymphocytes, was normal. These results thus suggested that a defect of B lymphocytes exists in active SLE patients. This B-cell defect and T suppressor cells apparently play an important role in the pathogenous of SLE.     

The effect of organic acids on phytohaemagglutinin-activated proliferation of human lymphocytes in vitro

The present study assesses the effects of 25 organic acids on in vitro proliferation of human peripheral lymphocyte stimulated with phytohaemagglutinin (PHA). Lymphocytes were cultured in flat-bottomed 96-well microplates at 37°C for 96 h in the presence of the mitogen and of one acid. The concentrations of organic acids tested in the cultures were from 1 to 5 mM, corresponding to those usually found in the blood of patients with organic acidaemias. Cellular growth was measured by the incorporation of 6[³H]-thymidine into cellular DNA. We observed that tiglic (2-methylcrotonic), α-keto-β-methylvaleric, aminoadipic, sebacic and alpha-ketoisocaproic acids strongly inhibited lymphocyte DNA synthesis, whereas α-ketoisovaleric, propionic, α-hydroxy-β-methylvaleric, α-methylbutyric and isobutyric acids moderately suppressed DNA synthesis. Lactic and ethylmalonic acids, however, stimulated DNA synthesis. The most inhibitory acids were added to cultures at different times after the beginning of the incubation period. Except for tiglic acid, whose action persisted even after 48 h from the onset of cultures, the others acted only when added during the first 24 h. The present study demonstrated that organic acids modulate DNA synthesis in mitogen-stimulated human lymphocytes.     

Studies of Anti-lymphocyte Antibody of Patients with Active SLE

Effect of anti-lymphocyte antibody of active systemic lupus erythematosus (SLE) on lymphocyte function was examined. Lymphocytes from normal individuals treated with anti-lymphocyte antibody and complement exhibited marked inhibition of response to concanavalin A (Con A), while the response of lymphocytes to phytohaemagglutinin M (PHA-M) and pokeweed mitogen (PWM) was slightly affected. In mixed lymphocyte culture response, both stimulator and responder cells were insensitive to anti-lymphocyte antibody. Treatment of sensitized lymphocytes with anti-lymphocyte antibody and complement caused a dose-dependent suppression of blastogenic response to purified protein derivatives (PPD). No effect, however, was noted on migration-inhibitory factor (MIF)-producing cells. In PWM-driven Ig synthesis, T lymphocytes lacking the anti-lymphocyte antibody-reactive T-cell subset enhanced PWM-driven Ig synthesis of autologous B lymphocytes. Con-A-induced suppressor function of lymphocytes was abolished by the treatment with anti-lymphocyte antibody and complement. The present study demonstrated that lymphocytes from normal individuals after treatment with anti-lymphocyte antibody and complement showed similar immunological reactivities with lymphocytes from active SLE, indicating that those anti-lymphocyte antibodies could play an important role in defective suppressor cell function.     

The influence of synovial fluids from patients with rheumatic diseases on chick embryo fibroblasts

Primary chick-embryo fibroblasts (PCEF) were used as target cells to measure the influence of synovial fluids of patients suffering from osteoarthritis (OA; n = 5), rheumatoid arthritis (RA; n = 12) and psoriatic arthritis (PA; n = 2). The following metabolic cell products were measured: DNA, RNA, glycosaminoglycans (GAG), sulfated glycosaminoglycans, protein and collagen, with the same joint effusions being used in each test. Since it is not a single substance that provokes a stimulating or inhibiting effect in the joint, the crude synovial fluids were applied in these preliminary experiments. It was found that each type of synovial fluid showed an influence on the biological processes in the PCEF. The DNA, RNA and GAG syntheses were strongly influenced by the joint effusions, in contrast to the protein, collagen and sulfated glycosaminoglycan syntheses which were less affected. Generally, the nucleic acid synthesis differed significantly between the OA, RA and PA synovial fluids. The addition of heparin to the synovial fluids caused an additive inhibiting effect on the DNA synthesis but did not influence the other biochemical parameters. The synovial fluids of RA patients, and to a much greater extent those of PA patients, inhibited the thymidine incorporation whereas OA synovial fluids had a less pronounced effect. This result indicates a disease-dependent composition of the synovial fluids. RNA synthesis was diminished in all three groups, but again this effect was strongest in the case of the PA synovial fluids. GAG synthesis was markedly stimulated by the PA synovial fluids and somewhat, though to a lesser extent, by the OA and RA synovial fluids. The sulfated glycosaminoglycan synthesis in the PCEF, as revealed by 35S incorporation into the GAG, was less influenced and on the whole stimulated by the OA and RA synovial fluids. The same trend could be observed with regard to the collagen synthesis. The intracellular protein synthesis was less influenced by the OA (91.9%) and more strongly suppressed by the RA (78.7%) and the PA (76.7%) synovial fluids. PCEF therefore appear to be a convenient and sensitive target cell system to study alterations of biochemical processes caused by crude synovial fluids and also of different origin by individual factors isolated from synovial fluids.     

Effect of stimulated neutrophils from the synovial fluid of patients with rheumatoid arthritis on lymphocytes—A possible role of increased oxygen radicals generated by the neutrophils

Neutrophils from the synovial fluid (SFN) of 10 patients with active rheumatoid arthritis (RA) were investigated to determine the generation of oxygen intermediates (OI) (O2-, H2O2, OH .), chemiluminescence, and lysosomal enzymes (lysozyme and beta-glucuronidase). Lymphocytes from healthy individuals were cocultured at 37 degrees C for 17 hr with SFN from the patients and the number of OKT4+, OKT8+, and OKT3+ cells and the response to mitogens were determined. A markedly increased OI and slightly elevated lysosomal enzyme levels were observed in SFN from patients. Coculture of lymphocytes with SFN resulted in a decreased number of OKT4+ and OKT8+ cells and a greatly reduced response to Con A and mildly diminished response to PHA, while OKT3+ cells were not affected. The simultaneous addition of superoxide dismutase and catalase restored the impairment of monoclonal antibody reaction and lymphocyte responsiveness almost to control levels. It is suggested that the disturbed immunoreactivity of synovial fluid lymphocytes from RA patients may be due to increased OI generated by stimulated neutrophils.     

Mixed lymphocyte reactions in the rabbit using peripheral blood cells: the effects of cell preparation and skin grafting.

The magnitude of mixed lymphocyte reactions using rabbit peripheral blood cells is subject to a number of variables including the method of cell preparation, the culture conditions employed, the genetic relationship of the cell donors, the number of bleedings immediately preceding the test bleed and the length of time that the cells are cultured. Mixed lymphocyte reactions (MLR) were carried out in microcultures and optimal responses were obtained when cultures contained 1 × 10⁶ cells and were fed daily. Both two-way and one-way MLR were done using irradiation or mitomycin-C to inhibit the response of one donor population. The responses between various pairs of rabbits fell into three categories: high responders with 6,000–9,000 cpm ¹²⁵IUdR incorporated into DNA, low responders with 1,000–3,000 cpm incorporated and non-responders with 20–70 cpm incorporated. In all cases the background incorporation was less than 70 cpm. There was no MLR prior to the third day of culture; the response peaked on the fifth or sixth day and declined thereafter. Repeated bleeding of the rabbits at short intervals resulted in decreased MLR. Lymphocytes derived from defibrinated blood were primarily responding cells whereas lymphocytes from heparinized blood were stimulatory cells. The responding cells were sensitive to killing by anti-thymocyte serum (ATS) and complement. There was some evidence that the magnitude of MLR is affected by genetic relatedness. The duration of skin graft survival in these studies was not related to the magnitude of MLR.     

T lymphocyte proliferation is suppressed by the opioid growth factor ([Met(5)]-enkephalin)-opioid growth factor receptor axis: implication for the treatment of autoimmune diseases

Opioid peptides function as immunomodulatory molecules. Reports have linked the opioid growth factor (OGF), [Met⁵]-enkephalin, and its receptor OGFr to autoimmune diseases. OGF repressed the incidence and magnitude of myelin oligodendrocyte-induced experimental autoimmune encephalomyelitis in mice. Given the extensive connection between the immune system and autoimmune diseases, the present study was conducted to examine the relationship of the OGF-OGFr axis and T lymphocyte proliferation. Splenic-derived mouse lymphocytes were stimulated with phytohemagglutin (PHA). All non-stimulated and PHA-stimulated T lymphocytes had immunoreactivity for OGF-like enkephalin and OGFr. OGF markedly suppressed T lymphocyte number in a dose-dependent manner. However, PHA-stimulated T lymphocytes were not altered in cell number by a variety of natural and synthetic opioid-related compounds, some specific for μ, δ, and κ opioid receptors. Persistent blockade of opioid receptors with the general opioid antagonist naltrexone (NTX), as well as antibody neutralization of OGF-like peptides, had no effect on cell number. Non-stimulated T lymphocytes exhibited no change in cell number when subjected to OGF or NTX. Treatment of T lymphocytes with siRNAs for μ, δ, or κ opioid receptors did not affect cell number, and the addition of OGF to these siRNA-exposed cultures depressed the population of cells. T lymphocytes treated with OGFr siRNA also had a comparable number of cells to control cultures, but the addition of OGF did not alter cell number. DNA synthesis in PHA-stimulated T lymphocytes exposed to OGF was markedly decreased from PHA-stimulated cultures receiving vehicle, but the number of cells undergoing apoptosis or necrosis in these cultures was similar to control levels. T lymphocytes subjected to siRNA for p16 and/or p21 had a comparable number of cells compared to controls, and treatment with OGF did not depress cell number in preparations transfected with both p16 and p21 siRNA. These data reveal that the OGF-OGFr axis is present in T lymphocytes and is capable of suppressing cell proliferation. However, T lymphocytes are not dependent on the regulation of cell proliferation by this system. The results showing that the OGF-OGFr axis is an immunosuppressant, offers explanation for reports that autoimmune diseases can be modulated by this system.     

Mitogenic responses to lipopolysaccharide by B lymphocytes from patients with systemic lupus erythematosus

Peripheral blood lymphocytes from 43 patients with systemic lupus erythematosus (SLE) and from age- and sex-matched normal controls were cultured with lipopolysaccharide (LPS) to examine the response to the polyclonal B-cell activator. Lymphocytes from active SLE patients incorporated 4840 +/- 471 (mean +/- SE) cpm in response to LPS, whereas lymphocytes from inactive SLE patients incorporated 6906 +/- 897 cpm. In contrast, lymphocytes from normal individuals incorporated 7452 +/- 1126 cpm. Ig synthesis of lymphocytes from active SLE in response to LPS stimulation was also less than that of normal individuals. The helper T-cell function of active SLE, as examined by co-culturing irradiated SLE lymphocytes with unirradiated normal lymphocytes, was normal. These results thus suggested that a defect of B lymphocytes exists in active SLE patients. This B-cell defect and T suppressor cells apparently play an important role in the pathogenesis of SLE.     

Effect of piroxicam therapy on granulocyte function and granulocyte elastase concentration in peripheral blood and synovial fluid of rheumatoid arthritis patients

The effect of piroxicam therapy (20 mg/day for 15 days) on various polymorphonuclear granulocyte (PMN) responses and on PMN elastase concentration was investigated in nine patients with active rheumatoid arthritis. Peripheral biood and synovial fluid samples were collected before starting therapy and 12 h after the last dose of the drug. All patients were evaluable for peripheral blood analysis and six for synovial fluid analysis. Piroxicam therapy had no effect on PMN random migration and phagocytosis, while it significantly reduced both FMLP-induced aggregation and FMLP-induced chemotaxis. This seems mainly due to an effect on FMLP binding, as no differences were observed after therapy in PMA- and PHA-induced aggregation as well as in serum-induced chemotaxis. In contrast, a marked impairment of NBT test and PMA- and FMLP-induced superoxide anion (O₂ ^–) production was found after piroxicam therapy. This effect was as evident in peripheral blood as in synovial fluid PMN. Also, a significant reduction in synovial fluid PMN number and synovial fluid PMN elastase concentration (elastase- ₁-proteinase complex) was found after treatment. It is concluded that piroxicam may act at different sites on various PMN responses-Its effect on O₂ ^– generation and PMN elastase concentration in synovial fluid may have an important role in reducing destruction of arthritic joint tissue.     

Sensitivity of cultured lymphocytes from patients with nevoid basal cell carcinoma syndrome to ultraviolet light and phytohemagglutinin stimulation

DNA repair and replication after in vitro UV irradiation were determined in cultured peripheral blood lymphocytes from 6 patients with nevoid basal cell carcinoma syndrome (NBCCS) and from a group of control donors. DNA repair synthesis (UDS) was measured in unstimulated lymphocytes by incubation with 3H-TdR in the presence of hydroxyurea for 3 and 6 h after UV irradiation (6-48 J/m2). DNA replication was measured in PHA-stimulated lymphocytes, UV-irradiated or mock-irradiated, by incubation with 3H-TdR for 24 h. The effect of the mitogen was followed during 5 days after stimulation by determining the incorporation of 3H-TdR, the increase of cell number, and the mitotic index. NBCCS and control lymphocytes showed equal sensitivity to UV light in terms of UDS and reduced response to PHA. On the contrary, the mitotic index and the number of cells in stimulated cultures were significantly lower in the affected subjects. These data suggest an altered progression along the cell cycle, which could be characteristic of stimulated NBCCS lymphocytes.