Suzanne C. Morris

IgG-blocking antibodies inhibit IgE-mediated anaphylaxis in vivo through both antigen interception and FcγRIIb cross-linking

Although it has long been hypothesized that allergen immunotherapy inhibits allergy, in part, by inducing production of IgG Abs that intercept allergens before they can cross-link mast cell Fc epsilonRI-associated IgE, this blocking Ab hypothesis has never been tested in vivo. In addition, evidence that IgG-allergen interactions can induce anaphylaxis by activating macrophages through Fc gammaRIII suggested that IgG Ab might not be able to inhibit IgE-mediated anaphylaxis without inducing anaphylaxis through this alternative pathway. We have studied active and passive immunization models in mice to approach these issues and to determine whether any inhibition of anaphylaxis observed was a direct effect of allergen neutralization by IgG Ab or an indirect effect of cross-linking of Fc epsilonRI to the inhibitory IgG receptor Fc gammaRIIb. We demonstrate that IgG Ab produced during the course of an immune response or administered passively can completely suppress IgE-mediated anaphylaxis; that these IgG blocking Abs inhibit IgE-mediated anaphylaxis without inducing Fc gammaRIII-mediated anaphylaxis only when IgG Ab concentration is high and challenge allergen dose is low; that allergen epitope density correlates inversely with the allergen dose required to induce both IgE- and Fc gammaRIII-mediated anaphylaxis; and that both allergen interception and Fc gammaRIIb-dependent inhibition contribute to in vivo blocking Ab activity.

Stat6 Regulation of In Vivo IL-4 Responses

Although in vitro development of a Th2 response from naive CD4+ T cells is Stat6 dependent, mice immunized with a goat Ab to mouse IgD have been reported to produce a normal primary IL-4 response in Stat6-deficient mice. Experiments have now been performed with mice immunized with more conventional Ags or inoculated with nematode parasites to account for this apparent discrepancy. The ability of an immunogen to induce a primary in vivo IL-4 response in Stat6-deficient mice was found to vary directly with its ability to induce a strong type 2 cytokine-biased response in normal mice. Even immunogens, however, that induce strong primary IL-4 responses in Stat6-deficient mice induce poor memory IL-4 responses in these mice. Consistent with this, Stat6-deficient CD4+ T cells make relatively normal IL-4 responses when stimulated in vitro for 3 days with anti-CD3 and anti-CD28, but poor IL-4 responses if they are later restimulated with anti-CD3. Thus, Stat6 signaling enhances primary IL-4 responses that are made as part of a type 0 cytokine response (mixed type 1 and type 2) and is required for normal development or survival of Th2 memory cells.

Stat6 signaling promotes protective immunity against Trichinella spiralis through a mast cell- and T cell-dependent mechanism.

Studies in mice infected with the gastrointestinal nematode parasite Nippostrongylus brasiliensis demonstrated that IL-4/IL-13 activation of Stat6 suppresses development of intestinal mastocytosis and does not contribute to IL-4/IL-13 production, but is still essential for parasite expulsion. Because expulsion of another gastrointestinal nematode, Trichinella spiralis, unlike N. brasiliensis expulsion, is mast cell dependent, these observations suggested that T. spiralis expulsion would be Stat6 independent. Instead, we find that Stat6 activation by IL-4/IL-13 is required in T. spiralis-infected mice for the mast cell responses that induce worm expulsion and for the cytokine responses that induce intestinal mastocytosis. Furthermore, although IL-4 induces N. brasiliensis expulsion in the absence of B cells, T cells, and mast cells, mast cells and T cells are required for IL-4 induction of T. spiralis expulsion. Thus, Stat6 signaling is required for host protection against N. brasiliensis and T. spiralis but contributes to expulsion of these two worms by different mechanisms. The induction of multiple effector mechanisms by Stat6 signaling provides a way for a cytokine response induced by most gastrointestinal nematode parasites to protect against most of these parasites, even though different effector mechanisms are required for protection against different nematodes.

Dependence of IL-4, IL-13, and Nematode-Induced Alterations in Murine Small Intestinal Smooth Muscle Contractility on Stat6 and Enteric Nerves

IL-4 and IL-13 promote gastrointestinal worm expulsion in part through effects on nonlymphoid cells, such as intestinal smooth muscle cells. The roles of Stat6 in IL-4-, IL-13-, and parasitic nematode-induced effects on small intestinal smooth muscle contractility were investigated in BALB/c wild-type and Stat6-deficient mice treated with a long-lasting formulation of recombinant mouse IL-4 (IL-4C) or IL-13 for 7 days. Separate groups of BALB/c mice were infected with Nippostrongylus brasiliensis or were drug-cured of an initial Heligmosomoides polygyrus infection and later reinfected. Infected mice were studied 9 and 12 days after inoculation, respectively. Segments of jejunum were suspended in an organ bath, and responses to nerve stimulation and to acetylcholine and substance P in the presence and absence of tetradotoxin, a neurotoxin, were determined. Both IL-4 and IL-13 increased smooth muscle responses to nerve stimulation in wild-type mice, but the effects were greater in IL-13-treated mice and were absent in IL-13-treated Stat6-deficient mice. Similarly, hypercontractile responses to nerve stimulation in H. polygyrus- and N. brasiliensis-infected mice were dependent in part on Stat6. IL-13, H. polygyrus, and N. brasiliensis, but not IL-4, also increased contractility to acetylcholine by mechanisms that involved Stat6 and enteric nerves. These studies demonstrate that both IL-4 and IL-13 promote intestinal smooth muscle contractility, but by different mechanisms. Differences in these effects correlate with differences in the relative importance of these cytokines in the expulsion of enteric nematode parasites.

The Role of IL-4 in Heligmosomoides polygyrus-Induced Alterations in Murine Intestinal Epithelial Cell Function

IL-4 and IL-13 promote gastrointestinal worm expulsion, at least in part, through effects on nonlymphoid cells, such as intestinal epithelial cells. The role of IL-4/IL-13 in the regulation of intestinal epithelial function during Heligmosomoides polygyrus (Hp) infection was investigated in BALB/c mice infected with Hp or treated with a long-lasting formulation of recombinant mouse IL-4/αIL-4 complexes (IL-4C) for 7 days. Separate groups of BALB/c mice were drug-cured of initial infection and later reinfected and treated with anti-IL-4R mAb, an antagonist of IL-4 and IL-13 receptor binding, or with a control mAb. Segments of jejunum were mounted in Ussing chambers, and short circuit current responses to acetylcholine, histamine, serotonin, PGE2, and glucose were determined. Although only modest changes in epithelial cell function were observed during primary Hp infection, IL-4C or a secondary Hp infection each induced more dramatic changes, including increased mucosal permeability, reduced sodium-linked glucose absorption, and increased Cl− secretory response to PGE2. Some, but not all, effects of IL-4C and Hp infection were dependent on enteric nerves. Hp-induced changes in epithelial function were attenuated or prevented by anti-IL-4R mAb. Thus, IL-4/IL-13 mediate many of the effects of Hp infection on intestinal epithelial cell function and do so both through direct effects on epithelial cells and through indirect, enteric nerve-mediated prosecretory effects. These immune system-independent effector functions of IL-4/IL-13 may be important for host protection against gastrointestinal nematodes.

Basophils Initiate IL-4 Production during a Memory T-dependent Response

Experiments were performed to characterize and identify the cellular sources of the secondary interleukin (IL)-4 response to a T cell–dependent antigen. Mice were primed by immunization with goat anti–mouse immunoglobulin (Ig)D antibody (GaMD), which stimulates naive CD4+ T cells to secrete IL-4 in 3–4 d. When challenged with goat serum 14 d after immunization, GaMD-primed mice generated an IL-4 response that exceeded the primary response by ∼100-fold, started in <2 h, and lasted for 4 d. Studies with 4get mice, in which cells with an accessible Il4 gene express a green fluorescent protein (GFP), revealed CD4+ memory T cells, natural killer T cells, basophils, mast cells, and eosinophils as possible rapid producers of IL-4. GFP+CD4+ T cells and basophils expanded more in the spleen than the other cell types during the primary response to GaMD. Quantitation of in vivo IL-4 production by the in vivo cytokine capture assay after individual cell types were selectively stimulated or deleted demonstrated that basophils and memory CD4+ T cells account for most of the secondary IL-4 response, with basophils initiating that response through IgE/FcɛRI-mediated signaling but secreting IL-4 for <4 h and memory T cells secreting IL-4 within 4 h and continuing to secrete this cytokine for 4 d.

STAT5 is critical to maintain effector CD8+ T cell responses.

During an immune response, most effector T cells die, whereas some are maintained and become memory T cells. Factors controlling the survival of effector CD4+ and CD8+ T cells remain unclear. In this study, we assessed the role of IL-7, IL-15, and their common signal transducer, STAT5, in maintaining effector CD4+ and CD8+ T cell responses. Following viral infection, IL-15 was required to maintain a subpopulation of effector CD8+ T cells expressing high levels of killer cell lectin-like receptor subfamily G, member 1 (KLRG1), and lower levels of CD127, whereas IL-7 and IL-15 acted together to maintain KLRG1lowCD127high CD8+ effector T cells. In contrast, effector CD4+ T cell numbers were not affected by the individual or combined loss of IL-15 and IL-7. Both IL-7 and IL-15 drove phosphorylation of STAT5 within effector CD4+ and CD8+ T cells. When STAT5 was deleted during the course of infection, both KLRG1highCD127low and KLRG1lowCD127high CD8+ T cells were lost, although effector CD4+ T cell populations were maintained. Furthermore, STAT5 was required to maintain expression of Bcl-2 in effector CD8+, but not CD4+, T cells. Finally, IL-7 and IL-15 required STAT5 to induce Bcl-2 expression and to maintain effector CD8+ T cells. Together, these data demonstrate that IL-7 and IL-15 signaling converge on STAT5 to maintain effector CD8+ T cell responses.

IL-13-Mediated Worm Expulsion Is B7 Independent and IFN-γ Sensitive

B7 costimulation is a required component of many type 2 immune responses, including allergy and protective immunity to many nematode parasites. This response includes elevations in Th2 cytokines and associated effector functions including elevations in serum IgG1 and IgE and parasite expulsion. In studies of mice infected with Trichuris muris, blocking B7 ligand interactions inhibited protective immunity, suppressed IL-4 production, and enhanced IFN-γ production, but unexpectedly did not inhibit production of the Th2 cytokine, IL-13. Blocking both IFN-γ and B7 restored protective immunity, which was IL-13 dependent, but did not restore IL-4 or associated IgE responses. Although IL-13 was required for worm expulsion in mice in which both IFN-γ and B7 were blocked, IL-4 could mediate expulsion in the absence of both IL-13 and IFN-γ. These studies demonstrate that 1) B7 costimulation is required to induce IL-4, but not IL-13 responses; 2) IL-13 is elevated in association with the IFN-γ response that occurs following inhibition of B7 interactions, but can only mediate IL-4-independent protection when IFN-γ is also inhibited; and 3) increased IL-13 production, in the absence of increased IL-4 production, is not associated with an IgE response, even in the absence of IFN-γ.

IL-4 Promotes the Migration of Circulating B Cells to the Spleen and Increases Splenic B Cell Survival

We report that IL-4 causes a redistribution of B cells and modestly increases B cell life span. Intravenous injection of a long-acting formulation of IL-4 induces increases in both spleen cell number and the percentage of splenic B cells. These effects are observed within 1 day of IL-4 administration and plateau after ∼3 days if IL-4 treatment is continued. The increase in splenic B cell number is IL-4 dose dependent, CD4+ T cell independent, FcγRII/FcγRIII independent, and Stat6 independent. Decreases in the number of B cells in the blood and the percentage of mature B cells in the bone marrow, concomitant with the increase in splenic B cell number, suggest that redistribution of circulating B cells to the spleen is partially responsible for IL-4 induction of splenic B cell hyperplasia. Considerable reduction in the effect of 5 days of IL-4 treatment on splenic B cell number when B lymphopoiesis is blocked with anti-IL-7 mAb suggests that generation of new B cells is also involved in IL-4-induced splenic B cell hyperplasia. 5-Bromo-2′-deoxyuridine labeling experiments demonstrate that IL-4 modestly prolongs the life span of newly generated splenic B cells, and experiments that measure B cell HSA (CD24) expression as an indicator of B cell age suggest that IL-4 may also prolong the life span of mature splenic B cells. Thus, IL-4 increases splenic B cell number through two Stat6-independent effects: increased net migration of circulating B cells to the spleen and increased B cell life span. Both effects may promote Ab responses to a systemic Ag challenge.

LOCAL TH1 AND TH2 RESPONSES TO PARASITIC INFECTION IN THE INTESTINE : REGULATION BY IFN-GAMMA AND IL-4

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.