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Regulation of the human immune response to Cryptococcus neoformans by interleukin-10
540
I Abstracts
CYTOKINE,
PI October 4: Lymphokinedcytokines
Vol. 6, No. 5 (September
1994: 539-582)
in Immune Regulation
A7
A10 The Effects of Cytokines and Cytokine Inducers with Recombinant Proteins. Huw P. A. Hughes, Pharmaceuticals, New York, NY, 10022-6030.
on Vaccination M6
Many cytokines have been used as adjuvants with killed, synthetic and recombinant vaccines. A number of studies have indicated that the dose of cytokine and its precise method of administration are of prime importance for optimal efficacy. There is increasing evidence that large doses of a single cytokine can result in undue side effects, such as autoimmune disease. Previously, low doses of recombinant IL-2 were found to be effective in enhancing immune responses to bovine herpes virus-l gD. However, this only occurred following the simultaneous use of Avridine, a potent IL-1 and IFN-gamma inducer. These studies indicate that low doses of cytokine can be effective vaccine adjuvants, but certain conditions may apply. These include (a) activation of antigen presenting cells, (b) sustained delivery of cytokine for a defined period of time and (c) delivery of cytokine to the same site (draining lymph node) as the antigen.
Estradiol and ProgesteroneInhibit the Induction of Nitic Oxide Synthase in Marine MacrophaSe. L. Miller. E.W. Alley. W. I. Murphy, S. W. Russell wd J. S. Hunl University of Kansas Medical Center. Kansas City, KS 66160-7400 Nitric oxide (NO) is synthesized by the inducible &form of nitric oxide syntbase ONOS) in activated murine manoohages and can act as a mtent mediator of cvmtoxic defense.Macmphages. present in i&i numbers in the uterus of pregnant mick, cantribute to the maintenance of an immunosuppressive environment believed to prot%zect the fetus from attack by the maternal immune system and perform various otba pregnancy-associated functions. Estrogen and progesterone have tab immunostimulatory and immunosuppressive effects an macrophage effecmr functions and monokine synthesis. The purpose of thtis study was to determine the role of esnadial-17b(E2) and progesterone.(P4) on iNOS gene activity and NO production in murim macrophaes. Stimulation of the macrouhaae cell lines RAW 2b4.7 and 1774 and bone marrow derived macrophages with l&pt?ysacchari& (LPS) and interferon gamma (1FN-y) for 24h resulted in an accumulationof nitrite (NCQ). a stable product of the reaction of NO wirh oxygen. Concomitant incubation of cells with E2 (IpM-10~M) and P4(l&b-lO!&M) caused a concenration-dependent inhibition of NO2- production. RAW 264.7 cells were transiently uansfected with a gene comtmct containing Ihe iNOS regulatory region inserted upstream of a luciferase reporter gene.Transfected macrophages,stimulated with LPS (IW n&l) and IFN7 (IOU/ml) in the presence of E2 and P4. displayed an averageof a 50% reduction in luciferase activity (8h) and in N@accumulation (24h) when compared with cells stimulatedwith LPS plus IF’Ny alone or LPS plus IPNy and vehicle &me. This study shows that the presence of bath E2 and p4 in cultures of stimulated manophages inhibit the induction of the iNOS promotor and reduce the production of NO suggesting a possible regulatory role for these steroids in uterine macrophage activation and function. This work was supponed by NIH grant HD2.4212to J.S.H. and PO1 CA55474 to S.W.R.
A8
All ROLE OF CHOLERA
TRANSFORMING TOXIN IN IgA
GROWTH ISOTYPE
FACTOR-RI SWITCHING
AND
Pyeung-Hyeun Kim Dept. Microbial. Kangwon Univ., Chunchon 200.701 S. Korea The aim of this study was whether TGF-Ol derived from intestinal Peyer’s patch (PP) T cells and cholera toxin (CT) have an activity for IgA isotype switching. Murine autoreactive PP T cell line was stimulated with Concanavalin A and the supematants (Sn) was added to surface IgA negative (s&A) B cell culture. The acid-activatedT cell Sn in the presence of IL-2 induced a lo-fold or greater increase in total IgA isotype production and secreting ceil numbers but not IgM or IgGl isotype. This specific activity of Sn was completely abrogated with the treatment of anti-TGF-Bl antibody. In addition, it was found that freshly isolated PP T cells synthesize TGF-Bl mRNA constitutively measured by using RT-PCR. Finally, B cells were stimulated with the B subunit of cholera toxin (CTB), in the presence or absence of IL-2, and then assayed for IgA producing cells using an ELISPOT assay. CTB in the presence of IL-2, significantly increased the precursor frequency of IgA producing cells. In contrast, CTB and IL-2, in combiantion, did not alter the burst size of IgA producing clones and did not increase the rate of IgA secretion per cell. Taken together, these results show that TGF-81 can he expressed by T cells at the mucosal sutface and it switches B cells to IgA producing cells as an endogenous source, while CT or CTB has the same activity for the IgA synthesis as an exogenous source.
SERUM ALBUMIN MODIFIES THE EFFECT OF BP1 ON SURVIVAL OF NEWBORN RATS WITH E. cohK1 SEPSIS. J. Mantilla, P. Garcia, J. Gait&n and C.A. Dinarello. Inst. Investigation en Inmunologia, Universidad de Guadalajara, A. P. 2-130, Jalisco 44280, Mexico. Human serum albumin (HSA) blocks in vitro bacterial killing by BP1 (bactericidal/permeability increasing protein), without preventing BP1 binding to bacteria nor increased membrane permeability. This study shows the effects of HSA on the in viva protective effects of BP1 in newborn (NB) rats with sepsis by E. coli Kl (EcKl). Survival of NB Wistar rats infected via ip at time zero with S-30 mg/kg BPI, and via SC with a ID70 (3x106 CFU) or a ID90 (5x106) EcKl, was improved, reaching the best protection at 15 mg/kg BPI. A IDso (1x106) EcKl, and 15 mg/kg HSA, or 15 and 30 mg/kg BP1 was injected at time zero. Six rats from each group were sacrificed at zero, 6, 12, 24 h, and every 24 h. Blood was drawn by aseptic cardiac puncture and CFU/ml and WBC/mm3 were determined. Bacteremia significantly decreased in NB rats treated with 30 mg/kg BPI; and leukopenia observed in controls at 12 h was prevented by 15 mg/kg BPI, and more significantly by 30 mg/kg BPI. Low HSA dose (10 mg/kg), but not a high dose (40 mg/kg), reduced the protective effect of exogenous BPI. Besides, HSA at 40 mg/kg improved survival to a high bacterial challenge, but increased mortality by a low bacterial inoculum. This suggests that controversial effects of albumin on the BP1 actions, might be due to the proportion between bacteria and albumin concentrations.
RECXJLAl-lON OF THE HUMAN IMMUNE RESPONSE TO Cryptococcus neoformans BY INTERLEUKIN-10. C. Mody, P. Warren. University of Calgary, Alberta, Canada, T2N 4Nl. C. neoformansis an encapsulated yeast that causes meningitis and pneumonia in HIV-infected patients. Since celI mediated immunity is important for the clearance of this pathogen, we are investigating the regulation of cell mediated immunity by C. tteofonmm. previously, we have found that little y-IFN is produced by C. neofotmumstimulated lymphocytes, which could be due to regulation by IL-IO. To determine whether IL-IO is produced during the response to C. neofonmzns, peripheral blood mononuclear cells (PBMC) from healthy adults were stimulated with the ceU wall of C. neoformuns and IL-10 production was measured by ELISA in culture supematants. Lymphocyte proliferation was determined in parallel cultures. IL-10 was released early in culture by PBMC (day 3= 306 pg/ml, n=l; day 5= 715 f 277 pg/ml; n=4), but decreased later in culture (day 7= 47 f 64 pg/ml, n=3; day 9= 98 + 35 pg/ml, n=5). Lymphocyte proliferation cccnrred in all cultcres, but did not correlate with the amount of IL10 produced. Since IL-10 is a potent inhibitor of TNI+x, we considered the possibility that TNF-a production would be suppressed by H-10. We found TNFwas produced at the same time as IL-lo, (day 3= 560 f 440 pg/ml, n=2; day 5= 605 + 395 pg/ml, n=2). We speculate that IL-10 is produced during infection with C. neoformzns, and plays a complex role in the celI mediated immune response to this opportunistic pathogen.
REGULATION OF INTERLEUIUN-4 EXPRESSION IN PORCINE LYMPHOCYTES. M. Murtaugh, Y. Zhou, and F. Zuckermsnn. Univ. Minnesota, St. Paul, MN 55108 and Univ. Illinois, Urbana, IL 61801 Intedeukin4 (H-4) gene expression was examined in Porcine peripheral blood mononuclear cells (PBMC) and in sorted populations of T cells under both non-specific and antigen-specific stimulation to gain insight into its role in host defense in swine. Reverse transcriptase-PCR analyses showed that resting cells do not express IL-4, but mRNA expression is induced by a variety of mitegem and immunomodulators, including concanavalin A, PMA, bacterial LPS, the superantigen Staphylococcus enterotoxin B, and tumor necrosis factor cx. In addition, IL4 strongly induced its own mRNA expression. Stimulation of pseudorabies virus (PRV)-primed PBMC with specific viral antigen also induced IL4 mRNA expression. Quantitative analysis by competitive PCR showed a @fold induction of IL-4 mRNA
expression in PRV-stimulated and double-positive
cells.
Sorted populations of CD4+,
CD8+
T cells showed similar patterns of IL-4 expression.
These results indicated that IL4 expression is regulated in Porcine PBMC in response to a variety of stimuli, consistent with previous observations of roles in humoral immunity and suppression of inflammatory responses. Supported by grants from the Minnesota Agricuhural Experiment Station.
I Abstracts
CYTOKINE,
PI October 4: Lymphokinedcytokines
Vol. 6, No. 5 (September
1994: 539-582)
in Immune Regulation
A7
A10 The Effects of Cytokines and Cytokine Inducers with Recombinant Proteins. Huw P. A. Hughes, Pharmaceuticals, New York, NY, 10022-6030.
on Vaccination M6
Many cytokines have been used as adjuvants with killed, synthetic and recombinant vaccines. A number of studies have indicated that the dose of cytokine and its precise method of administration are of prime importance for optimal efficacy. There is increasing evidence that large doses of a single cytokine can result in undue side effects, such as autoimmune disease. Previously, low doses of recombinant IL-2 were found to be effective in enhancing immune responses to bovine herpes virus-l gD. However, this only occurred following the simultaneous use of Avridine, a potent IL-1 and IFN-gamma inducer. These studies indicate that low doses of cytokine can be effective vaccine adjuvants, but certain conditions may apply. These include (a) activation of antigen presenting cells, (b) sustained delivery of cytokine for a defined period of time and (c) delivery of cytokine to the same site (draining lymph node) as the antigen.
Estradiol and ProgesteroneInhibit the Induction of Nitic Oxide Synthase in Marine MacrophaSe. L. Miller. E.W. Alley. W. I. Murphy, S. W. Russell wd J. S. Hunl University of Kansas Medical Center. Kansas City, KS 66160-7400 Nitric oxide (NO) is synthesized by the inducible &form of nitric oxide syntbase ONOS) in activated murine manoohages and can act as a mtent mediator of cvmtoxic defense.Macmphages. present in i&i numbers in the uterus of pregnant mick, cantribute to the maintenance of an immunosuppressive environment believed to prot%zect the fetus from attack by the maternal immune system and perform various otba pregnancy-associated functions. Estrogen and progesterone have tab immunostimulatory and immunosuppressive effects an macrophage effecmr functions and monokine synthesis. The purpose of thtis study was to determine the role of esnadial-17b(E2) and progesterone.(P4) on iNOS gene activity and NO production in murim macrophaes. Stimulation of the macrouhaae cell lines RAW 2b4.7 and 1774 and bone marrow derived macrophages with l&pt?ysacchari& (LPS) and interferon gamma (1FN-y) for 24h resulted in an accumulationof nitrite (NCQ). a stable product of the reaction of NO wirh oxygen. Concomitant incubation of cells with E2 (IpM-10~M) and P4(l&b-lO!&M) caused a concenration-dependent inhibition of NO2- production. RAW 264.7 cells were transiently uansfected with a gene comtmct containing Ihe iNOS regulatory region inserted upstream of a luciferase reporter gene.Transfected macrophages,stimulated with LPS (IW n&l) and IFN7 (IOU/ml) in the presence of E2 and P4. displayed an averageof a 50% reduction in luciferase activity (8h) and in N@accumulation (24h) when compared with cells stimulatedwith LPS plus IF’Ny alone or LPS plus IPNy and vehicle &me. This study shows that the presence of bath E2 and p4 in cultures of stimulated manophages inhibit the induction of the iNOS promotor and reduce the production of NO suggesting a possible regulatory role for these steroids in uterine macrophage activation and function. This work was supponed by NIH grant HD2.4212to J.S.H. and PO1 CA55474 to S.W.R.
A8
All ROLE OF CHOLERA
TRANSFORMING TOXIN IN IgA
GROWTH ISOTYPE
FACTOR-RI SWITCHING
AND
Pyeung-Hyeun Kim Dept. Microbial. Kangwon Univ., Chunchon 200.701 S. Korea The aim of this study was whether TGF-Ol derived from intestinal Peyer’s patch (PP) T cells and cholera toxin (CT) have an activity for IgA isotype switching. Murine autoreactive PP T cell line was stimulated with Concanavalin A and the supematants (Sn) was added to surface IgA negative (s&A) B cell culture. The acid-activatedT cell Sn in the presence of IL-2 induced a lo-fold or greater increase in total IgA isotype production and secreting ceil numbers but not IgM or IgGl isotype. This specific activity of Sn was completely abrogated with the treatment of anti-TGF-Bl antibody. In addition, it was found that freshly isolated PP T cells synthesize TGF-Bl mRNA constitutively measured by using RT-PCR. Finally, B cells were stimulated with the B subunit of cholera toxin (CTB), in the presence or absence of IL-2, and then assayed for IgA producing cells using an ELISPOT assay. CTB in the presence of IL-2, significantly increased the precursor frequency of IgA producing cells. In contrast, CTB and IL-2, in combiantion, did not alter the burst size of IgA producing clones and did not increase the rate of IgA secretion per cell. Taken together, these results show that TGF-81 can he expressed by T cells at the mucosal sutface and it switches B cells to IgA producing cells as an endogenous source, while CT or CTB has the same activity for the IgA synthesis as an exogenous source.
SERUM ALBUMIN MODIFIES THE EFFECT OF BP1 ON SURVIVAL OF NEWBORN RATS WITH E. cohK1 SEPSIS. J. Mantilla, P. Garcia, J. Gait&n and C.A. Dinarello. Inst. Investigation en Inmunologia, Universidad de Guadalajara, A. P. 2-130, Jalisco 44280, Mexico. Human serum albumin (HSA) blocks in vitro bacterial killing by BP1 (bactericidal/permeability increasing protein), without preventing BP1 binding to bacteria nor increased membrane permeability. This study shows the effects of HSA on the in viva protective effects of BP1 in newborn (NB) rats with sepsis by E. coli Kl (EcKl). Survival of NB Wistar rats infected via ip at time zero with S-30 mg/kg BPI, and via SC with a ID70 (3x106 CFU) or a ID90 (5x106) EcKl, was improved, reaching the best protection at 15 mg/kg BPI. A IDso (1x106) EcKl, and 15 mg/kg HSA, or 15 and 30 mg/kg BP1 was injected at time zero. Six rats from each group were sacrificed at zero, 6, 12, 24 h, and every 24 h. Blood was drawn by aseptic cardiac puncture and CFU/ml and WBC/mm3 were determined. Bacteremia significantly decreased in NB rats treated with 30 mg/kg BPI; and leukopenia observed in controls at 12 h was prevented by 15 mg/kg BPI, and more significantly by 30 mg/kg BPI. Low HSA dose (10 mg/kg), but not a high dose (40 mg/kg), reduced the protective effect of exogenous BPI. Besides, HSA at 40 mg/kg improved survival to a high bacterial challenge, but increased mortality by a low bacterial inoculum. This suggests that controversial effects of albumin on the BP1 actions, might be due to the proportion between bacteria and albumin concentrations.
RECXJLAl-lON OF THE HUMAN IMMUNE RESPONSE TO Cryptococcus neoformans BY INTERLEUKIN-10. C. Mody, P. Warren. University of Calgary, Alberta, Canada, T2N 4Nl. C. neoformansis an encapsulated yeast that causes meningitis and pneumonia in HIV-infected patients. Since celI mediated immunity is important for the clearance of this pathogen, we are investigating the regulation of cell mediated immunity by C. tteofonmm. previously, we have found that little y-IFN is produced by C. neofotmumstimulated lymphocytes, which could be due to regulation by IL-IO. To determine whether IL-IO is produced during the response to C. neofonmzns, peripheral blood mononuclear cells (PBMC) from healthy adults were stimulated with the ceU wall of C. neoformuns and IL-10 production was measured by ELISA in culture supematants. Lymphocyte proliferation was determined in parallel cultures. IL-10 was released early in culture by PBMC (day 3= 306 pg/ml, n=l; day 5= 715 f 277 pg/ml; n=4), but decreased later in culture (day 7= 47 f 64 pg/ml, n=3; day 9= 98 + 35 pg/ml, n=5). Lymphocyte proliferation cccnrred in all cultcres, but did not correlate with the amount of IL10 produced. Since IL-10 is a potent inhibitor of TNI+x, we considered the possibility that TNF-a production would be suppressed by H-10. We found TNFwas produced at the same time as IL-lo, (day 3= 560 f 440 pg/ml, n=2; day 5= 605 + 395 pg/ml, n=2). We speculate that IL-10 is produced during infection with C. neoformzns, and plays a complex role in the celI mediated immune response to this opportunistic pathogen.
REGULATION OF INTERLEUIUN-4 EXPRESSION IN PORCINE LYMPHOCYTES. M. Murtaugh, Y. Zhou, and F. Zuckermsnn. Univ. Minnesota, St. Paul, MN 55108 and Univ. Illinois, Urbana, IL 61801 Intedeukin4 (H-4) gene expression was examined in Porcine peripheral blood mononuclear cells (PBMC) and in sorted populations of T cells under both non-specific and antigen-specific stimulation to gain insight into its role in host defense in swine. Reverse transcriptase-PCR analyses showed that resting cells do not express IL-4, but mRNA expression is induced by a variety of mitegem and immunomodulators, including concanavalin A, PMA, bacterial LPS, the superantigen Staphylococcus enterotoxin B, and tumor necrosis factor cx. In addition, IL4 strongly induced its own mRNA expression. Stimulation of pseudorabies virus (PRV)-primed PBMC with specific viral antigen also induced IL4 mRNA expression. Quantitative analysis by competitive PCR showed a @fold induction of IL-4 mRNA
expression in PRV-stimulated and double-positive
cells.
Sorted populations of CD4+,
CD8+
T cells showed similar patterns of IL-4 expression.
These results indicated that IL4 expression is regulated in Porcine PBMC in response to a variety of stimuli, consistent with previous observations of roles in humoral immunity and suppression of inflammatory responses. Supported by grants from the Minnesota Agricuhural Experiment Station.