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Interleukin-10 Is expressed by dermal fibroblasts but not exhibiting regulatory effects on human eosinophils
J ALLERGY CLIN IMMUNOL VOLUME 109, NUMBER 1
51 Effect of Norepinephrine on the Production of Macrophage Inflammatory Protein 1 c( (MIP-Ic0 in Thermally Injured Patients Hitoshi Takahashi, Makiko Kobayashi, Steven E Wolf, David N Herndon, Richard B Pollard, Fujio Suzuki University of Texas Medical Branch and Shriners Burns Hospital, Galveston, TX Infection is a major concern in the mortality of burned patients. MIP- 1c~ has a function to induce host's protective immunity against microbial infections. In our previous studies, the production of MIP-10c was markedly impaired in burned patients. Also, the production of M I P - l a was not demonstrated in cultures of T cells from spleens of thermally injured mice. On the other hand, serum norepinephrine (NE) levels have been increased in patients immediately after thermal injuries. In the present study, therefore, the relationship between burn-associated production of NE and the impairment of MIP-1 c~ production was investigated in a murine model of thermal injury. Seven to eight-weeks old BALB/c mice were used in these experiments. Thermally injured mice (3rd degree, 15% of total body surface area) were produced through exposure of gas flame, as described previously. Serum specimens were obtained from mice 18 hrs after thermal injury. The amounts of NE in serum specimens were measured, as described previously. T cells were prepared from spleens of normal and thermally injured mice by T cell enrichment columns. To induce MIP- 1~, T cells (2 × 106 cells/ml) were treated with or without various concentrations of NE (10-8~10 -5 M) for 18 hrs. Cells were then washed and stimulated with anti-CD3 mAb (2.5 ~tg/ml) for 72 hrs. In some experiments, T cells were treated with 10-8 to 10-5 M of NE and 10-5 M ofpropranolol (J3-blocker) for 18 hrs, and stimulated for the MIP- loc production by anti-CD3 mAb for 72 hrs. Culture fluids harvested were assayed for MIP- lcx by ELISA. After the anti-CD3 mAb stimulation, amounts at 4 to 7 ng/ml of MIP-lo~ were produced in cultures of 2 x 106 cells/ml of splenic T cells derived from normal mice. However, the MIP- l a production was not demonstrated in cultures of splenic T cells from thermally injured mice. NE at amounts of 10-8 to 10-6 M was detected in serum of mice 1 to 12 hrs after thermal injury, while serum specimens for normal mice contained < 10.9 M of NE consistently. The M I P - l a production was markedly inhibited by NE in cultures of normal splenic T cells stimulated with anti-CD3 mAb. Only 0.1 ng/ml of MIP10t was produced when mAb-stimulated normal splenic T cells were cultured with 10-8 M of NE. The MIP-10c production suppressed by NE was recovered to normal levels when normal splenic T cells were cultured together with 10-8 M of NE and 10-5 M of propranolol, an antagonist of NE. These results suggest that the burn-associated production of NE has a role on the impairment of the MIP- 1o~production in thermally injured patients.
le) Interleukin-10 Is Expressed by Dermal Fibroblasts But Not
l~~Jlt- ExhibitingRegulatory Effects on Human Eosinophils
Holger Petering, Daniela Kimmig, Regina Smolarski, Alexander Kapp, Jgrn Elsner Hannover Medical University, Hannover, Germany Allergic diseases such as bronchial asthma or atopic dermatitis involves recruitment and activation of a variety of inflammatory cells at the site of antigen stimulation. Especially the recruitment of eosinophils may lead to a long-term damage of the mucosal tissue as a result of the production of reactive oxygen species (ROS). Therefore, the characterization of antiinflammatory cytokines such as IL- 10 seems to be of importance. The present study was performed to investigate whether IL-10 is able to affect directly or indirectly effector functions of human eosinophils. Since the interaction between eosinophils and dermal fibroblasts is known to be important in the pathogenesis of chronic inflammatory disease, RT-PCR analysis was performed demonstrating that dermal fibroblasts express mRNA specific for IL-10. The activation marker CD69 is expressed on activated eosinophils and flow cytometry with an anti-CD69 mAb was performed with GM-CSF plus IFNgamma stimulated cells indicating that the expression of CD69 is
Abstracts
$65
not influenced directly or indirectly by IL-10. Furthermore, the expression of the C5a anaphylatoxin receptor and CCR3 as the predominant CC chemokine receptor on eosinophils was investigated. Preincubation with IL-10 exhibited no differences in the expression of both receptors. To analyze whether IL-10 modulates directly or indirectly the respiratory burst of human eosinophils, lucigenin-dependent chemiluminescence was performed showing that IL- 10 does not induce directly the ROS production. To rule out indirect inhibitory effects of IL-10, purified eosinophils were preincubated with IL-10 and subsequently stimulated with C5a or eotaxin (CCL 11 ) and MCP-4 (CCL 13). No modulatory effects of IL- 10 could be observed. To find whether the IL-10 receptor is expressed on human eosinophils, transient changes of [CaSUP2+SUP]SUBiSUB were measured without showing any activity for IL- 10. In addition, transient changes of [CaSUP2+SUP]SUBiSUB in eosinophils induced by C5a or CC chemokines were not influenced by priming cells with IL- 10. Furthermore, RT-PCR analysis was performed demonstrating that eosinophils failed to express IL-10R mRNA. Our study is giving evidence that IL-10 is rather directly modulating eosinophil effector functions than regulating eosinophil activation indirectly via affecting other inflammatory cells.
1 ~ l , ~ Albumin-Depleted Human Factor VIII (hFVIII) LessImmunogenic ~1~t Than hFVIII With Albumin, But Oral Tolerance Achievable With Both Preparations Elizabeth Ann Secord*, Ping Gao§, Joseph Kaplan§ *Children's Hospital of Michigan, Wayne State University, Detroit, MI §Wayne State University, Children's Hospital of Michigan, Detroit, MI Factor VIII antibodies occur in approximately 20% to 40% of persons with hemophilia A. We have previously reported induction of tolerance to recombinant human Factor VIII (rhfVIII) in neonatal mice in two different model systems: via breastmilk of hFVIII-treated mothers, and via direct high dose feeds in 14 day old mice. Concern about contamination with infectious agents has prompted the manufacturers of rhfVIII to make product depleted of human albumin. Attempts to establish oral tolerance to hfVIII with this new preparation have yielded results suggesting lower immunogenicity of albumin-depleted product. In the first model system, 250 ug of albumin-containing (a.c.) or albumin-depleted (a.d.) rhfVIII was injected intravenously (i.v.) into lactating Balb/c mice 1-2 days after giving birth. Control mothers were treated with saline. In the second model system, 14 day old mice were fed 300 ugm of hfVIII (a.c. or a.d.) by gavage. Control neonates were fed saline. All pups were infused i.v. rhfVIII at six weeks of age, bled 10 days later, re-infused with hFVIII within 24 hours of the first bleed, and then bled again 14 days later. Sera were analyzed for anti-FVIII antibody levels by ELISA. In the lactation experiments, the progeny of test and control mice treated with a.d. hfVIII failed to mount a significant primary response. The response to secondary immunization with hFVIII was relatively weak in the control a.d. progeny (O.D. @ 1:30 dilution = 0.963+ .86) compared to that of the a.c. control progeny in parallel experiments. Nevertheless, it was significantly greater (p=0.014) than the secondary response of the a.d. test progeny (.O.D. @ 1:30 = 0.292+ .lg), indicating that oral tolerance was induced. When a single high oral dose of a.d. hFVIII was given at age 14 days both the test and control mice failed to mount a significant primary response to hFVIII. The response to secondary immunization with a.d. hFVIII in test and control mice was weak (O.D. @ 1:30 = 0.88+ 1.15 and 0.38+ .67, respectively) compared to that of mice treated with a.c. hfVIII. Although the difference was not statistically significant the average secondary response of control mice was greater than that of the test mice, indicating tolerance induction. Albumin-depleted rhfVIII appears to be less immunogenic than a.c. rhfVIII, but oral tolerance to the product can be demonstrated in a mouse model. This product may make long term tolerance induction easier to accomplish.
51 Effect of Norepinephrine on the Production of Macrophage Inflammatory Protein 1 c( (MIP-Ic0 in Thermally Injured Patients Hitoshi Takahashi, Makiko Kobayashi, Steven E Wolf, David N Herndon, Richard B Pollard, Fujio Suzuki University of Texas Medical Branch and Shriners Burns Hospital, Galveston, TX Infection is a major concern in the mortality of burned patients. MIP- 1c~ has a function to induce host's protective immunity against microbial infections. In our previous studies, the production of MIP-10c was markedly impaired in burned patients. Also, the production of M I P - l a was not demonstrated in cultures of T cells from spleens of thermally injured mice. On the other hand, serum norepinephrine (NE) levels have been increased in patients immediately after thermal injuries. In the present study, therefore, the relationship between burn-associated production of NE and the impairment of MIP-1 c~ production was investigated in a murine model of thermal injury. Seven to eight-weeks old BALB/c mice were used in these experiments. Thermally injured mice (3rd degree, 15% of total body surface area) were produced through exposure of gas flame, as described previously. Serum specimens were obtained from mice 18 hrs after thermal injury. The amounts of NE in serum specimens were measured, as described previously. T cells were prepared from spleens of normal and thermally injured mice by T cell enrichment columns. To induce MIP- 1~, T cells (2 × 106 cells/ml) were treated with or without various concentrations of NE (10-8~10 -5 M) for 18 hrs. Cells were then washed and stimulated with anti-CD3 mAb (2.5 ~tg/ml) for 72 hrs. In some experiments, T cells were treated with 10-8 to 10-5 M of NE and 10-5 M ofpropranolol (J3-blocker) for 18 hrs, and stimulated for the MIP- loc production by anti-CD3 mAb for 72 hrs. Culture fluids harvested were assayed for MIP- lcx by ELISA. After the anti-CD3 mAb stimulation, amounts at 4 to 7 ng/ml of MIP-lo~ were produced in cultures of 2 x 106 cells/ml of splenic T cells derived from normal mice. However, the MIP- l a production was not demonstrated in cultures of splenic T cells from thermally injured mice. NE at amounts of 10-8 to 10-6 M was detected in serum of mice 1 to 12 hrs after thermal injury, while serum specimens for normal mice contained < 10.9 M of NE consistently. The M I P - l a production was markedly inhibited by NE in cultures of normal splenic T cells stimulated with anti-CD3 mAb. Only 0.1 ng/ml of MIP10t was produced when mAb-stimulated normal splenic T cells were cultured with 10-8 M of NE. The MIP-10c production suppressed by NE was recovered to normal levels when normal splenic T cells were cultured together with 10-8 M of NE and 10-5 M of propranolol, an antagonist of NE. These results suggest that the burn-associated production of NE has a role on the impairment of the MIP- 1o~production in thermally injured patients.
le) Interleukin-10 Is Expressed by Dermal Fibroblasts But Not
l~~Jlt- ExhibitingRegulatory Effects on Human Eosinophils
Holger Petering, Daniela Kimmig, Regina Smolarski, Alexander Kapp, Jgrn Elsner Hannover Medical University, Hannover, Germany Allergic diseases such as bronchial asthma or atopic dermatitis involves recruitment and activation of a variety of inflammatory cells at the site of antigen stimulation. Especially the recruitment of eosinophils may lead to a long-term damage of the mucosal tissue as a result of the production of reactive oxygen species (ROS). Therefore, the characterization of antiinflammatory cytokines such as IL- 10 seems to be of importance. The present study was performed to investigate whether IL-10 is able to affect directly or indirectly effector functions of human eosinophils. Since the interaction between eosinophils and dermal fibroblasts is known to be important in the pathogenesis of chronic inflammatory disease, RT-PCR analysis was performed demonstrating that dermal fibroblasts express mRNA specific for IL-10. The activation marker CD69 is expressed on activated eosinophils and flow cytometry with an anti-CD69 mAb was performed with GM-CSF plus IFNgamma stimulated cells indicating that the expression of CD69 is
Abstracts
$65
not influenced directly or indirectly by IL-10. Furthermore, the expression of the C5a anaphylatoxin receptor and CCR3 as the predominant CC chemokine receptor on eosinophils was investigated. Preincubation with IL-10 exhibited no differences in the expression of both receptors. To analyze whether IL-10 modulates directly or indirectly the respiratory burst of human eosinophils, lucigenin-dependent chemiluminescence was performed showing that IL- 10 does not induce directly the ROS production. To rule out indirect inhibitory effects of IL-10, purified eosinophils were preincubated with IL-10 and subsequently stimulated with C5a or eotaxin (CCL 11 ) and MCP-4 (CCL 13). No modulatory effects of IL- 10 could be observed. To find whether the IL-10 receptor is expressed on human eosinophils, transient changes of [CaSUP2+SUP]SUBiSUB were measured without showing any activity for IL- 10. In addition, transient changes of [CaSUP2+SUP]SUBiSUB in eosinophils induced by C5a or CC chemokines were not influenced by priming cells with IL- 10. Furthermore, RT-PCR analysis was performed demonstrating that eosinophils failed to express IL-10R mRNA. Our study is giving evidence that IL-10 is rather directly modulating eosinophil effector functions than regulating eosinophil activation indirectly via affecting other inflammatory cells.
1 ~ l , ~ Albumin-Depleted Human Factor VIII (hFVIII) LessImmunogenic ~1~t Than hFVIII With Albumin, But Oral Tolerance Achievable With Both Preparations Elizabeth Ann Secord*, Ping Gao§, Joseph Kaplan§ *Children's Hospital of Michigan, Wayne State University, Detroit, MI §Wayne State University, Children's Hospital of Michigan, Detroit, MI Factor VIII antibodies occur in approximately 20% to 40% of persons with hemophilia A. We have previously reported induction of tolerance to recombinant human Factor VIII (rhfVIII) in neonatal mice in two different model systems: via breastmilk of hFVIII-treated mothers, and via direct high dose feeds in 14 day old mice. Concern about contamination with infectious agents has prompted the manufacturers of rhfVIII to make product depleted of human albumin. Attempts to establish oral tolerance to hfVIII with this new preparation have yielded results suggesting lower immunogenicity of albumin-depleted product. In the first model system, 250 ug of albumin-containing (a.c.) or albumin-depleted (a.d.) rhfVIII was injected intravenously (i.v.) into lactating Balb/c mice 1-2 days after giving birth. Control mothers were treated with saline. In the second model system, 14 day old mice were fed 300 ugm of hfVIII (a.c. or a.d.) by gavage. Control neonates were fed saline. All pups were infused i.v. rhfVIII at six weeks of age, bled 10 days later, re-infused with hFVIII within 24 hours of the first bleed, and then bled again 14 days later. Sera were analyzed for anti-FVIII antibody levels by ELISA. In the lactation experiments, the progeny of test and control mice treated with a.d. hfVIII failed to mount a significant primary response. The response to secondary immunization with hFVIII was relatively weak in the control a.d. progeny (O.D. @ 1:30 dilution = 0.963+ .86) compared to that of the a.c. control progeny in parallel experiments. Nevertheless, it was significantly greater (p=0.014) than the secondary response of the a.d. test progeny (.O.D. @ 1:30 = 0.292+ .lg), indicating that oral tolerance was induced. When a single high oral dose of a.d. hFVIII was given at age 14 days both the test and control mice failed to mount a significant primary response to hFVIII. The response to secondary immunization with a.d. hFVIII in test and control mice was weak (O.D. @ 1:30 = 0.88+ 1.15 and 0.38+ .67, respectively) compared to that of mice treated with a.c. hfVIII. Although the difference was not statistically significant the average secondary response of control mice was greater than that of the test mice, indicating tolerance induction. Albumin-depleted rhfVIII appears to be less immunogenic than a.c. rhfVIII, but oral tolerance to the product can be demonstrated in a mouse model. This product may make long term tolerance induction easier to accomplish.