Tatyana Orekhova

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.

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.

Diesel Exhaust Particles Suppress In Vivo IFN-γ Production by Inhibiting Cytokine Effects on NK and NKT Cells

Diesel exhaust particles (DEP) have strong, selective Th2 adjuvant activity when inhaled with conventional Ags. We used a novel technique for measuring in vivo cytokine production to investigate possible mechanisms by which DEP might promote a Th2 response. Injection of DEP i.p. stimulated IL-6 secretion, but failed to increase IL-4, IL-10, or TNF-α secretion, and decreased basal levels of IFN-γ. When injected with or before LPS, DEP had little effect on the LPS-induced TNF-α responses, but partially inhibited the LPS-induced IL-10 response and strongly inhibited the LPS-induced IFN-γ response. DEP also inhibited the IFN-γ responses to IL-12, IL-12 plus IL-18, IL-2, and poly(I · C). DEP treatment had little effect on the percentages of NK and NKT cells in the spleen, but inhibited LPS-induced IFN-γ production by splenic NK and NKT cells. In contrast, DEP failed to inhibit the IFN-γ response by anti-CD3 mAb-activated NKT cells. Taken together, these observations suggest that DEP inhibit Toll-like receptor ligand-induced IFN-γ responses by interfering with cytokine signaling pathways that stimulate NK and NKT cells to produce IFN-γ. Our observations also suggest that DEP may promote a Th2 response by stimulating production of inflammatory cytokines while simultaneously inhibiting production of IFN-γ, and raise the possibility that the same mechanisms contribute to the association between DEP exposure and asthma.

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.

IL-4 Induces In Vivo Production of IFN-γ by NK and NKT Cells

Although IL-4 and IFN-γ often have opposite effects and suppress each other’s production by T cells, IL-4 can stimulate IFN-γ production. To characterize this, we injected mice with IL-4 and quantified IFN-γ production with the in vivo cytokine capture assay. IL-4 induced Stat6-dependent IFN-γ production by NK and, to a lesser extent, NKT cells, but not conventional T cells, in 2–4 h. Increased IFN-γ production persisted at a constant rate for >24 h, but eventually declined, even with continuing IL-4 stimulation. This eventual decline in IFN-γ production was accompanied by a decrease in NK and T cell numbers. Consistent with a dominant role for NK cells in IL-4-stimulated IFN-γ secretion, IL-4 induction of IFN-γ was B and T cell-independent; suppressed by an anti-IL-2Rβ mAb that eliminates most NK and NKT cells; reduced in Stat4-deficient mice, which have decreased numbers of NK cells; and absent in Rag2/γc-double-deficient mice, which lack T, B, and NK cells. IL-4-induced IFN-γ production was not affected by neutralizing IL-12p40 and was increased by neutralizing IL-2. IL-13, which signals through the type 2 IL-4R and mimics many IL-4 effects, failed to stimulate IFN-γ production and, in most experiments, suppressed basal IFN-γ production. Thus, IL-4, acting through the type 1 IL-4R, induces Stat6-dependent IFN-γ secretion by NK and NKT cells. This explains how IL-4 can contribute to Th1 cytokine-associated immune effector functions and suggests how IL-13 can have stronger proallergic effects than IL-4.

The In Vivo Cytokine Capture Assay for Measurement of Cytokine Production in the Mouse

Because most cytokines are utilized, catabolized, or excreted shortly after they are produced, it has been difficult to directly measure in vivo cytokine production. Consequently, it has been necessary to infer in vivo cytokine secretion levels from the results of ex vivo assays of cytokine secretion, assays that measure tissue levels of cytokine mRNA, or assays that stain tissues for cytokine protein levels. Results of these assays provide important and useful information, but do not necessarily reflect in vivo cytokine secretion. To better determine in vivo cytokine production, the in vivo cytokine capture assay (IVCCA) was developed. IVCCA facilitates measurement of cytokines in serum by increasing their in vivo half‐lives. This increases the sensitivity of measurement of in vivo cytokine production 30‐ to 1,000‐fold. The first protocol described in this unit is for luminescence‐based ELISA, while the second is for an absorbance‐based method.

In Vivo Survival of Autoreactive B Cells: Characterization of Long-Lived B Cells

To determine the effects of chronic Ag stimulation on B cell survival and phenotype, we compared survival and surface markers of hen egg lysozyme (HEL)-specific B cells in Ig transgenic (Tgn) mice, which lack HEL, and in HEL-Ig transgenic mice, which express soluble HEL. Serum HEL levels were maximized in HEL-Ig Tgn mice by feeding them zinc, which activates the metallothionein promoter that regulates HEL expression. B cell age was characterized by expression of heat-stable Ag, and B220 and B cell survival was studied by evaluating changes in B cell number when lymphopoiesis was suppressed with anti-IL-7 mAb and by identifying newly generated B cells through 5-bromo-2′-deoxyuridine incorporation. Our observations show that the mean B cell life span is considerably reduced in HEL-Ig Tgn compared with Ig Tgn mice, but also demonstrate that some HEL-Ig Tgn B cells survive to maturity. Some of these surviving B cells have undergone receptor editing (substitution of an endogenous Ig light chain for the transgenic Ig light chain), so that their ability to bind HEL is decreased or absent. Surviving HEL-Ig Tgn B cells that retain HEL specificity express decreased mIgD and little or no mIgM. mIgD expression progressively decreases with increasing HEL-Ig Tgn B cell age. These observations suggest that self Ag-specific B cells can survive in the presence of soluble self Ag by down-regulating mIg expression, which should limit B cell signaling by Ag that might otherwise cause deletion of these cells.