Search

An estimated 300-500 million new infections and 1.5-2.7 million deaths attributed to malaria occur annually in the developing world, and every year tens of millions of travelers from countries where malaria is not transmitted visit countries with malaria. Because the parasites that cause malaria have developed resistance to many antimalarial drugs, new methods for prevention are required. Intraperitoneal injection into mice of one dose of 150 ng (approximately 7.5 micrograms per kg body weight) recombinant mouse interleukin-12 (rmIL-12) 2 days before challenge with Plasmodium yoelii sporozoites protects 100% of mice against malaria. We report that one subcutaneous injection of 10 micrograms/kg recombinant human IL-12 (rhIL-12) 2 days before challenge with P. cynomolgi sporozoites protected seven of seven rhesus monkeys. Protection was associated with marked increases in plasma levels of interferon-gamma (IFN-gamma), and relative increases of lymphoid cell messenger RNA coding for IFN-gamma and several other cytokines. We speculate that rIL-12 protects monkeys through IFN-gamma-dependent elimination of P. cynomolgi-infected hepatocytes. This first report of rIL-12-induced protection of primates against an infectious agent supports assessment of rhIL-12 for immunoprophylaxis of human malaria.

Studies with rodents infected with Trichinella spiralis, Heligmosomoides polygyrus, Nippostronglyus brasiliensis, and Trichuris muris have provided considerable information about immune mechanisms that protect against parasitic gastrointestinal nematodes. Four generalizations can be made: 1. CD4+ T cells are critical for host protection; 2. IL-12 and IFN-gamma inhibit protective immunity; 3. IL-4 can: (a) be required for host protection, (b) limit severity of infection, or (c) induce redundant protective mechanisms; and 4. Some cytokines that are stereotypically produced in response to gastrointestinal nematode infections fail to enhance host protection against some of the parasites that elicit their production. Host protection is redundant at two levels: 1. IL-4 has multiple effects on the immune system and on gut physiology (discussed in this review), more than one of which may protect against a particular parasite; and 2. IL-4 is often only one of multiple stimuli that can induce protection. Hosts may have evolved the ability to recognize features that characterize parasitic gastrointestinal nematodes as a class as triggers for a stereotypic cytokine response, but not the ability to distinguish features of individual parasites as stimuli for more specific protective cytokine responses. As a result, hosts deploy a set of defense mechanisms against these parasites that together control infection by most members of that class, even though a specific defense mechanism may not be required to defend against a particular parasite and may even damage a host infected with that parasite.

We studied whether long-lived IgE+ memory B cells develop following three types of primary IgE immune responses. Immunization of mice with anti-IgD antibody induced a T cell-dependent, interleukin (IL)-4-dependent primary IgE response and the formation of IgE isotype switched (IgE+) memory B cells. These IgE+ memory B cells could be stimulated in vivo by injection with goat anti-IgE antibodies to produce a profound IL-4-independent memory IgE response. By contrast, both infection of mice with Nippostrongylus brasiliensis or repeated immunization with benzylpenicilloyl-keyhole limpet hemocyanin (BPO-KLH) in alum stimulated good primary IgE responses and profound memory T cell-dependent antigen-specific IgE responses, but failed to induce the development of long lived IgE+ memory B cells because they could not be recalled with goat anti-IgE antibodies. Mice receiving double immunizations combining anti-IgD with either N. brasiliensis infection or BPO-KLH immunization mounted significant goat anti-IgE-induced secondary IgE responses, but no N. brasiliensis or BPO-KLH-specific IgE could be detected. This indicates that the N. brasiliensis and BPO-KLH induced immune responses do not suppress the development of IgE+ B cells, but rather, do not provide the necessary conditions for their formation. Taken together these data indicate that long-lived IgE+ B cells fail to develop during the primary IgE response to N. brasiliensis infection or BPO-KLH immunization. By contrast, significant numbers of IgE+ memory B cells form during the primary IgE immune response induced by anti-IgD immunization. Our observations suggest that immunization protocols involving membrane IgD cross-linking and limited duration of cognate T cell help are necessary for the formation of IgE+ memory B cells. It will be important to determine the relevance of membrane IgD interaction with allergens, as this would influence the design of new therapies for the treatment of allergy and asthma.

Control of parasitic infections is dependent on the production of cytokines that activate mechanisms which limit invasion, reproduction or survival of the parasite. In contrast, conditions that induce inappropriate cytokine responses facilitate the spread of infection and ultimately exacerbate the level of disease. Measurement of local cytokine responses to different gastrointestinal parasites, such as the intracellular protozoan, Cryptosporidium parvum, and luminal dwelling nematodes like Nippostrongylus brasiliensis and Heligmosomoides polygyrus, reveal stereotype response patterns. In general, intracellular parasites stimulate type 1 responses where IFN-gamma is the predominant immune activator, while extracellular parasites stimulate type 2 responses where IL-4 plays a prominent role in elevating humoral immune mechanisms. Cytokines alter cellular function and the milieu of the intestinal lumen to affect the outcome of an infection. The importance of a particular response during the course of an infection can be studied by selective enhancement with an excess of exogenous recombinant cytokine or cytokine antagonists. For example, exogenous IL-12 enhances resistance to C.parvum, but suppresses the normally rapid cure of an infection with N. brasiliensis. Both mechanisms are dependent on expression of IFN-gamma. At the molecular level, exogenous IL-12 stimulates IFN-gamma production which elevates a protective type 1 response to C. parvum but converts the normally anti-worm type 2 response to a type 1 response that inappropriately regulates the infection. Alternatively, excess IL-4 plays a prominent role in modulating effector elements that change intestinal physiology to create a hostile environment for worm parasites. Exogenous IL-4 can cure chronic worm infection, while IL-4 antagonists interfere with protective responses to infection. These observations provide a paradigm for analysis of stereotype responses to different gastrointestinal parasites, and demonstrate how cytokine-induced immune system-dependent and independent effector mechanisms can limit parasitic infection, while inappropriate cytokine responses can exacerbate the state of disease.

Immunoglobulin (Ig) class switching in B cells is regulated by stimuli transduced by cytokines and cell-cell contact. Among these stimuli, interleukin (IL)-4 has been considered an absolute prerequisite for class switching to IgE in the mouse. Here we report that IL-4-deficient (IL-4-/-) and wildtype mice had comparably elevated serum IgE levels during the course of a murine retrovirus-induced immunodeficiency syndrome, MAIDS. IgE switching in IL-4-/- mice was also induced by injection of anti-IgD antibody. Treatment with anti-IgD induced germline epsilon (g epsilon) transcripts with comparable efficiency in IL-4-/- mice and controls, but the levels of productive epsilon transcripts (p epsilon) were lower by a factor of 200 and serum IgE levels were lower by a factor of 300 in IL-4-/- mice as compared with controls. Induction of g epsilon after anti-IgD treatment of IL-4-/- mice was unaffected by simultaneous treatment with monoclonal antibodies to IL-4 and IL-4 receptor alpha chain. Infection of IL-4-/- mice with Nippostrongylus brasiliensis, a potent stimulus for IgE production, resulted in induction of g epsilon transcripts; however, p epsilon transcripts were barely detectable and serum IgE was not detected. These findings establish a novel IL-4-independent pathway for IgE switching in the mouse that is strongly activated in retroviral infection but weakly in nematode infection. This pathway appears to be dependent on distinct factors that separately control induction of g epsilon transcription and switch recombination to p epsilon.

The gastrointestinal nematode parasite, Heligmosomoides polygyrus, has been used extensively in experimental studies of host immunity. The pronounced type 2 primary immune response to H. polygyrus is associated with elevated CD4+, TCR-alpha/beta + T cell IL-4 production and elevated serum IgE levels that are blocked by inhibiting CD28/ CTLA4-B7 interactions following in vivo administration of the chimeric fusion protein, CTLA4Ig. In the present study, we have examined the in vivo effects of blocking CTLA4Ig ligands on the secondary type 2 mucosal host protective immune response to this parasite. Our results show that although CD4+, TCR-alpha/beta + cells remain the primary source of elevated IL-4 during the secondary response, the protective immune response and the effector cell activity associated with it is B7-independent as CTLA4Ig administration at the time of challenge does not block (1) elevations in T cell IL-4 gene expression or protein secretion; (2) elevations in serum IgE levels, mucosal mastocytosis, or eosinophilia; or (3) host protection, as measured by adult worm burden and fecundity. These findings suggest that memory T helper cells do not require CD28-B7 interactions for their activation to effector cells that can mediate a host protective type 2 immune response.

We have shown that systemic administration of rmIL-12 could trigger Th1-type responses to a protein antigen delivered orally with CT as mucosal adjuvant. The most striking finding was that IL-12 could retain its regulatory effects when orally administered and could redirect the immune response to the oral vaccine toward a Th1-type. However, regulation by orally administered IL-12 differed from parenteral treatment with IL-12 since only the latter treatment affected mucosal S-IgA responses. These findings have important implications for the development of mucosal vaccines that induce the desired immune response.

Dendritic cells (DC) are unmatched among APCs in their ability to bind, process, and present Ag. Presentation by such potent APCs, if always immunogenic and never tolerogenic, might stimulate pathogenic autoimmune responses. To determine whether Ag presentation by DC can induce tolerance, mice were injected with a rat IgG2b anti-splenic DC mAb, 33D1, and challenged 13 to 28 days later with a stimulatory rat IgG2b mAb. Injection of mice with 1 ng/100 micrograms of 33D1 rarely induced an anti-rat IgG2b Ab response and, in most mice, induced rat IgG2b-specific T cell and B cell tolerance. Tolerant mice had decreased ability to secrete Ab and make both type 1 and type 2 cytokine mRNA and protein in response to immunization with rat IgG2b. 33D1 was 100- to 1000-fold more potent as a tolerogen than an isotype-matched control rat IgG2b mAb. Injecting mice with aggregated 33D1, 33D1 plus anti-IgD mAb, or 33D1 plus IL-1 induced an IgG1 anti-rat IgG2b Ab response rather than tolerance. IL-1 injected 3 days after 33D1 still induced an Ab response rather than tolerance. Not all anti-DC mAbs are tolerogenic. Injection of a DC-specific hamster anti-CD11c mAb (N418) stimulates an IgG anti-hamster response, and injection of 33D1 plus N418 stimulates both anti-hamster and anti-rat IgG2b responses. These observations indicate that DCs can present Ag in either a tolerogenic or stimulatory manner and suggest that inflammatory stimuli can convert an otherwise tolerogenic signal to a stimulatory signal.

CD40/CD40 ligand interactions are required for the development of T cell-dependent Ab responses in vivo. The role of these cell surface molecules in contributing to T cell cytokine production and the development of effector populations other than B cells and T cells is, however, less well defined. We have examined the in vivo effects of blocking CD40/CD40 ligand interactions on the type 2 mucosal immune response that follows oral inoculation of mice with the nematode parasite, Heligmosomoides polygyrus. Administration of anti-gp39 (CD40L) mAb (MR1) blocked H. polygyrus-induced elevations in serum IgG1 levels and inhibited elevations in blood eosinophils and mucosal mast cells at day 14 after inoculation. Anti-gp39 mAb markedly inhibited B cell blastogenesis 8 days after H. polygyrus inoculation but did not inhibit elevations in B cell class II MHC expression. Maximal elevations in B7-2 expression required signaling through both CD40 and the IL-4R. Elevations in T cell cytokine gene expression and elevations in the number of IL-4-secreting cells were unaffected by treatment with anti-gp39 mAb, although IL-4 production was inhibited by anti-IL-4R mAb. These results suggest that CD40/CD40L interactions are not required to activate T cells to produce cytokines but are required for the activation and proliferation of other effector cells associated with the type 2 response.

Recent evidence suggests that T cells and their associated cytokines critically influence outcome in mice experimentally infected with Borrelia burgdorferi (Bb), the causative agent of human Lyme disease. In vivo T cell subset and cytokine depletion studies suggest that CD4+ T cell-derived IL-4 plays a critical role in control of spirochete growth in vivo, whereas CD8+ T cell-derived IFN-gamma appears to promote disease, particularly in susceptible mouse strains. To further investigate the immunologic basis of protection and the role of IL-4, we have examined the effects of early rIL-4 treatment on outcome in susceptible mice infected with Bb. In this study, we show that administration of rIL-4 to susceptible C3H mice during the first week of infection with Bb leads to early control of their infections, as evidenced by significant reductions in joint swelling at wk 5, 6, and 7 postinfection, and in the numbers of spirochetes recovered from their joints and skin at wk 7 when compared with sham-treated mice. Increased resistance in rIL-4-treated mice was accompanied by significant reductions in their in vitro splenic Bb-specific IFN-gamma responses and in serum levels of specific IgG2a and IgG3 Abs and significant increases in specific IgG1 Abs. We also show that the inherent susceptibility of Ab-deficient, C57BL/6-IgM knockout (B6-MKO) mice to Rh infection is intermediate relative to C57BL/6 severe combined immunodeficient (B6-SCID) mice (susceptible) or normal C57BL/6 mice (resistant), confirming the importance of both Ab-dependent and Ab-independent, T cell-dependent immune mechanisms in control of Bb infections. The additional finding that early treatment with rIL-4 significantly reduced the severity of Bb infections in B6-MKO mice indicates that IL-4 may augment anti-spirochetal immunity via an Ab-independent mechanism.

We have assessed during B cell development, the regulation and function of bcl-x, a member of the bcl-2 family of apoptosis regulatory genes. Here we show that Bcl-xL, a product of bcl-x, is expressed in pre-B cells but downregulated at the immature and mature stages of B cell development. Bcl-xL but not Bcl-2 is rapidly induced in peripheral B cells upon surface immunoglobulin M (IgM) cross-linking, CD40 signaling, or LPS stimulation. Transgenic mice that overexpressed Bcl-xL within the B cell lineage exhibited marked accumulation of peripheral B cells in lymphoid organs and enhanced survival of developing and mature B cells. B cell survival was further increased by simultaneous expression of bcl-xL and bcl-2 transgenes. These studies demonstrate that Bcl-2 and Bcl-xL are regulated differentially during B cell development and activation of mature B cells. Induction of Bcl-xL after signaling through surface IgM and CD40 appears to provide mature B cells with an additional protective mechanism against apoptotic signals associated with antigen-induced activation and proliferation.

The protozoan parasite Cryptosporidium parvum (Cp) causes diarrhea that can be acutely severe in immunocompetent persons and can become chronic and life-threatening in immunocompromised individuals. Because studies in mice have implicated IFN-gamma in protection against this parasite, and IL-12 can induce IFN-gamma production, we examined the ability of IL-12 to prevent or cure Cp infection in neonatal BALB/c and SCID mice. Treatment of both immunocompetent and immunodeficient mice with IL-12 before experimental inoculation with Cp oocysts prevented or greatly reduced the severity of infection. Intestinal epithelial cell invasion and/or early intracellular development of Cp was inhibited by exogenous IL-12. The protective effect of IL-12 was completely blocked by anti-IFN-gamma mAb. Established infections were associated with elevated IFN-gamma gene expression and were not ameliorated by IL-12 treatment, even though such treatment further enhanced IFN-gamma gene expression. The severity of Cp infection was, however, exacerbated by treatment with anti-IL-12 Ab. These observations provide the first evidence that treatment with exogenous IL-12 can prevent Cp infection through an IFN-gamma-dependent, specific immune system-independent mechanism, and that endogenous IL-12 production has a role in limiting Cp infection.

Allergic asthma is characterized by airway hyperresponsiveness and pulmonary eosinophilia, and may be mediated by T helper (Th) lymphocytes expressing a Th2 cytokine pattern. Interleukin (IL) 12 suppresses the expression of Th2 cytokines and their associated responses, including eosinophilia, serum immunoglobulin E, and mucosal mastocytosis. We have previously shown in a murine model that antigen-induced increases in airway hyperresponsiveness and pulmonary eosinophilia are CD4+ T cell dependent. We used this model to determine the ability of IL-12 to prevent antigen-induced increases in airway hyperresponsiveness, bronchoalveolar lavage (BAL) eosinophils, and lung Th2 cytokine expression. Sensitized A/J mice developed airway hyperresponsiveness and increased numbers of BAL eosinophils and other inflammatory cells after single or repeated intratracheal challenges with sheep red blood cell antigen. Pulmonary mRNA and protein levels of the Th2 cytokines IL-4 and IL-5 were increased after antigen challenge. Administration of IL-12 (1 microgram/d x 5 d) at the time of a single antigen challenge abolished the airway hyperresponsiveness and pulmonary eosinophilia and promoted an increase in interferon (IFN) gamma and decreases in IL-4 and IL-5 expression. The effects of IL-12 were partially dependent on IFN-gamma, because concurrent treatment with IL-12 and anti-IFN-gamma monoclonal antibody partially reversed the inhibition of airway hyperresponsiveness and eosinophilia by IL-12. Treatment of mice with IL-12 at the time of a second antigen challenge also prevented airway hyperresponsiveness and significantly reduced numbers of BAL inflammatory cells, reflecting the ability of IL-12 to inhibit responses associated with ongoing antigen-induced pulmonary inflammation. These data show that antigen-induced airway hyperresponsiveness and inflammation can be blocked by IL-12, which suppresses Th2 cytokine expression. Local administration of IL-12 may provide a novel immunotherapy for the treatment of pulmonary allergic disorders such as atopic asthma.

The principal pathological manifestation of murine Schistosoma mansoni infection is the egg-induced granuloma. Synchronous pulmonary granulomas forming around intravenously injected schistosome eggs are widely used to study the immunopathology of schistosomiasis. A number of anticytokine antibody treatments have a remarkable effect in modulating granulomas in this model but little effect on the size of hepatic granulomas around laid eggs during experimental infection. To examine this discrepancy, we examined the effects of anticytokine antibodies on liver and lung granulomas around injected eggs and around eggs laid during infection in both locations. Anti-interleukin-4 (IL-4) treatment greatly reduced the volume of granulomas around eggs injected into the liver via the portal vein and around eggs injected into the lung via the tail vein. On the contrary, granulomas around eggs laid by worms in either the liver or the lung during the course of infection were not significantly decreased in size by anti-IL-4 treatment. Thus, site is not important for the disparate effects of anti-IL-4 in granuloma formation around injected versus laid eggs. This effect is seen in naive and sensitized animals and is most probably due to differences in the quality of injected eggs versus those laid in situ by the worms.

We examined the ability of a long acting formulation of IL-4 (IL-4/anti-IL-4 mAb complexes) to limit established infections of normal and immune deficient mice with two nematode parasites: Heligmosomoides polygyrus and Nippostrongylus brasiliensis. IL-4, at a dose of 5 to 20 micrograms every 3 to 4 days, rapidly decreased egg production and, over a period of 6 to 9 days, terminated infection in H. polygyrus-inoculated BALB/c mice. IL-4 treatment also considerably decreased egg production in H. polygyrus-inoculated CB.17 severe combined immunodeficient (SCID) mice and terminated infection in N. brasiliensis-inoculated SCID mice and anti-CD4 mAb-treated BALB/c mice. IL-4 was less effective at limiting H. polygyrus infection if administrated when parasites were in larval stages than if administered after adult worms had developed. The effects of IL-4 were inhibited completely by an mAb that specifically blocks the mouse IL-4R. These observations demonstrate that IL-4 can limit the fecundity and survival of gastrointestinal nematode parasites through effects on the host that are independent of the specific immune system.

Anti-interleukin-4 (IL-4) treatment of Schistosoma japonicum-infected mice markedly inhibited in vitro secretion of the Th2 cytokines IL-4 and IL-5 from antigen-stimulated spleen cells, but enhanced the secretion of the Th1 cytokine IFN-gamma. IL-2 secretion was unaffected. Hepatic fibrosis was markedly diminished in anti-IL-4-treated-mice at ten weeks of infection while granulomas around S. japonicum eggs in the livers were slightly-to-moderately increased in size. The number of eggs per worm pair in the tissues and feces did not differ significantly in treated and untreated mice. These findings suggest that Th2 cytokine responses are important in the genesis of schistosomal hepatic fibrosis.

In vivo experiments were performed to determine whether the cross-linking of membrane immunoglobulin (mIg) D on mature B cells, in the absence of T cell help, leads to B cell death. Mice were injected with either a monoclonal antibody (mAb) that cross-links mIgD effectively or a mAb that binds to mIgD avidly but cross-links it to a limited extent, and effects on B cell number and B cell Ia, mIgM, and mIgD expression were observed. In most experiments, mice were pretreated with anti-interleukin 7 mAb to prevent the generation of new bone marrow B cells, and with anti-CD4 mAb to prevent the generation of T cell help. In some experiments, mice also received anti-Fc gamma RII mAb to prevent cross-linking of mIgD with Fc gamma RII, and cobra venom factor to prevent possible mIg-complement receptor interactions and complement-mediated killing of B cells. The results of these studies demonstrate that (a) even limited cross-linking of mIgD on mature B cells can lead to B cell death; (b) increased cross-linking of mIgD leads to increased B cell death; (c) the loss of B cells is first detected 2 d after anti-IgD mAb injection and increases during the subsequent 3 d; (d) sustained modulation of mIgD may be necessary to cause B cell death; (e) mIgMdull but not mIgMbright B cells are lost in mice injected with anti-IgD mAbs; and (f) T cell help prevents or minimizes B cell death.

The CD28/CTLA-4 costimulatory signal is required for TCR-mediated T cell activation resulting in increased IL-2 production in vitro, but its role in IL-4 production is unclear and few studies have examined the function of CTLA-4/CD28 in the in vivo immune response. We have examined the in vivo effects of blocking the interaction of B7 with its ligands, CTLA-4 and CD28, in an IL-4 dominant in vivo immune response to goat anti-mouse IgD. This response is characterized by elevations in serum Igs preceded by elevations in IL-2 and the Th2 cytokines: IL-4, IL-9, and IL-10. The fusion protein CTLA4-Ig administered during the in vivo immune response to goat anti-mouse IgD caused an inhibition in elevations of IL-2, IL-4, and IL-9 gene expression at both day 3 and day 6 after immunization. In contrast, IL-10 cytokine gene expression as late as day 6 after immunization was not decreased. Cell sorting analysis demonstrated that TCR-alpha beta +, CD4+ T cells were the primary source of the elevated IL-10, suggesting that T cell activation leading to IL-10 gene expression may not require CTLA-4 ligand interactions. Similarly CTLA4-Ig completely blocked elevations in the number of IL-4- but not IL-10-secreting cells, as measured by ELISPOT, in both unsorted splenic cells and sorted CD4+, TCR-alpha beta+ T cells. In situ staining of spleen sections also showed inhibition of IL-4-producing cells. These results suggest that, with the notable exception of IL-10, interaction, of B7 with its ligands is required for elevated Th2 cytokine gene expression and secretion during a primary systemic IL-4-dominant response.

Among other effects, prostaglandins (PG) of the E series are known to inhibit several acute and chronic inflammatory conditions in vivo and proinflammatory cytokine production by activated macrophages in culture. The research presented here demonstrates that the inhibitory effect of PGE2 on tumor necrosis factor alpha (TNF-alpha) and interleukin 6 (IL-6) production by lipopolysaccharide (LPS)-stimulated murine peritoneal macrophages involves IL-10. In a dose-dependent manner, PGE2 inhibits LPS-induced release of TNF-alpha and IL-6, but not of lactate or nitric oxide. The decrease in the level of these cytokines is inversely proportional to the increase in immunoreactive IL-10. This differential inhibitory effect of PGE2 is mimicked by agents that elevate intracellular levels of cAMP, but not cGMP. Neutralizing anti IL-10 antibody but not neutralizing antibodies against other macrophage secretory products (IL-6, leukemia inhibitory factor, and transforming growth factor beta [TGF-beta]), significantly reverse the potent inhibitory effect of PGE2. In vivo, the administration of PGE2 before LPS challenge significantly reduces circulating TNF-alpha and IL-6 levels. Anti-IL-10 antibody substantially enhanced the LPS-induced TNF-alpha and IL-6 levels in mice that received either LPS alone or LPS plus PGE2. These results suggest that the anti-inflammatory effect of PGE2 on mononuclear phagocytes is mediated in part by an autocrine feedback mechanism involving IL-10.

IL-12, a potent stimulator of hemopoietic progenitor cells, was evaluated as a potential protector against 60Co-gamma radiation-induced lethal hemopoietic syndrome in mice. Administration of IL-12 before lethal irradiation of genetically distinct strains of mice, B6D2F1 and C3H/HeJ, protected a significant fraction of both strains of mice from death. Radioprotection was associated with a fivefold increase in the number of bone marrow cells at 6 days after irradiation. Even at supralethal doses of radiation (1200 cGy), the number of c-kit+ bone marrow cells 3 days after irradiation was twofold greater in IL-12-treated mice than in saline-treated mice. However, mice that received IL-12 and 1200 cGy (B6D2F1) or 900 cGy (C3H/HeJ) died of the gastrointestinal syndrome, as was evident by gross necroscopy and histologic evaluation, within 4 to 6 days after irradiation. Induction of the gastrointestinal syndrome in mice not treated with IL-12 required radiation doses of 1500 cGy or greater in both strains. Thus, at doses of radiation at which IL-12 still protects c-kit+ hemopoietic cells, it sensitizes the intestinal tract to damage. Radioprotection with IL-12 was abrogated by anti-IL-1R or anti-stem cell factor Ab. Anti-IFN-gamma Ab did not affect IL-12-induced hemopoietic radioprotection, but abrogated sensitization of the intestinal tract by IL-12. The sensitizing effect of IL-12 may be related to its ability to prime mice to subsequent inflammatory challenge, as demonstrated by an almost 100-fold increase in circulating TNF and IL-6 levels in normal B6D2F1 mice challenged with IL-12 and LPS. This priming effect of IL-12 also was abrogated by anti-IFN-gamma Ab.

The injection of DBA/2 (D2) spleen cells into (C57BL/6 x DBA/2)F1 mice (BDF1) induces a chronic, autoimmune graft-vs-host disease (GVHD) that is characterized by: increased production of Th2-associated cytokines; increased levels of serum Ig, including IgE; increased production of IgG anti-DNA Abs; and no detectable antihost CTL activity. Experiments were performed to determine if treatment with the cytokine IL-12, which stimulates the production of Th1-associated cytokines and inhibits Th2-associated cytokine production, would inhibit humoral autoimmunity in this system. Treatment of mice with 100 ng IL-12 per day for 5 days, starting on the day of cell transfer, resulted in: 1) near complete suppression of autoantibody production; 2) decreased serum Ig levels; 3) detectable donor antihost CTL activity; and 4) greatly reduced numbers of host splenic B and T cells. Treatment of mice with a neutralizing anti-IFN-gamma mAb did not reverse these effects of IL-12. Thirty nanograms per day resulted in reduced numbers of host B cells and reduced serum anti-DNA levels, but no detectable antihost CTL activity. IL-12 treatment initiated 7 days after cell transfer had little effect on the development of autoimmune GVHD. These observations suggest the following: 1) IL-12 inhibits humoral autoimmunity in a murine parent-->F1 GVHD model by inducing the activation of host-reactive CTLs that reject the host immune system. 2) This effect is IFN-gamma-independent. 3) IL-12 needs to be present during the initial differentiation of T cells in this system to have this effect.

Intraperitoneal injection of recombinant Interleukin 12 (rIL-12) at 30 ng/day for 5 days beginning 1 to 2 days before sporozoite challenge or administration of a single dose of 150 ng of rIL-122 days before challenge protected 100% of BALB/c mice against challenge with 10(2) Plasmodium yoelii sporozoites. rIL-12-induced protection was eliminated in all mice by administration of a monoclonal antibody against interferon gamma and in 50% of mice by administration of NG-monomethyl-L-arginine, a competitive inhibitor of nitric oxide synthase. rIL-12 protected BALB/c mice treated with cytotoxic anti-CD4 and anti-CD8 monoclonal antibodies, as well as T-cell- and B-cell-deficient severe combined immunodeficiency mice. These data suggest that rIL-12 stimulates non-B, non-T cells to produce interferon gamma that kills intrahepatic parasites by stimulating nitric oxide production. If rIL-12 proves to be well tolerated by humans, our findings support consideration of rIL-12 as an immunoprophylactic against malaria.

In view of our observation that anti-immunoglobulin antibody conjugated to high-molecular-weight dextran stimulates high levels of B-cell activation (Brunswick et al. J. Immunol. 1989, 143, 1239), we coupled T cell-dependent antigens to dextran. When mice were immunized, in the absence of adjuvant, with a BSA-dextran conjugate (BSA-dex), a persistent, high-titre anti-BSA IgG1 response was induced. Titres were dose-dependent and seen with as little as 10 micrograms of conjugated protein. Anti-BSA titres were detected as early as day 7, usually peaked at about day 14 and persisted for at least 4 weeks. Anti-hapten antibodies were also elicited in mice that were immunized with haptenated BSA covalently bound to dextran, and secondary responses could be induced even after inoculation of the unconjugated protein. Covalent attachment of the protein to the polymer was necessary, and the response was specific, as coinjection of BSA-dex and an unrelated antigen, goat IgG, did not elicit detectable anti-goat antibodies. The immunogenic potential of these conjugates did not depend on the ability of the dextran carrier to induce antibody, inasmuch as they stimulated high levels of anti-protein antibody in mice unresponsive to dextran. A minimum size dextran polymer was required for enhanced immunogenicity as conjugates of BSA with dextran of molecular mass 500 or 2000 kDa but not of 70 kDa gave detectable anti-BSA titres.

The costimulatory signal provided to T cells through CTLA-4-ligand interactions is required for T cell activation resulting in increased interleukin 2 (IL-2) production in vitro, but its role in the production of IL-4 and other cytokines is unclear and few in vivo studies have been performed to confirm results of in vitro experiments. We have examined the in vivo effects of blocking CTLA-4 ligands on the T helper cell 2 (Th2)-associated mucosal immune response that follows oral infection of mice with the nematode parasite, Heligmosomoides polygyrus. CTLA-4Ig administration inhibited H. polygyrus-induced increases in mesenteric lymph node (MLN) B cell major histocompatibility complex class II expression and size and T cell-derived IL-4 gene expression. In addition, CTLA-4 immunoglobulin (Ig) partially blocked increased IL-3, IL-5, and IL-9 cytokine gene expression in Peyer's patch (PP) and MLN 8 d after primary inoculation of mice with the parasite. Increases in the number of IL-4- but not IL-5-secreting cells were also inhibited by CTLA-4Ig. H. polygyrus-induced elevations in serum IgE levels but not blood eosinophils, were markedly inhibited by CTLA-4Ig. These results suggest that stimulation of CD28 and/or CTLA-4 is required for T cell priming leading to IL-4 cytokine production, B cell activation, and IgE secretion during a Th2-like, mucosal immune response to a nematode parasite.

Increasing evidence suggests that schistosome egg granulomas are primarily Th2 cellular reactions. Mice infected with Schistosoma mansoni were treated with a neutralizing mAb against IL-4 to evaluate the role of this cytokine in the generation of parasite egg-induced cell-mediated responses and hepatic pathology. Animals treated with anti-IL-4 before egg deposition showed decreased IL-4, IL-5, and IL-10 production in response to in vitro antigenic stimulations and decreased IL-5 and IL-13 mRNA levels in the liver. As observed previously, non-B, non-T cells were a major source of IL-4 in infected mice treated with control mAb, and the diminished IL-4 response in anti-IL-4-treated animals was shown to be caused at least in part by a reduction in the number of these cells, as well as by decreased secretion of IL-4 per cell. In contrast, production of the Th1 cytokines IL-2 and IFN-gamma was elevated in anti-IL-4-treated infected mice in vitro, and the corresponding mRNAs in the liver were increased. Anti-IL-4 treatment did not consistently reduce the size of hepatic granulomas around S. mansoni eggs, but markedly inhibited granuloma formation in the lungs of the same animals after i.v. egg injection. Nevertheless, anti-IL-4-treated infected mice showed consistent and marked reductions in hepatic collagen deposition. These findings indicate that IL-4 plays a major role in the development of the Th2 response in S. mansoni-infected mice and contributes to the pathogenesis of hepatic fibrosis.

We investigated the role of soluble interleukin-4 receptor (sIL-4R) as a regulator of IL-4 mediated activities in vivo. Administration of recombinant sIL-4R to mice resulted in (i) prolonged survival of heterotopic cardiac allografts; (ii) decreased popliteal lymph node enlargement in response to allogeneic cells; and (iii) inhibition of IgE secretion in response to anti-IgD treatment. Transgenic mice constitutively expressing elevated levels of biologically active sIL-4R displayed prolonged cardiac allograft survival compared with control animals. However the sIL-4R transgenic mice were capable of mounting normal antigen-specific IgE responses despite the presence in serum of up to 3 micrograms/ml sIL-4R. Surprisingly, coadministration of IL-4/sIL-4R or IL-4/anti-IL-4 mAb complexes caused a superinduction of IgE secretion in anti-IgD-treated normal mice and subsequently in other IL-4-dependent biological activities. Thus, recombinant sIL-4R can not only antagonize functions mediated by endogenous IL-4, but also potentiate the biological activity of exogenously administered IL-4. These dual roles may have possible clinical implications for the recombinant molecule, as well as for natural sIL-4R immunoregulation.

The pathogenesis of asthma remains unclear. An in vivo murine model of antigen-induced airway hyperreactivity and inflammation was developed to investigate the possibility, suggested by a wealth of descriptive human data, that alterations in immunoregulation are important in the genesis of airway hyperreactivity. A/J mice developed airway hyperreactivity and markedly increased numbers of pulmonary inflammatory cells following intraperitoneal sensitization and intratracheal challenge with sheep red blood cells. Notably, eosinophils were a prominent component of the inflammatory infiltrate. The dependence of these phenomena, both pathologic and functional, on CD4+ T lymphocytes was investigated by in vivo depletion of CD4+ cells using the anti-CD4 mAb GK1.5. When administered before antigen challenge, GK1.5 completely prevented both airway hyperreactivity and the infiltration of eosinophils. This model provides the first direct demonstration of the dependence of airway hyperreactivity upon CD4+ T lymphocytes, and the results are consistent with the possibility that eosinophils are effectors of this response.

The cytokine interleukin (IL) 12 stimulates T cell and natural killer cell production of interferon (IFN) gamma and inhibits T cell production of IL-4. We investigated the effects of IL-12 on cytokine gene expression, immunoglobulin (Ig)E, mucosal mast cell, and eosinophil responses, and the course of infection in mice inoculated with the nematode parasite Nippostrongylus brasiliensis, as well as the IFN-gamma dependence of these effects. IL-12 stimulated IFN-gamma and IL-10 gene expression during primary and secondary N. brasiliensis infections and inhibited IL-3, IL-4, IL-5, and IL-9 gene expression during primary infections but had little inhibitory effect during secondary infections. IL-12 inhibited IgE, mucosal mast cell, and blood and tissue eosinophil responses during primary infections, but only eosinophil responses during secondary infections. IL-12 enhanced adult worm survival and egg production during primary, but not secondary infections. IL-12 needed to be administered by day 4 of a primary infection to inhibit IgE and mucosal mast cell responses, and by day 6 to strongly inhibit eosinophil responses and to enhance worm survival and fecundity. Anti-IFN-gamma mAb inhibited the effects of IL-12 on IgE secretion, intestinal mucosal mastocytosis, and parasite survival and fecundity, but did not affect IL-12 inhibition of eosinophilia. These observations indicate that IL-12, if administered during the initiation of eosinophilia. These observations indicate that IL-12, if administered during the initiation of an immune response, can change the response from one that is characterized by the production of T helper (Th)2-associated cytokines to one characterized by the production of Th-1 associated cytokines. However, IL-12 treatment has less of an effect once the production of Th2-associated cytokines has become established. In addition, our results provide evidence that Th2-associated responses protect against, and/or Th1-associated responses exacerbate, nematode infections.

Targeting Ag to the Fc epsilon RII by Ag-specific IgE has been shown to be an efficient means of enhancing Ag presentation by B cells to Ag-specific T cells. To take advantage of the Fc epsilon RII as a targeting molecule and to investigate whether IgE was required for mediation of the enhanced stimulation, Ag was covalently coupled to anti-Fc epsilon RII by using heterobifunctional crosslinking reagents. These Ag-Ab conjugates were used with T cell lines specific for the Ags, OVA (BB6.5) or rabbit gamma-globulin (CDC35 and D1.6), and splenic B cells to examine both B cell and T cell proliferation in vitro. Significant presentation of Ag-anti-Fc epsilon RII conjugates was apparent at doses of Ag 1,000- to 10,000-fold lower than seen with unconjugated Ag alone. Ag presentation with the use of anti-Fc epsilon RII-Ag conjugates was as good as or better than conjugates with Ab to the adhesion molecule Pgp-1 or control Ab in T cell proliferation and better than those conjugates in B cell proliferation assays (10- to 100-fold). Anti-Fc epsilon RII-Ag conjugates were clearly more effectively presented than Ag-anti-Fc gamma RII conjugates (> 100-fold). Mouse Fc epsilon RII is presently known to be expressed on B cells and follicular dendritic cells and these in vitro results suggest that the conjugates would be useful tools for investigating the role of IgE-mediated B cell Ag presentation in vivo. BALB/c mice immunized with OVA-anti-Fc epsilon RII conjugates made a quite significant OVA-specific IgG1 response and a detectable IgE response. No detectable Ab was produced in response to OVA alone and a minimal response was seen when an isotype-matched control conjugate was used. Thus, the results indicate that Fc epsilon RII targeting is operative both in vivo and in vitro.

In vivo experiments were performed to determine if the cross-linking of mlg on antigen-presenting B cells could induce them to present Ag to naive T cells in a stimulatory rather than a tolerogenic fashion. Mice were injected with a foreign mAb to a B cell Ag (Fc epsilon RII or CR1), and/or a self-anti-IgD mAb. Injection of either mAb alone failed to induce an Ab response; however, simultaneous injection of the foreign anti-B cell mAb plus the self-anti-IgD mAb stimulated a large response. Inasmuch as injection of the anti-IgD mAb should not have facilitated transfer of the foreign mAb to dendritic cells, this observation suggests that cross-linking of B cell mlg can induce B cells to acquire the ability to present Ag to naive T cells in an activating manner. Furthermore, when injected with anti-IgD mAb, both foreign anti-B cell mAbs were more potent in inducing Ab responses in this system than were isotype-matched control mAbs, consistent with the hypothesis that T cells were activated by Ag-presenting B cells. Results of dose-response and cell transfer studies, however, suggested that stringent cross-linking of B cell mlg on large numbers of B cells is required for B cell Ag presentation to induce T cell activation rather than tolerance. Therefore, these observations suggest that professional APCs usually are required to activate naive T cells, and that B cell Ag presentation can only activate naive T cells under unusual circumstances.

T cell tolerance can be induced by B cell presentation of Ags to naive T cells. To further characterize this mechanism of T cell tolerance induction, we have investigated the effects of injecting mice with an intact rat IgG2a Ab, which binds to the B cell low-affinity Fc epsilon receptor (CD23), on the responsiveness of B cells and T cells to rat IgG2a. Our observations indicate that 1) intravenous, subcutaneous, or intraperitoneal injection of this Ab induces antigen-specific B cell and T cell tolerance; 2) both forms of tolerance are induced more completely by injection of rat IgG2a anti-CD23 mAb than by injection of an equal dose of a control rat IgG2a Ig; and 3) reduced responsiveness to Ag is seen as early as 1 to 3 days after anti-CD23 mAb injection and reaches maximum levels by 7 days after injection. Although tolerance induced by the injection of soluble proteins has been reported to be characterized by reduced production of IL-2 and IFN-gamma, but normal production of IL-4, injection of mice with rat IgG2a anti-mouse CD23 mAb greatly decreases the IL-4 response to a rat IgG2a immunogen that normally induces a large IL-4 response.

In experimental Leishmania donovani infection in BALB/c mice, initial susceptibility gives way to T-cell-dependent acquired resistance and eventual control over visceral infection. Since various cytokines appear to underlie the host response to Leishmania infection, we examined infected liver tissue for gene expression of cytokines associated with Th1 (gamma interferon [IFN-gamma] and interleukin-2 [IL-2]) and Th2 cells (IL-4 and IL-10). By Northern (RNA) blot analysis, only IFN-gamma mRNA expression was detected in livers of infected euthymic mice. To determine whether activation of Th1 cells develops selectively in this model, qualitative PCR analysis was used. These results indicated that mRNAs for IFN-gamma, IL-2, IL-4, and IL-10 were all induced by L. donovani infection. The potentially negative Th2 cell-associated response did not appear to play a functional role, however, since resistance was acquired, anti-IL-4 monoclonal antibody treatment did not accelerate control over visceral infection, and serum immunoglobulin E levels remained low. As judged by PCR analysis, IL-4 and IL-10 mRNAs were also expressed under three other conditions without apparent effect: in naive euthymic mice treated with IL-2, which induces leishmanicidal activity; in rechallenged immune mice, which resist reinfection; and in nude mice, which fail to control L. donovani. These results suggest that, like other Leishmania species, L. donovani infection may trigger a potentially suppressive Th2 cell-associated cytokine response. However, in T-cell-intact mice able to control L. donovani, this response either is insufficient to influence outcome or more likely is overshadowed by the Th1 cell response.

The effects of murine rIL-12 on cytokine gene expression and Ig secretion were studied in vivo. In untreated mice IL-12 enhanced IFN-gamma and IL-10 gene expression and protein secretion, reduced base line IL-3 and IL-4 gene expression, and increased serum IgG2a concentration. In mice that had been injected with goat anti-mouse IgD antibody (G alpha M delta) to induce increases in IL-3, IL-4, and IL-10 gene expression and serum IgE, IgG1, IgG2a, and IgG3 concentrations, the simultaneous injection of IL-12 enhanced IFN-gamma and IL-10 gene expression and suppressed IL-3 and IL-4 gene expression and serum IgG and IgE responses. Anti-IFN-gamma mAb neutralized most, but not all, IFN-gamma produced by mice treated with G alpha M delta and IL-12. Anti-IFN-gamma mAb enhanced IL-3 and IL-4 gene expression, did not affect IL-10 or IFN-gamma gene expression, and increased serum IgG1, IgG2a, and IgG3 levels, but had relatively little effect on serum IgE in these mice. In contrast to its effects in G alpha M delta-treated mice, IL-12 failed to inhibit the IgE response to G alpha M epsilon antibody, which stimulates mIgE+ B cells to secrete IgE. These observations demonstrate that: 1) IL-12 may limit its own effects by inducing the production of a cytokine (IL-10) that down-regulates both IL-12 production and IL-12-induced IFN-gamma production; 2) IL-12 inhibits the production of at least one cytokine, IL-3, that is not generally regarded to be strictly Th1- or Th2-associated; 3) IL-12 inhibits switching to IgE secretion to a greater extent than it inhibits switching to other Ig isotypes; and 4) the in vivo effects of IL-12 are, to a large extent, IFN-gamma-dependent.

Eosinophils obtained by bronchoalveolar lavage (BAL) from the lungs of mice infected with Toxocara canis were characterized by flow cytometry with respect to cytophilic antibodies and surface Fc receptors. Freshly harvested BAL eosinophils were negative for sIgM, sIgA, sIgE, and Fc epsilon RII. These eosinophils were positive for sIgG1 and Fc gamma RII, although not all Fc gamma RIIs contained bound ligand. Culturing eosinophils for 24 or 48 hr with exogenous IgE and/or IL-4 did not induce IgE binding capacity or Fc epsilon RII expression. IL-4 did not decrease Fc gamma RII expression but did decrease ligand binding capacity by Fc gamma RII. These findings are in marked contrast to the results of studies characterizing the surface of both human and rat eosinophils and may indicate different functional activities for mouse BAL eosinophils in helminth infections.

The specificity of antibody (Ab) responses depends on focusing helper T (Th) lymphocyte signals to suitable B lymphocytes capable of binding foreign antigens (Ags), and away from nonspecific or self-reactive B cells. To investigate the molecular mechanisms that prevent the activation of self-reactive B lymphocytes, the activation requirements of B cells specific for the Ag hen egg lysozyme (HEL) obtained from immunoglobulin (Ig)-transgenic mice were compared with those of functionally tolerant B cells isolated from Ig-transgenic mice which also express soluble HEL. To eliminate the need for surface (s)Ig-mediated Ag uptake and presentation and allow the effects of sIg signaling to be studied in isolation, we assessed the ability of allogeneic T cells from bm12 strain mice to provide in vivo help to C57BL/6 strain-transgenic B cells. Interestingly, non-tolerant Ig-transgenic B cells required both allogeneic Th cells and binding of soluble HEL for efficient activation and Ab production. By contrast, tolerant self-reactive B cells from Ig/HEL double transgenic mice responded poorly to the same combination of allogeneic T cells and soluble HEL. The tolerant B cells were nevertheless normally responsive to stimulation with interleukin 4 and anti-CD40 Abs in vitro, suggesting that they retained the capacity to respond to mediators of T cell help. However, the tolerant B cells exhibited a proximal block in the sIg signaling pathway which prevented activation of receptor-associated tyrosine kinases in response to the binding of soluble HEL. The functional significance of this sIg signaling defect was confirmed by using a more potent membrane-bound form of HEL capable of triggering sIg signaling in tolerant B cells, which markedly restored their ability to collaborate with allogeneic Th cells and produce Ab. These findings indicate that Ag-specific B cells require two signals for mounting a T cell-dependent Ab response and identify regulation of sIg signaling as a mechanism for controlling self-reactive B cells.

Most inbred strains of mouse infected with the intestinal nematode Trichuris muris are resistant to infection expelling the parasite before adult worms establish. However, a few susceptible strains exist that are incapable of worm expulsion and harbor chronic infections of mature adult worms. Analyses of in vitro cytokine production by cells from the draining lymph node (mesenteric lymph node) have indicated that expulsion phenotype is tightly correlated with the selective expansion of helper T cells (Th) of the Th1 or Th2 cell subset within the mesenteric lymph node, resulting in susceptibility and resistance to T. muris, respectively. We have now confirmed and extended our in vitro observations in a series of experiments involving the in vivo manipulation of host cytokine levels. Depletion of interferon (IFN)-gamma in normally susceptible mice resulted in expulsion of the parasite, representing the first evidence for a role for IFN-gamma in the establishment of chronic helminth infection. Blocking interleukin (IL)-4 function in normally resistant animals prevented the generation of a protective immune response allowing adult stages of the parasite to develop. Conversely the administration of IL-4 to a normally susceptible host facilitated expulsion and indeed enabled established adult worms to be expelled when administered late in infection. In all cases assessment of a variety of in vivo parameters indicative of a Th1- or Th2-type response (parasite-specific immunoglobulin (Ig) G2a and the parasite-specific IgG1, total IgE levels and intestinal mastocytosis, respectively) demonstrated that the in vivo modulation of a Th1- or Th2-specific cytokine allowed the reciprocal Th cell subset to expand and become dominant with dramatic consequences for worm expulsion.

Mice infected with the gastrointestinal nematode parasite Nippostrongylus brasiliensis (Nb) develop responses associated with enhanced production of IL-4 (increased serum IgE levels and intestinal mucosal mastocytosis) and IL-5 (tissue and peripheral blood eosinophilia). The antagonistic effects of IFN on IL-4-mediated responses prompted an examination of the effects of IFN on the host response to Nb. Treatment with rIFN-alpha and rIFN-gamma induced a marked increase in parasite egg production (fecundity) in BALB/c mice infected with Nb and delayed intestinal expulsion of adult worms. Treatment with rIFN-alpha or rIFN-gamma also inhibited the rise in peripheral blood eosinophilia that follows inoculation with Nb, and the intensity of pulmonary perivascular tissue eosinophilia. However, Nb-induced increases in serum IgG levels and intestinal mastocytosis were only temporarily delayed by IFN. Induction of endogenous IFN production by injection of fixed Brucella abortus into mice infected with Nb also resulted in an increased worm fecundity and delayed adult worm expulsion. These effects were ablated when mice given Brucella abortus also received injections of neutralizing anti-IFN antibodies. Thus, IFN inhibit host protective immunity to Nb, perhaps by interfering with the production and effects of Th2 cytokines.

In vitro studies have established that Ig isotype switching typically involves deletion of CH genes that are located between VDJ and the CH gene that will be expressed, and is preceded by transcription of a germline (g) form of that CH gene. Increases in g epsilon transcript levels are induced by the cytokine IL-4, and always precede switching to IgE. To evaluate whether a similar relationship occurs in vivo, we examined IL-4 mRNA, g epsilon RNA, productive (p) epsilon mRNA, and serum IgE levels in two in vivo systems: one in which the injection of anti-IgD antibody induces mIgD+ B cells to switch to the expression of IgE and to secrete this isotype, and a second in which the injection of anti-IgE antibody stimulates IgE secretion by B cells that had been induced to express membrane IgE by earlier treatment with anti-IgD antibody. Increases in IL-4 transcript levels in anti-IgD-injected mice were followed within 24 h by increases in g epsilon RNA, and, one to two days later, by increased p epsilon mRNA and serum IgE levels. IL-4 antagonists blocked the g epsilon and p epsilon RNA and serum IgE responses in these mice, whereas the injection of otherwise untreated mice with IL-4 stimulated, within 24 h, a large increase in g epsilon RNA levels, followed 1-2 days later by a small increase in p epsilon mRNA. Injection of anti-IgD-primed mice with anti-IgE antibody also stimulated increases in IL-4, g epsilon and p epsilon RNA levels; however, the increases in IL-4 and g epsilon RNA were considerably smaller, and the increases in p epsilon mRNA and serum IgE considerably larger, than those observed in anti-IgD antibody-injected mice. IL-4 antagonists blocked the anti-IgE antibody-induced g epsilon RNA response, but not the p epsilon mRNA or serum IgE responses. Thus, IL-4 is required for the induction of g epsilon RNA in at least two in vivo systems, increased g epsilon RNA levels precede increases in p epsilon RNA levels in vivo as in vitro, and neither IL-4 nor g epsilon RNA is required to induce B cells that have already switched to IgE expression to differentiate into IgE-secreting cells.

Immunization of BALB/c mice with killed Brucella abortus (BA) has previously been shown to increase serum IgG2a levels and long-term T cell clones from these mice secrete Th1-associated cytokines: IFN-gamma and IL-2 but not IL-4 or IL-5. We analyzed cytokine gene expression following primary immunization with BA to determine when CD4+ T cells first express cytokine genes and whether specific hypothesized cytokine patterns (e.g. Th precursor, Th0) could be identified prior to a Th1-like pattern. Our results demonstrated a highly consistent and novel pattern of Th1/Th2 cytokine gene expression characterized by elevated IL-10 and IFN-gamma in CD4+ T cells which rapidly manifests itself and is sustained for at least 10 days after immunization. No elevation in IL-2 cytokine gene expression was observed and treatment of BA-immunized mice with blocking anti-IL-2 antibodies had no effect on the cytokine gene expression pattern, although treatment with anti-IFN antibodies resulted in increased IL-4, IL-5, and IL-9 cytokine gene expression, in the absence of any change in IFN-gamma or IL-10 as early as 4 days after immunization. These results suggest that a whole pathogen may trigger sufficient costimulatory signals to rapidly induce effector T cells in the absence of elevated IL-2 and that IL-10 is specifically elevated in certain Th1-like responses.

Anti-cytokine antibodies that block interactions between cytokines and cytokine receptors have been used to inhibit endogenous cytokine function. However, injection of mice with mixtures of IL-4 and either of two neutralizing anti-IL-4 mAb, at a cytokine/anti-cytokine mAb molar ratio of approximately 2:1, enhances and prolongs in vivo IL-4 activity, as measured by induction of increased spleen cell Ia expression. Although splenocyte Ia expression returns to baseline two days after mice are injected with free IL-4, soluble IL-4-anti-IL-4 mAb complexes still induce several-fold increases in Ia expression 3 days after injection. Complexes that contain as little as 400 ng of IL-4 have considerable in vivo stimulatory activity, and a maximal effect on splenocyte Ia expression is induced by injection of 2 micrograms of complexed IL-4. The stimulatory effect of IL-4-containing complexes on splenocyte Ia expression can be blocked by increasing the ratio of anti-IL-4 mAb to IL-4, by injection of anti-IL-4R mAb, and by in vivo aggregation of the complexes. Complexes of IL-4 with a non-neutralizing anti-IL-4 mAb do not have increased IL-4 agonist activity in vivo. These observations are most consistent with the possibility that neutralizing anti-IL-4 mAb act as carrier proteins that increase the in vivo half-life of IL-4 by preventing its excretion, and possibly, by preventing modification of its active site. The enhanced agonist effect of IL-4-anti-IL-4 mAb complexes is not unique; complexes of IL-3 with a neutralizing anti-IL-3 mAb have a greatly increased ability, compared with free IL-3, to stimulate mucosal mastocytosis, and complexes of IL-7 with a neutralizing anti-IL-7 mAb have a greatly increased ability, compared with free IL-7 or IL-7 complexed with a non-neutralizing anti-IL-7 mAb, to stimulate an increase in pre-B cell number. These observations suggest that complexes of cytokines and neutralizing anti-cytokine mAb may provide a generally useful way to increase the magnitude and duration of cytokine effects in vivo.

The effects of interleukin 7 (IL-7) on the growth and differentiation of murine B cell progenitors has been well characterized using in vitro culture methods. We have investigated the role of IL-7 in vivo using a monoclonal antibody that neutralizes IL-7. We find that treatment of mice with this antibody completely inhibits the development of B cell progenitors from the pro-B cell stage forward. We also provide evidence that all peripheral B cells, including those of the B-1 and conventional lineages, are derived from IL-7-dependent precursors. The results are consistent with the rapid turnover of B cell progenitors in the marrow, but a slow turnover of mature B cells in the periphery. In addition to effects on B cell development, anti-IL-7 treatment substantially reduced thymus cellularity, affecting all major thymic subpopulations.

Prior work using the parent-into-F1 model of graft-versus-host disease (GVHD) has shown that production of IL-2 by donor CD4+ T cells is an early, initiating event in either acute or stimulatory forms of GVHD. The aim of the present study was to determine whether acute GVHD could be prevented by blocking the effects of IL-2 in vivo during the first 2 weeks of disease. Acute GVHD was induced in B6D2F1 mice by the injection of C57BL/6 spleen cells i.v. Mice were untreated or received either anti-IL-2 mAb (S4B6) or control mAb at the time of GVHD induction and 1 week later. At 2 weeks, untreated or control mAb treated GVHD mice exhibited findings typical of acute GVHD, i.e. (i) in vitro donor anti-host cytotoxic T lymphocyte (CTL) activity, (ii) significant reductions in both numbers and function of splenic lymphocytes, and (iii) in vitro suppressor cell activity which profoundly blocked IL-2 production by normal syngeneic spleen cells. All of these findings were reversed or significantly inhibited in acute GVHD mice receiving anti-IL-2 treatment. Additionally, anti-IL-2 treated GVHD mice exhibited features previously observed in stimulatory GVHD such as: (i) B cell hyperactivity, (ii) a mild defect in CD4+ T cell production of IL-2 in vitro, and (iii) the ability to induce in co-culture a similar CD4+ cell defect in normal, syngeneic F1 spleen cells.(ABSTRACT TRUNCATED AT 250 WORDS)

IgD receptor (IgD-R) bearing CD4+ T cells with immunoaugmenting properties in vivo are induced in mice within 24 h after a single injection of dimeric or aggregated IgD. In the present study, we sought to identify the region(s) of IgD responsible for upregulation of IgD-R and for the immunoaugmenting effect of IgD. IgD-R can be upregulated on CD4+ T cells in vitro and in vivo by glutaraldehyde-aggregated mutant IgD or by fragments of enzymatically digested IgD molecules possessing either the C delta 1 domain (Fd delta) or the C delta 3 domain (Fc delta). Neoglycoproteins (D-galactose--BSA and N-acetyl-D-glucosamine--BSA), can competitively block upregulation of IgD-R by IgD in vitro. Furthermore, when injected 1 day before antigen, the aggregated IgD derived molecules, KWD1 (which lacks C delta 1), KWD6 (which lacks C delta 1 plus C delta-hinge), and Fab delta can all cause augmentation of antigen-specific primary and secondary antibody responses comparable to that achieved with intact aggregated IgD. Moreover, the immunoaugmenting effect of intact oligomeric IgD molecules in primary antibody responses is competitively blocked by simultaneous injection of monomeric forms of KWD6 and Fab delta. These results suggest that the binding of IgD to IgD-R, previously shown to be dependent on N-glycans present on Fd delta and Fc delta regions, also contributes to the upregulation of IgD-R and immunoagumentation.

Surface immunoglobulin (sIg)-mediated stimulation of B lymphocytes induces a tyrosine kinase-dependent sequence of events leading to rapid and large elevations in intracellular ionized calcium ([Ca2+]i). These early biochemical events do not necessarily lead to proliferation of B cells, however, and conversely, the absence of or inhibition of these events does not necessarily prevent cellular proliferation. We now show by digital image analysis of single B cells that conditions which lead to B cell proliferation are associated with low-level but persistent sustained or cyclic elevations in [Ca2+]i. In marked contrast, early and nonsustained elevations in [Ca2+]i are induced in B cells by stimuli that lead to G1 transition but fail to progress to DNA synthesis. Thus, when B cells were stimulated with mitogenic and nonmitogenic anti-IgD antibodies, both of which induce entry of cells into G1 and early calcium transients of comparable magnitude, persistent low-level calcium elevations were only detected in cells stimulated with the mitogenic antibody. Furthermore, persistent calcium elevations were also seen when B cells were stimulated with a multivalent dextran-anti-Ig conjugate which induced very high levels of B cell proliferation in the absence of detectable phosphatidylinositol 4,5-biphosphate hydrolysis or elevations in [Ca2+]i as detected by flow cytometry. Finally, B cells from X-linked B cell-defective mice, which do not proliferate in response to anti-Ig antibody, show marked and early increases in [Ca2+]i, but do not show persistent calcium elevations. These data suggest that the rapid and large increases of [Ca2+]i seen in lymphocytes within seconds after antigen receptor ligation may be associated with entry in G1, whereas low-level but persistent elevations may be the hallmark of a cell destined to synthesize DNA.

The immune response that is characteristic of parasitic helminth infections includes components associated with immediate-type hypersensitivity: elevated serum IgE, eosinophilia, and intestinal mast cell hyperplasia. In infection with the parasitic nematode, Heligmosomoides polygyrus, IL-4 mediates protective immunity, suggesting the presence of a host-protective Th2 response. In this investigation, we examined early stages of immune responsiveness to H. polygyrus infection to determine whether and at what stage a specific Th2-like pattern first appears. Using a quantitative reverse transcriptase-polymerase chain reaction assay, we analyzed changes in IL-2, IFN-gamma, IL-3, IL-4, IL-5, IL-6, IL-9, and IL-10 gene expression in the spleen, mesenteric lymph node, and Peyer's patch at various time points after infection. Our results demonstrate a highly specific and reproducible pattern of cytokine gene expression that remains localized to the enteric region. By 6 h after infection, IL-5 and IL-9 mRNA were elevated in the Peyer's patch and IL-3 was elevated by 12 to 24 h after infection. IL-4 RNA became elevated by 4 to 6 days after infection, but little change was observed in IFN-gamma, IL-2, or IL-10 mRNA levels. The early increases in IL-3, IL-5, and IL-9 gene expression after infection were probably T cell-independent, inasmuch as they were observed in Peyer's patches of congenitally athymic mice and anti-CD4, anti-CD8 mAb-treated conventional mice. However, treatment with these mAb considerably decreased cytokine gene expression 6 days after infection, and 8 days after infection, increased IL-4 gene expression in mesenteric lymph node cells was restricted to the CD4+ population. Thus, H. polygyrus infection induces cytokine gene expression that is restricted to some Th2-associated cytokines, is initiated by a T-independent response, and culminates in a T-dependent response.

Schistosoma japonicum-infected mice were injected with antibodies to interleukin-2 (IL-2) and/or IL-2 receptor to clarify the role of IL-2 on the granulomatous reaction around schistosome eggs in the liver. Granulomas were of normal or slightly increased size in animals subjected to IL-2 blockade, but hepatic fibrosis was markedly decreased in treated animals 10 weeks after infection. Anti-IL-2 treatment significantly decreased the in vitro secretion of IL-5 by antigen-stimulated spleen cells, and peripheral eosinophilia and tissue eosinophilia were diminished. Secretion of IL-2, IL-4, and gamma interferon was unaffected. Our results indicate that IL-2 is not an essential determinant of granuloma size in S. japonicum-infected mice but that, as in Schistosoma mansoni infection, the development of hepatic fibrosis is critically dependent on IL-2 levels and granuloma size and hepatic fibrosis are differentially regulated.

This study examines the effects of soluble IL-4R (sIL-4R) administration on IgE production in vivo by using an anti-IgD injection model. Anti-IgD-treated mice were given various doses of sIL-4R or anti-IL-4 mAb over a 3-day period and serum IgE levels were determined by ELISA on day 9. The sIL-4R inhibited IgE production by up to 85%. Anti-IL-4 mAb administration resulted in comparable levels of inhibition at considerably lower doses. The disparity in efficacy between sIL-4R and anti-IL-4 mAb was likely the result of differences in the biodistribution and in vivo half-life of the two IL-4-binding proteins. The specificity of the sIL-4R inhibitory effect was assessed by mixing sIL-4R with various concentrations of IL-4 before injection. Exogenous IL-4 partially overcame the inhibitory effect of high-dose sIL-4R or anti-IL-4 mAb. Unexpectedly, coadministration of suboptimal concentrations of anti-IL-4 mAb or sIL-4R with IL-4 resulted in superinduction of the IgE response. This stimulatory effect was dose dependent for both IL-4 and the IL-4 cognates and was not seen in the absence of exogenous IL-4 over the entire concentration range tested for either sIL-4R or anti-IL mAb. The results indicate that sIL-4R can block IgE secretion by neutralizing endogenous IL-4. Furthermore, sIL-4R can enhance, in a dose-dependent manner, the biologic effects of exogenously administered IL-4, presumably by altering the biodistribution of the cytokine. These findings suggest two alternative applications for cytokine-binding proteins, i.e., 1) as antagonists of biologic activities of endogenously produced cytokines and, 2) as vehicles for cytokine delivery.