ABSTRACT
This study evaluated the pharmacokinetics, the sedative and anti-nociceptive effects of intravenous hydromorphone in dogs. Five adult dogs were administered hydromorphone (0.1 mg/kg and 0.2 mg/kg) and morphine (0.5 mg/kg and 1 mg/kg) at weekly intervals. Blood samples were drawn before and at 1, 2, 5, 15, 30, 60 and 120 min after drug administration. Plasma hydromorphone only was measured by high pressure liquid chromatography (HPLC) with electrochemical detection and pharmacokinetic parameters calculated. Anti-nociceptive and sedation scores were submitted to Kruskal-Wallis one-way anova on ranks and post-hoc Bonferroni test with 5% significance level. The data fitted a two-compartment model with a fast distribution (<1 min for both doses) and slower elimination rate. Mean elimination half-life was 80 +/- 52.7 and 57.7 +/- 30.4 min for the high and low dose, respectively. The apparent mean volumes of distribution at steady-state were 7.2 +/- 3 and 4.5 +/- 2.4 L/kg, while the clearance was 74.7 +/- 19 and 68.1 +/- 20 mL/kg/min for the high and low doses, respectively. Compared to saline, hydromorphone and morphine produced significant anti-nociception and sedation of similar magnitude for 120 min. In conclusion, intravenous hydromorphone has a large volume of distribution, and high clearance rate that exceeds hepatic blood flow. In dogs, it produced mechanical anti-nociception and sedation of a magnitude similar to morphine.
Subject(s)
Analgesia , Analgesics, Opioid/pharmacology , Hydromorphone/pharmacokinetics , Analgesics, Opioid/administration & dosage , Animals , Area Under Curve , Body Temperature/drug effects , Dogs , Dose-Response Relationship, Drug , Female , Half-Life , Hydromorphone/administration & dosage , Hydromorphone/pharmacology , Injections, Intravenous , Metabolic Clearance Rate , Models, Biological , Morphine/administration & dosage , Morphine/pharmacokinetics , Receptors, Opioid, mu/antagonists & inhibitors , Respiration/drug effectsABSTRACT
This study compared plasma histamine concentrations, behavioral and cardiovascular parameters following intravenous administration of hydromorphone and morphine in conscious dogs. Five adult female dogs received a 15-sec bolus injection of saline, hydromorphone (0.1 and 0.2 mg/kg) or morphine (0.5 and 1.0 mg/kg) randomly at weekly intervals. Blood samples were collected from the jugular vein before and at 1, 2, 5, 15, 30, 60 and 120 min after drug administration. Plasma histamine concentration, noninvasive oscillometric blood pressure, heart rate and rhythm were evaluated. Data were analyzed with repeated measures anova and Tukey-Kramer post hoc test with a 5% significance level. Median plasma histamine increased significantly only after the higher dose of morphine. Maximum plasma histamine measured was 0.8 ng/mL after saline and, after the lower and higher doses, respectively, 10.2 and 9.7 ng/mL for hydromorphone, and 440 and 589 ng/mL for morphine. One dog became hypotensive immediately after receiving the highest dose of morphine. Occasional ventricular premature contractions occurred in one dog after both opioids and dosages. No dogs vomited or defecated, but all salivated profusely with both opioids. Neuroexcitation occurred in four dogs following each opioid. In conclusion, intravenous hydromorphone induced minimal histamine release and was well tolerated by these conscious healthy dogs.
Subject(s)
Analgesics, Opioid/pharmacology , Histamine/blood , Hydromorphone/pharmacology , Morphine/pharmacology , Analgesics, Opioid/administration & dosage , Animals , Behavior, Animal/drug effects , Dogs , Dose-Response Relationship, Drug , Female , Heart Rate/drug effects , Hydromorphone/administration & dosage , Infusions, Intravenous/veterinary , Morphine/administration & dosageABSTRACT
This study examined the pharmacokinetics and physiologic effects of two infusions rates of morphine in conscious dogs. Five adult dogs were randomly studied at weekly intervals. An initial dose of either 0.3 or 0.6 mg/kg were each followed by infusions of 0.17 and 0.34 mg/kg/h. Plasma morphine concentrations, physiological parameters, sedation and mechanical antinociception were evaluated during each infusion. Morphine was assayed by high pressure liquid chromatography (HPLC) with electrochemical coulometric detection and pharmacokinetic parameters were calculated. Data were fitted to a bi-compartment model with a rapid distribution (<1 min for both doses) and slower termination rate. For the high and low doses, respectively, mean+/-SD terminal half-life was 38+/-5 and 27+/-14 min, apparent volumes of distribution at steady-state were 1.9+/-0.5 and 1.3+/-0.8 L/kg, with clearances of 50+/-15 and 67+/-20 mL/kg/min. Steady-state plasma concentrations ranged from 93 to 180 ng/mL and 45 to 80 ng/mL in the high and low doses, respectively. Respiratory rate increased significantly, pulse oximetry remained>95% and body temperature decreased significantly during both infusions. No vomition or neuroexcitation occurred. Sedation and mechanical antinociception were both mild during the lower infusion rate, and mild to moderate during the higher infusion rate. In conclusion, morphine pharmacokinetics was not altered by increasing infusion rates, producing stable, long-lasting plasma concentrations.
Subject(s)
Analgesics, Opioid/pharmacokinetics , Dogs/metabolism , Morphine/pharmacokinetics , Analgesics, Opioid/administration & dosage , Analgesics, Opioid/blood , Animals , Behavior, Animal , Consciousness , Cross-Over Studies , Female , Infusions, Intravenous/veterinary , Morphine/administration & dosage , Morphine/blood , Pain/prevention & control , Pain/veterinary , Pain Measurement/veterinarySubject(s)
DNA-Binding Proteins/immunology , Nuclear Proteins , Th2 Cells/immunology , Transcription Factors/immunology , Transcription, Genetic , Animals , Antigens, CD/analysis , Antigens, Differentiation, T-Lymphocyte/analysis , CD4-Positive T-Lymphocytes/immunology , Cell Count , DNA-Binding Proteins/genetics , Gene Deletion , Hyaluronan Receptors/analysis , L-Selectin/analysis , Lectins, C-Type , Lymphocytes/cytology , Lymphokines/biosynthesis , Lymphokines/genetics , Mice , NFATC Transcription Factors , Spleen/cytology , Spleen/immunology , Transcription Factors/geneticsABSTRACT
A destructive joint disease can be induced in susceptible DBA/1 mice by immunization with type II collagen emulsified with oil and either killed Mycobacterium tuberculosis or IL-12 as adjuvant. Cellular and humoral anti-collagen immune mechanisms appear to be involved in the pathogenesis of arthritis. We have characterized the adjuvant effect or IL-12 in more detail and addressed the question whether mycobacteria might act via the induction of endogenous IL-12. Injections of IL-12 into collagen-immunized DBA/1 mice promoted the development of IFN-gamma-producing CD4+ T cells and strongly upregulated the production of complement-fixing IgG2a and IgG2b antibodies resulting in severe arthritis. Neutralization of IFN-gamma in vivo largely inhibited the increase in antibody synthesis and prevented joint disease in IL-12-treated mice. However, collagen-specific IFN-gamma synthesis by T cells was further enhanced in these animals. Furthermore, IL-12 treatment promoted the development of IFN-gamma-producing T cells but failed to enhance antibody synthesis and to induce arthritis in C57BL/6 or BALB/c mice immunized with collagen in oil. These results indicate that the induction (by IL-12) of a strong collagen-specific T-cell response alone is not sufficient to trigger arthritis. Attempts to show a role for endogenous IL-12 in DBA/1 mice immunized with collagen with mycobacteria as adjuvant gave no reliable results. Whereas anti-IL-12 treatment delayed the onset and ameliorated the disease in some experiments, it failed to do so in other experiments, or, control reagents also had some effect. A slight inhibition of collagen-specific IgG2a synthesis was observed in most experiments in the sera of anti-IL-12-treated mice. Taken together, the results show that exogenous IL-12 can promote arthritis via its direct effect on T cells and its effect on antibody production, which is at least in part IFN-gamma-dependent. On the other hand, whether or not endogenous IL-12 is involved in the adjuvant effect of mycobacteria needs further clarification.
Subject(s)
Arthritis/immunology , Autoimmune Diseases/immunology , Collagen/immunology , Interleukin-12/antagonists & inhibitors , Interleukin-12/immunology , Adjuvants, Immunologic , Animals , Antibody Formation , Immunity, Cellular , Interferon-gamma/physiology , Mice , Mice, Inbred C57BL , Mice, Inbred DBA , Spleen/metabolismABSTRACT
DBA/1 (H-2q) and C57BL/6 (H-2b) mice develop an intermediate immune responses when immunized with chicken type II collagen (CII) emulsified with incomplete Freund's adjuvant (IFA). Only a few animals develop a mild form of arthritis. As reported before and confirmed herein, administration of IL-12 to DBA/1 mice immunized with CII in IFA strongly enhances the cellular and humoral (auto)immune response to CII and induces severe destructive joint disease with an incidence of 80-100%. In contrast, the same treatment did not promote joint disease in C57BL/6 mice. Characterization of the IL-12 effect on the CII-specific immune response of C57BL/6 mice revealed that IL-12 promoted the development of CII-specific T cells producing IFN-gamma in DBA/1 and C57BL/6 mice equally well. However, whereas treatment with IL-12 in DBA/1 mice strongly up-regulated the synthesis of CII-specific antibodies, especially of the IgG2a and IgG2b subclasses, it rather slightly down-regulated the CII-specific IgG2a and IgG2b synthesis in C57BL/6 mice. This may indicate that the effect of IL-12 on the CII-specific antibody synthesis is of crucial importance in the pathogenesis of type II collagen-induced arthritis (CIA). The failure of IL-12 to up-regulate IgG2a and IgG2b synthesis in C57BL/6 mice is specific for CII as antigen and not a general property of this strain because the keyhole limpet hemacyanin-specific antibody response is up-regulated by IL-12 in C57BL/6 mice. Furthermore, it is not the H-2b haplotype of C57BL/6 mice but rather the genetic background (DBA/1 versus BL/6 or BL/10) that limits the effect of IL-12 on the CII-specific antibody response because IL-12 treatment of CII-immunized B10.Q (H-2q) mice also failed to induce arthritis and to enhance CII-specific IgG2a and IgG2b synthesis. However, as in the two other strains, injection of IL-12 promoted the development of splenic T cells producing IFN-gamma upon activation with CII. These results indicate that an enhancement of the cellular and humoral anti-CII response by IL-12 is required for inducing arthritis.
Subject(s)
Arthritis/etiology , Collagen/immunology , Immunity, Cellular , Interleukin-12/pharmacology , Th1 Cells/immunology , Animals , Antibody Formation , Antigens/administration & dosage , Arthritis/immunology , Chickens , Collagen/administration & dosage , Female , Freund's Adjuvant/administration & dosage , Hemocyanins/administration & dosage , Hemocyanins/immunology , Immunization , Immunoglobulin G/biosynthesis , Male , Mice , Mice, Inbred C57BL , Mice, Inbred DBAABSTRACT
Co-activation of CD4+ T cells by anti-CD4 mAb strongly enhances the proliferation of these T cells. We have analyzed the influence of CD4-mediated co-activation on Th cell differentiation. Our data demonstrate that activation of high density (HD)-CD4+ T cells by immobilized anti-CD3 mAb in combination with immobilized anti-CD4 mAb led to a strong inhibition of Th1 differentiation and to a variable but significant induction of Th2 development. Priming of highly enriched Mel-14highCD4+ T cells in the presence of anti-CD4 mAb also resulted in a pronounced suppression of secondary IFN-gamma production, indicating that the Th1 development of naive CD4+ T cells is inhibited by co-activation via CD4. In contrast to HD-CD4+ T cells, CD4-induced costimulation of MEL-14highCD4+ T cells did not result in a primary and secondary IL-4 production. Hence, these results suggest that a MEL-14low population within the HD-CD4+ T cell fraction was the source of the endogenous IL-4 and imply, in addition, that co-activation via CD4 inhibits the development of Th1 cells from naive CD4+ T cells independently from endogenous IL-4. This assumption is further corroborated by the fact that neither the application of neutralizing anti-IL-4 mAb nor the use of T cells from IL-4 knockout mice could prevent CD4-mediated inhibition of Th1 development. The Th1-inhibiting effect of anti-CD4 mAb could not be reversed by the application of the Th1 inducer IL-12. On the contrary, the secondary IL-4 production of HD-CD4+ T cells as an indicator of Th2 differentiation, which was promoted by anti-CD4 mAb, was enhanced even in the presence of IL-12. Therefore, our results suggest that co-activation of naive CD4+ T cells by anti-CD4 mAb directly and selectively inhibits Th1 differentiation of naive dense CD4+ T cells.
Subject(s)
Antibodies, Monoclonal/pharmacology , CD4 Antigens/immunology , CD4-Positive T-Lymphocytes/immunology , Growth Inhibitors/immunology , Lymphocyte Activation , Th1 Cells/immunology , Animals , CD3 Complex/immunology , CD4 Lymphocyte Count , Cell Differentiation/immunology , Drug Synergism , Female , Growth Inhibitors/pharmacology , Immunophenotyping , Immunosuppressive Agents/pharmacology , Interferon-gamma/antagonists & inhibitors , Interferon-gamma/biosynthesis , Interleukin-12/pharmacology , Interleukin-4/physiology , Lymphocyte Activation/drug effects , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Inbred CBA , Mice, Mutant StrainsABSTRACT
Cytokines were found to play a key role in Th cell differentiation. Among them IL-12 was shown to be a potent differentiation factor for Th1 cells, whereas IL-4 is the only known cytokine that promotes the development of Th2 cells. Upon addition of comparable amounts of IL-4 and IL-12 to a primary culture of naive CD4+ T cells activated by immobilized anti-CD3 mAb, it was found that the Th1-inducing capacity of IL-12 is dominated by the Th2-promoting effect of IL-4. However, high amounts of IL-12 (10,000 U/ml) in combination with low amounts of IL-4 (100 U/ml) led to the development of a Th cell population that, upon rechallenge, showed a substantial secondary IFN-gamma (Th1 cytokine) production concomitantly with the production of high amounts of IL-4 (Th2 cytokine). This can be due to the coexistence of Th1 and Th2 cells or to the development of Th0 cells producing a mixed pattern of cytokines. Immunofluorescence double staining of intracellular IL-4 and IFN-gamma in combination with flow cytometry (FACS) revealed that most of the emerging Th cells produced either IL-4 or IFN-gamma. Only a few double producers could be detected. This finding indicates that individual naive CD4+ T cells can differentiate under the same conditions towards Th1 or Th2 cells and implicates that the development of Th1 and Th2 cells is not necessarily mutually exclusive.
Subject(s)
CD4-Positive T-Lymphocytes/cytology , Interleukin-12/pharmacology , Interleukin-4/pharmacology , Th1 Cells/cytology , Th2 Cells/cytology , Animals , CD4-Positive T-Lymphocytes/drug effects , CD4-Positive T-Lymphocytes/metabolism , Cell Differentiation/drug effects , Cell Differentiation/immunology , Cells, Cultured , Drug Combinations , Female , Interferon-gamma/biosynthesis , Interleukin-12/biosynthesis , Interleukin-12/immunology , Interleukin-4/biosynthesis , Interleukin-4/immunology , Male , Mice , Mice, Inbred BALB C , Mice, Inbred CBA , Mice, Inbred DBA , Th1 Cells/drug effects , Th1 Cells/metabolism , Th2 Cells/drug effects , Th2 Cells/immunologyABSTRACT
Collagen-induced arthritis (CIA) is an (autoimmune) joint disease readily elicited in DBA/1 mice by immunization with type II collagen (CII) emulsified with complete Freund's adjuvant. It is a destructive arthritis involving about 50% of the limbs and occurs with an incidence of 70% to 100%. In this study we evaluated the effect of mouse recombinant interleukin-12 (mrIL-12) on CIA. Administration of mrIL-12 at high doses (1 micrograms/mouse, daily) for 2 or 3 weeks delayed the onset and reduced the incidence of CIA. Furthermore, the severity of CIA was much milder and in most cases restricted to single digits of the paws. Short-term administration of high doses of IL-12 exerted some, but less pronounced, disease-suppressing effect. In contrast, 10-fold lower doses of IL-12 given during the first 3 weeks, or high doses of IL-12 administered therapeutically proved to be ineffective. Only those regimens of IL-12 treatment that ameliorated CIA were associated with a down-regulation of the CII-specific antibody response. A strong inhibition of CII-specific IgG1 antibodies (10- to 20-fold) and a moderately (2- to 6-fold) suppressed IgG2b response was observed, whereas the level of CII-specific IgG2a antibodies remained high. Taken together, the results indicate that some initial events in the induction of CIA in DBA/1 mice injected with CII emulsified with CFA are suppressed by treatment with high doses of IL-12.
Subject(s)
Arthritis, Experimental/immunology , Arthritis, Experimental/prevention & control , Collagen , Freund's Adjuvant , Interleukin-12/therapeutic use , Animals , Arthritis, Experimental/epidemiology , Autoantibodies/biosynthesis , Autoantibodies/drug effects , Dose-Response Relationship, Immunologic , Drug Administration Schedule , Incidence , Interleukin-12/administration & dosage , Male , Mice , Mice, Inbred DBAABSTRACT
Interleukin (IL)-12 was originally identified as a factor produced by human Epstein-Barr virus-transformed B cell lines. It was detected by one group as cytotoxic lymphocyte maturation factor, a cytokine that synergized with IL-2 in the induction of lymphokine-activated killer cells and cytotoxic T lymphocytes. A second group characterized it as a natural killer (NK) cell stimulatory factor, due to the enhancement of cytotoxicity and IFN-gamma synthesis by NK cells. Human IL-12 was purified to homogeneity and cloned by both groups. We had identified a murine factor, provisionally termed T cell-stimulating factor (TSF), which was involved in the proliferation, synthesis of IFN-gamma and cell adhesion of CD4+ Th1 cells. TSF was produced in the antigen-specific interaction between Th1 cells and macrophages as antigen-presenting cells, partially purified from supernatants of such cultures, and shown to be identical to IL-12. Monocytes/macrophages appear to be the major source of IL-12. It is rapidly produced by phagocytic cells after stimulation with several bacteria/bacterial products and other microorganisms. In the light of its effects on NK cells as well as CD4+ and CD8+ T cells, IL-12 can be regarded as a cytokine that connects the innate immune system with the acquired immunity. IL-12 has a broad range of activities already reviewed in three papers. These include the regulation of cytokine synthesis and proliferation of T and NK cells, the promotion of Th1 cell development, the differentiation of CD8+ T cells and effects on hematopoiesis. When applied in vivo, IL-12 was shown to enhance the resistance to bacterial and parasitic infections, to promote antitumor immunity, and to influence antiviral responses including HIV in vivo or in vitro. This review will briefly summarize these effects, but mainly focus on recent results concerning the regulation of the production and the activity of IL-12, its role in the differentiation of Th cells and the implications for delayed- and immediate-type hypersensitivity reactions, its importance for organ-specific autoimmune diseases, and the possible role of the IL-12p40 homodimer as a specific inhibitor of the IL-12 heterodimer.
Subject(s)
Interleukin-12/physiology , Animals , Antibody Formation , Autoimmune Diseases/physiopathology , Cell Differentiation , Communicable Diseases/physiopathology , Hematopoiesis , Humans , Hypersensitivity/physiopathology , Interleukin-12/chemistry , Killer Cells, Natural/physiology , Receptors, Interleukin/physiology , Receptors, Interleukin-12 , Shock, Septic/physiopathology , Signal Transduction , T-Lymphocytes/physiologyABSTRACT
The synthesis of antibodies of the IgE isotype in mice largely depends on IL-4, a cytokine that is released by T lymphocytes of the Th2 subtype. IL-12 is a cytokine considered to direct Th cell development into a Th1 direction and to suppress Th2 responses including the synthesis of IgE. Here we report about the influence of IL-12 on the IgE responses of mice immunized with protein antigens adsorbed to aluminum hydroxide. To avoid problems with the detection of IgE caused by an excess of competitive IgG antibodies produced in IL-12-treated mice, serum IgE was first extracted from the serum by plate-bound anti-IgE mAb and then determined either as total IgE or as antigen-specific IgE by using biotinylated anti-IgE or biotinylated antigen. Depending on the strain of mice and the dose of IL-12 injected together with the antigen, IL-12 can either temporarily suppress or augment the synthesis of (antigen-specific) IgE antibodies. This applies for CBA/J mice immunized six times in biweekly intervals with minute (0.1 micrograms/injection) or three-times with large (5 micrograms/injection) amounts of the bee venom allergen phospholipase A2 (PLA2). Under both conditions the antibody response is characterized by the production of predominantly IgG1 as well as IgE but very little IgG2a, IgG2b and IgG3 antibodies. Simultaneous application of low doses of IL-12 (1 or 10 ng/day) led to a 2- to 4-fold enhancement of IgE production (PLA2-specific IgE or total IgE). Only a high dose of 1 micrograms IL-12/day resulted in a 3- to 10-fold reduction of the IgE response. This suppression was not stable, however, because the synthesis of IgE antibodies was stimulated to a high level when these mice subsequently received a second course of immunizations in the absence of IL-12. Likewise, the synthesis of IgE was only temporarily suppressed by IL-12 treatment in CBA/J mice immunized with keyhole limpet hemocyanin (KLH) as antigen. However, application of low (10 ng/day) or high (1 microgram/day) doses of IL-12 during the primary course of immunizations of CBA/J mice with KLH suppressed the IgE response slightly or strongly respectively. In striking contrast, the KLH-specific IgE response of BALB/c mice was upregulated even when high doses of IL-12 (1 microgram/day) were injected simultaneously with the immunizations. Thus, these results demonstrate a great variability regarding the influence of IL-12 treatment on ongoing IgE responses in vivo.
Subject(s)
Immunoglobulin E/biosynthesis , Interleukin-12/pharmacology , Th2 Cells/drug effects , Animals , Antibodies, Anti-Idiotypic/immunology , Antibodies, Monoclonal/immunology , Antibody Specificity , Cricetinae , Hemocyanins/immunology , Immunoglobulin E/immunology , Immunoglobulin G/biosynthesis , Interleukin-12/administration & dosage , Mice , Mice, Inbred BALB C , Mice, Inbred CBA , Phospholipases A/immunology , Phospholipases A2 , Rats , Recombinant Proteins/pharmacology , Th1 Cells/immunology , Th2 Cells/immunologyABSTRACT
Encainide treatment in patients after myocardial infarction is associated with increased risk of sudden cardiac death. This may relate to drug-induced changes in the electrophysiologic milieu, thus predisposing the patient to sustained ventricular tachyarrhythmias. The goals of this study were to first develop a model of class Ic-induced ventricular tachycardia (VT) and then to design treatments to oppose this prodysrhythmic activity. Dogs with time-dependent loss of inducible sustained VT in the antiarrhythmic drug-free state were studied late after infarction. These dogs received a series of three loading and maintenance infusions of O-demethyl encainide (ODME) to achieve concentrations of 60 +/- 31, 136 +/- 46 and 339 +/- 171 ng/ml. Drug maintenance continued until programmed stimulation induced monomorphic sustained VT. When ODME infusion allowed this induction, barium chloride infusions were added. ODME treatment allowed induction of monomorphic sustained VT in 9 of 10 dogs studied. Prodysrhythmic monomorphic VT was significantly related (P < .01) to prolongation of conduction velocity in the peri-infarct zone. ODME modestly increased ventricular refractoriness at some but not all peri-infarct sites. Infusion of barium chloride in the above nine dogs caused their hearts to return to the noninducible state. Prolongation of refractoriness in the peri-infarct zone was correlated to this suppression of prodysrhythmia. Prolongation of conduction velocity in the absence of substantial prolongation of refractoriness may underlie ODME-facilitated induction of monomorphic VT. Prolongation of refractoriness in the peri-infarct zone by combination treatment with barium chloride reversed prodysrhythmic VT in all of the dogs.
Subject(s)
Barium Compounds/therapeutic use , Chlorides/therapeutic use , Encainide/therapeutic use , Myocardial Infarction/drug therapy , Tachycardia, Ventricular/prevention & control , Animals , Death, Sudden , Dogs , Drug Therapy, Combination , Electric Stimulation , Encainide/adverse effects , Myocardial Infarction/complications , Tachycardia, Ventricular/chemically inducedABSTRACT
The induction of arthritis in DBA/1 mice usually requires immunization with the antigen type II collagen emulsified with Mycobacterium tuberculosis in oil. Here we describe that interleukin 12 (IL-12) can replace mycobacteria and cause severe arthritis of DBA/1 mice when administered in combination with type II collagen. Immunization of DBA/1 mice with type II collagen emulsified in oil alone resulted in a weak immune response, and only a few animals (10-30%) developed arthritis. Administration of IL-12 for 5 days simultaneously with each immunization strongly enhanced the anti-type II collagen immune response. Collagen-specific interferon gamma (IFN-gamma) synthesis by ex vivo activated spleen cells was enhanced 3- to 10-fold. IFN-gamma was almost completely produced by CD4+ T cells. Furthermore, the production of collagen-specific IgG2a and IgG2b antibodies was upregulated 10- to 100-fold. As a consequence, the incidence of arthritis in the group of mice immunized with collagen plus IL-12 was very high (80-100%). The developing arthritis was severe, involving approximately 50% of all limbs with strongly increased footpad thickness in most cases. Furthermore, histological examination revealed massive, mainly polymorphonuclear cell infiltration, synovial hyperplasia, cartilage and bone destruction, as well as new bone formation. In many cases, this resulted in the complete loss of joint structure. Neutralization of IFN-gamma in vivo prevented the development of arthritis in collagen-immunized and IL-12-treated mice. In conclusion, our data show that in vivo administered IL-12 can profoundly upregulate a T helper I-type autoimmune response, resulting in severe joint disease in DBA/1 mice.
Subject(s)
Arthritis, Experimental/chemically induced , Arthritis, Experimental/immunology , CD4-Positive T-Lymphocytes/immunology , Collagen , Interleukin-12 , Animals , Antibody Formation , Arthritis, Experimental/pathology , Chickens , Drug Interactions , Hindlimb , Inflammation , Interferon-gamma/biosynthesis , Mice , Mice, Inbred DBA , Time FactorsABSTRACT
Activation of naive dense CD4+ T cells by plate-bound anti-CD3 antibodies favors the development of Th1 cells which, upon re-stimulation, produce significant amounts of IFN-gamma but no IL-4. However, co-activation of such naive T cells in the presence of IgE [anti-dinitrophenyl (DNP)]-loaded bone marrow-derived mast cells (BMMC) on plates coated with anti-CD3 antibodies and DNP-BSA led to the development of IL-4-producing Th2 cells. The same result could be observed if irradiated (800 rad) BMMC were applied as co-stimulators. Moreover, BMMC could be replaced by the supernatant of IgE-activated BMMC suggesting that a soluble mediator, presumably IL-4, was responsible for this effect. This assumption was substantiated using neutralizing anti-IL-4 antibodies which abolished the BMMC-mediated Th2 development in all cases. Addition of IL-12, a cytokine that was shown to antagonize the Th2-promoting effect of IL-4 in vivo, could not inhibit the development of IL-4-producing T cells, but gave rise to a T cell population which produced relatively high amounts of IL-4 and IFN-gamma. Since BMMC represent the in vitro equivalent of mucosal mast cells these data suggest that IgE-activated mucosal mast cells can bias an emerging T cell dependent immune response towards a Th2 dominated reaction by the initial production of IL-4.
Subject(s)
CD4-Positive T-Lymphocytes/physiology , Interleukin-4/physiology , Lymphocyte Activation , Mast Cells/physiology , Th2 Cells/immunology , Animals , Bone Marrow Cells , CD3 Complex/immunology , Cells, Cultured , Cytokines/biosynthesis , Female , Interleukin-4/biosynthesis , Ionomycin/pharmacology , Male , Mast Cells/cytology , Mice , Mice, Inbred BALB C , Receptors, IgE/physiologyABSTRACT
The influence of the cytokine interleukin-12 (IL-12) on humoral immune responses was studied in vivo. CBA/J mice immunized with protein antigens (keyhole limpet hemocyanin, phospholipase A2) adsorbed to aluminum hydroxide (Alum) develop a Th2-like immune response characterized by the production of large amounts of IgG1 as well as some IgE but little IgG2a, IgG2b and IgG3 antibodies. IL-12 is a cytokine that promotes the development and the activation of Th1 cells. Th1 cells are involved in the induction of cellular immunity, which is characterized by low or absent antibody production. On the other hand, some Th1-like immune responses are associated with a strong antibody production of the IgG2a, IgG2b and IgG3 subclasses. Thus, we investigated whether treatment with IL-12 would down-regulate the humoral immune response or stimulate antibody production of the IgG2a, IgG2b and IgG3 subclasses. We observed that: 1) administration of IL-12 to mice together with protein antigens adsorbed to Alum strongly enhanced the humoral immune response by increasing the synthesis of antigen-specific antibodies of the IgG2a, IgG2b and IgG3 subclasses 10- to 1000-fold. The synthesis of IgG1 was not or only slightly (2-5-fold) enhanced, whereas that of the IgE isotype was suppressed. 2) These effects of IL-12 were observed when high (10 micrograms, 100 micrograms) or low doses (0.1 microgram) of antigen were used for immunization. 3) Titration of IL-12 in vitro revealed that IgG2a is strongly up-regulated over a wide dose range of IL-12 (10 to 1000 ng/day). 4) The effects of IL-12 in vivo are at least partially interferon (IFN)-gamma-dependent because an anti-IFN-gamma mAb in combination with IL-12 prevented most of the enhanced IgG2a production. 5) Mice receiving IL-12 showed a strong up-regulation of IFN-gamma but no inhibition of IL-5 synthesis by spleen cells activated ex vivo with antigen. These results suggest that IL-12 is a potent adjuvant for enhancing humoral immunity to protein antigens adsorbed to Alum, primarily by inducing the synthesis of the complement-fixing IgG subclasses 2a, 2b and 3.
Subject(s)
Antigens/immunology , Complement System Proteins/immunology , Immunoglobulin G/biosynthesis , Interleukin-12/immunology , Aluminum Hydroxide/immunology , Animals , Complement Fixation Tests/methods , Haptens/immunology , Hemocyanins/immunology , Immunoglobulin G/classification , Interferon-gamma/immunology , Interleukin-5/immunology , Mice , Mice, Inbred CBA , Phospholipases A/immunology , Phospholipases A2 , Spleen/cytology , Up-Regulation/immunologySubject(s)
Antigen Presentation/physiology , Dendritic Cells/immunology , Animals , Antibodies, Monoclonal , Antigens/metabolism , Antigens, Protozoan/metabolism , B-Lymphocytes/immunology , In Vitro Techniques , Leishmania major/immunology , Mice , Mice, Inbred BALB C , Ovalbumin/immunology , Ovalbumin/metabolism , Spleen/cytology , Spleen/immunology , Th2 Cells/immunologyABSTRACT
The antibody 4F7 was reported to recognize an epitope expressed on dendritic cells (DC) from various tissues. To study the ability of splenic 4F7+ dendritic cells to process antigen for presentation to CD4+ T cells, DC were enriched using a separation procedure avoiding overnight culture which could lead to an altered phenotype. These DC were used as antigen-presenting cells (APC) in stimulation cultures of major histocompatibility complex class II-restricted T cells. It was found that they induce antigen-dependent lymphokine production by T cells and therefore could present exogenous antigens. These processing takes place intracellularly, because fixation abrogates presentation to T cells. Moreover, antigen presentation needs intracellular processing within endo- or lysosomes as chloroquine-treatment prevents T cell activation. Titration of APC numbers revealed that contaminating APC most likely did not account for antigen-specific T cell activation by DC. No evidence was found for release of antigenic peptides or for partial antigen processing possibly done by cell surface located enzymes on DC. In conclusion, these results indicate that freshly enriched DC are able to process antigens similarly to other APC.