Effect of corticosteroids on the human immune response. Suppression of mitogen-induced lymphocyte proliferation by "pulse" methylprednisolone.
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Biomedical subjects
Publications and source records attributed to D T Yu.
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In spite of the increasing use of single and multiple pharmacologic intravenous pulses of MPS for immunosuppression in various diseases, their immunosuppressive effects have not been documented. We treated two groups of six patients with classic RA unresponsive to conventional therapy with either one or three daily 1 gm intravenous doses of MPS and measured the immune response and clinical activity over 16 weeks. Lymphocytopenia with selective T lymphocyte suppression was noted 2 hr following each infusion, which was maximal at 6 hr with complete recovery 24 hr after each dose beyond which no lymphocytopenia or T lymphocyte depletion was seen. Preservation of skin test positivity to recall antigens such as PPD and histoplasmin, rise in antibody titers to the secondary antigens tetanus and typhoid, and primary antibody response to KLH were found in both groups after treatment. Serum gamma globulin concentrations were unchanged. Five of six patients receiving 3 doses and three of six receiving 1 dose had satisfactory improvement in clinical parameters, with maximal benefit seen within the first 4 days. Six patients still felt better at 4 weeks, and one patient in each group entered a clinical remission greater than 16 weeks. We conclude that higher and repeated doses of MPS caused neither greater lymphocytopenia nor more prolonged suppression of recirculating lymphocytes than the conventional oral doses. The clinical benefits stem from reduction of inflammation, and it is doubtful that pulse therapy by itself induced significant generalized immunosuppression.
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When human lymphocytes were incubated with rabbit red blood cells (RRBC) in vitro at 4 degrees C overnight, they adhered to form rosettes. 44 +/- 4.5% of thymocytes and o.7 +/- o.3% of peripheral blood lymphocytes (PBL) formed these rosettes. The rosette-forming cells (RFC) increased to 10--40% when the PBL were stimulated by mitogens. The increases were detected 4 h after culture and reached a maximum at 24 h, thus preceding significant increases in DNA syntheses. In another set of experiments, PBL were stimulated by a range of concentrations of each of 5 mitogens. The concentrations whick led to high DNA synthesis rates also generated high percentages of the rosettes. Lastly, PBL were activated by mitogens and the effect of inhibitors of DNA synthesis, protein synthesis and ATPase activity investigated. Only the latter could inhibit the generation of the RFC. In conclusion, 1y RRBC rosettes in mitogen stimulated human PBL were markers of activated lymphocytes, 2) the percentages of RFC could constitute a reliable index of mitogenic responses, and 3) this index was independent of the usual criteria of protein and DNA synthesis.
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Peripheral blood T (SRBC rosette) and B (AgG- and C-receptor) lymphocyte subpopulations and responsiveness to phytohaemagglutinin (PHA) were assayed in 40 patients with ankylosing spondylitis and in 55 normal subjects. There was no significant difference in the lymphocyte concentrations or responsiveness to PHA between the two groups. However, the percentages of T lymphocytes were significantly lower in the patients irrespective of their HLA typing. This was probably due to an increase in the 'null' population since the percentages of both the AgG- and C-receptor cells were normal.
Rosette formation between human lymphocytes and horse red blood cells could be promoted by a low pH medium, overnight incubation and a temperature of 4 degrees C. The percent of sheep, horse and human rosette-forming cells in the peripheral blood were 71.7 +/- 1.8, 30.5 +/- 2.8 and 28.3 +/- 3.4 respectively. However, their percentages in thymuses were 97.1 +/- 1.1, 91.4 +/- 2.4 and 89.0 +/- 3.4. Using preparations of isolated subpopulations, it was observed that the horse and human red cell rosette-forming cells were probably also "early" sheep red cell rosette-forming cells. Rosette formation with all three types of red blood cells were inhibited by a preparation of Fetuin-glycopeptide.
Eight subjects ran on a treadmill at 8 miles per hr for 10 min. All developed lymphocytosis with predominant effect on the B cells. This was repeated 5 hr after receiving 60 mg of Prednisone. The lymphocytosis of both T and B cells were suppressed. When this was repeated 2 hr after receiving Prednisone, only lymphocytosis of the T subpopulation was suppressed. It was concluded that corticosteroid administration could suppress entry of lymphocytes into the circulation, with preponderant effect on the T subpopulation.
Human peripheral blood mononuclear cell preparations, after treatment by neuraminidase plus galactose oxidase, stimulated untreated lymphocytes. The increases in tritiated thymidine incorporation in the responder lymphocytes were observed after 48 h of mixed cell cultures. Monocytedepleted lymphocyte preparations were equally effective stimulator cells. Both purified T and B fractions were effective stimulator cells. On the other hand, only the T but not the B fractions could respond to the stimulation. The response of the cells to this type of stimulation was suppressed by 10(4)-10(7) M of the corticosteroid preparation methylprednisolone. When the cells treated with neuraminidase plus galactose oxidase were cultured alone for 48 h, they lost their stimulating capacity. However this loss could not be prevented by the presence in the culture of methylprednisolone. Hence the drug has selective suppressive activity on one type of lymphocyte activity but not the other.
Human peripheral blood lymphocytes were stimulated by concanavalin A, sodium periodate, and neuraminidase plus galactose oxidase. Response to mitogens was measured by the amount of tritiated thymidine incorporated as well as the percent of "giant sheep red blood cell rosettes" generated. The thymidine incorporation was diminished by the absence of monocytes or the presence of corticosteroids. The percent of giant rosettes generated was not influenced by either change. This finding suggested that considerable lymphocyte activation could still take place in the presence of corticosteroids. When subjects received 60 mg of prednisone, they developed lymphopenia 5 hr later. The circulating lymphocytes at that time responded less well to mitogen stimulation when measured by both thymidine incorporation and percent giant rosettes, suggesting a selective sequestration of mitogen-responsive lymphocytes outside the circulatory compartment.
A rosette-type assay of the physical interaction between lymphocytes and monocytes after treatment with neuraminidase-galactose oxidase (NGAO) is reported. Monocyte-lymphocyte (ML) rosette formation and subsequent lymphocyte proliferation occurred when either lymphocytes or homologous monocytes were treated with NGAO and cultured together. Maximal ML rosette formation took place at 37 degrees C 4 hr after culture in media containing 10% serum at lymphocyte to monocyte ratios of 10:1 to 20:1. The percentage of rosette formation correlated with the extent of thymidine incorporation when increasing concentrations of NGAO were used. When NGAO-treated monocytes were added to untreated T and non-T lymphocytes, they bound preferentially to T lymphocytes and induced proliferation only in the T subpopulation. These results indicate that the ML rosette assay measures a highly specific monocyte-lymphocyte physical interaction after a mitogenic stimulus which is an early event in lymphocyte activation since it reflects the degree of subsequent lymphocyte proliferation.
The data indicated that 2 populations of thymocytes existed: immature and mature types, identifiable by their rosette characters. The immature type was capable of changing spontaneously to the mature type, but was partially suppressed in vivo by the high concentration of thymic hormone present in the intrathymic environment. The mature types of thymocytes emigrated to the peripheral organ, accounting for their high percentage of small rosettes. Alternately, cells of peripheral organs might have originated from the immature type in the thymus. Once emigrated and exposed to a lower concentration of thymic hormone, they changed into the mature pattern. The fact that lymph node rosettes were less affected by culture in vitro indicated that once cells have changed to the mature pattern they have less ground for further differentiation.
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Peripheral blood lymphocytes from pregnant and nonpregnant females were studied for the presence of the following surface receptors: (i) Receptor for heat-aggregated human IgG (AggIgG); (ii) Receptor(s) for complement components; (iii) Receptor for sheep red blood cells (SRBC). Absolute numbers of lymphocytes with receptors for complement and those with receptors for SRBC were present in equal numbers in both groups. However, in the pregnant females there was a significantly lower number of lymphocytes with receptors for AggIgG. It is hypothesized that some of the immunological changes which occur during pregnancy may be mediated partly through changes in the total numbers of certain lymphocyte subpopulations.
The effect of administration of 0-2 mg of epinephrine (Parke-Davis) on circulatory lymphocytes was investigated in fifteen normal subjects. Blood samples were taken prior to, 10 and 20 min after, subcutaneous injections. Thymus-derived (T) cells were assayed by sheep red blood cell rosettes, bone marrow-derived (B) cells by their complement receptors and immunofluorescent detection of Fc receptors plus surface immunoglobulins. Their percentages prior to injections were 72-2 +/- 1-4, 13-8 +/- 1-1, and 20-3 +/- 1-3. Ten minutes after injections, the absolute concentrations of these cells increased to 164 +/- 14%, 326 +/- 57%, and 272 +/- 45% respectively of the values prior to injection (averages +/- standard errors). Further, when cells with receptors for both sheep red blood cells and complement were assayed by simultaneous rosette technique, they increased from 2-5 +/- 0-4% to 10-5 +/- 1-3% of the lymphocytes. Such changes were also observed in three subjects who had undergone splenectomy more than 2 years previously, but not in four subjects receiving injections of saline instead of epinephrine.
Giant SRBC rosette-forming cells were detected in samples of mitogen-stimulated human peripheral blood lymphocytes. When the concentrations of mitogens were varied, the amount of [3H]TdR incorporated by the lymphocytes also varied. In general, the higher the amount of [3H]TdR incorporated, the higher were the percentages of giant rosettes. Hence the percentages of rosettes constituted a reliable index of mitogenic responses. Lymphocytes were stimulated by mitogens and cultured in the presence of cardiac glycosides or inhibitors of the synthesis of DNA, RNA or protein. The generation of giant SRBC rosette-forming cells was found to be dependent on RNA and protein synthesis and the integrity of membrane Na+ K+ ATPase, but not on DNA synthesis.
Human thymus-derived lymphocytes have the ability to form rosettes with sheep red blood cells (SRBC) in vitro. In the investigation of rosettes of peripheral blood lymphocytes of 10 normal subjects, the number of SRBC adhering to the lymphocyte in each of 100 rosettes was assessed. The percentage of rosettes with SRBC greater than or equal to 36 per rosette was only 1.2 +/- 0.5. These were defined as giant SRBC rosettes. Peripheral blood lymphocytes were stimulated in vitro by four mitogens: sodium periodate, neuraminidase plus galactose oxidase, pokeweed mitogen, and concanavalin A. The lymphocytes were then cultured at 37 degrees C. The giant rosette-forming lymphocytes became significantly increased 4 to 24 hr after stimulation, prior to the appearance of lymphoblasts or increased incorporation of tritiated thymidine. The giant rosettes were not caused by the hemagglutinating properties of pokeweed mitogen and concanavalin A that were adsorbed on the lymphocyte surfaces. This was shown by the fact that, on removal of the receptors by trypsinization, they were regenerated on culture in vitro in the absence of the mitogens. It was concluded that giant SRBC rosettes constituted a marker for some of the activated lymphocytes. Their appearance was independent of the increase in size of the cells or of DNA synthesis. These receptors were intrinsic to lymphocytes and not caused by mitogens adsorbed on their surfaces.
In the process of forming SRBC rosettes of human lymphocytes, two types were distinguished, the early and late rosettes. The former appeared immediately whereas the latter required further incubation at 4degreesC. In this study, the two types of cells were separated by gradient centrifugation and studied separately. It was found that the proportion of caps in the late rosettes was much greater than in the early ones. Treatment of the former by trypsin, AET, and neuraminidase decreased this proportion so that it resembled the late one. It was postulated that some of the differences in the early and late RFC lie in the differences in the content of membrane sulphydryl and sialic acid groups and distribution of SRBC receptors.