Signal Transducer and Activator of Transcription 6 Is Essential in the Induction of Contact Hypersensitivity

Contact hypersensitivity (CHS) is thought to be mainly associated with the activation of T helper type 1 (Th1) cells. However, there is also evidence that Th2 cells or Th2 cytokines play a role in the development of CHS. To analyze the functional contribution of Th2 cytokines interleukin (IL)-4 and IL-13, signal transducer and activator of transcription 6 (STAT6)-deficient (STAT6−/−) and wild-type (wt) control C57BL/6 mice were contact sensitized with 5% 2,4,6-trinitrochlorobenzene (TNCB), 0.5% 2,4-dinitrofluorobenzene, or 5% 4-ethoxyl methylene-2-phenyl-2-oxazolin-5-one, and any skin reactions were examined. Ear swelling was significantly reduced with a delayed peak response in STAT6−/− mice compared with wt mice. A histological analysis revealed that the infiltration of both eosinophils and neutrophils in the skin challenged after 24 h in STAT6−/− mice decreased substantially compared with that in wt mice. The expression of Th2 cytokines (IL-4, IL-5) in TNCB-challenged skin tissues and the supernatants from T cells stimulated by 2,4,6-trinitrobenzene sulfonate–modified spleen cells, as well as the immunoglobulin (Ig)E and IgG1 response after challenge, were also profoundly reduced in STAT6−/− mice, whereas the expression of interferon γ was the same in STAT6−/− and wt mice after challenge. Furthermore, adoptive transfer experiments revealed that STAT6−/− mice induced CHS after injection of lymph node cells obtained from sensitized wt mice. Our data suggest that the STAT6 signal plays a critical role in the induction phase of CHS.

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title: Interleukin-4 is a critical cytokine in contact sensitivity.
abstract: This study demonstrates an essential role for interleukin-4 (IL-4) in the delayed hypersensitivity reaction, as illustrated by contact sensitivity (CS) to trinitrochlorobenzene (TNCB). Injection of mice with monoclonal antibody to IL-4, but not with control antibody, reduced CS after active immunization by 75%, as judged by ear swelling. The histological alterations of CS were also reduced. IL-4 was essential to the effector stage, as inhibition of its production or action blocked the passive transfer of CS. In particular, treatment of immune lymph node cells with antisense oligonucleotide to IL-4 inhibited the systemic transfer of CS. Transfer was also inhibited by monoclonal antibody to IL-4 given to the recipient. The present results indicate that IL-4 is an essential cytokine at the effector stage of the CS reaction.

title: Role of IL‐4 in delayed type hypersensitivity
abstract: IL‐4 plays a key role in the contact sensitivity skin reaction. This has several implications. First, the view that contact sensitivity (CS) is only mediated by cells with a Th1 profile of cytokine secretion needs modification, in the light of the essential role of IL‐4 at the effector stage. Second, the concept of a single cell involved in the systemic transfer of CS is no longer tenable, as it is known that both αβ and γδ cells are required. Studies with the cell lines (which contain both αβ and a few γδ cells) suggest that this double requirement may involve the action of IL‐4 on γδ cells, which bear receptors for IL‐4. Finally, the view that T cell lines only transfer CS when injected locally, but not when injected intravenously (systemic transfer), is correct but incomplete, as T cell lines actually give systemic transfer of CS, providing the cell line or the recipient is treated with IL‐4.

title: Diminished Contact Hypersensitivity Response in IL‐4 Deficient Mice at a Late Phase of the Elicitation Reaction
abstract: Contact hypersensitivity (CHS) is thought to depend on the activation of T cells of Th1 and/or Tc1 type. The role of Th2/Tc2 cells in the contact allergic reaction is not clear. The aim of this study was to analyse the functional contribution of Th2/Tc2 cells in CHS using the interleukin‐4 (IL‐4) deficient mouse model. Interleukin‐4 deficient (IL4T) and control (wt) mice were sensitized by epicutaneous application of 2,4‐dinitrofluorobenzene. The ear swelling response measured 24 h after challenge was similar in IL4T and control mice. However, from 48 h onwards, ear swelling values were significantly reduced in IL4T mice. The stimulatory capacity of freshly isolated as well as 3‐day cultured epidermal cells, prepared from IL4T and wt mice, for allogeneic T cells in a primary and secondary response, was comparable. The reduced number of T cell receptor (TCR) γδ+ cells observed in epidermal sheets prepared from IL4T mice was not responsible for the decreased ear swelling response in IL4T mice, because the use of TCR δ deficient mice lacking TCR γδ+ cells revealed a down‐regulatory role of this cell population in the CHS response. The data indicate that the effector stage of the CHS response can be subdivided into two phases. The first phase proceeds efficiently in IL‐4 deficient mice indicating the dependence on Th1/Tc1 cells, while the second phase does not develop in mice lacking IL‐4, suggesting the possibility that Th2/Tc2 cells intensify the reaction.

title: Interleukin 10 but not interleukin 4 is a natural suppressant of cutaneous inflammatory responses
abstract: We have examined the role of endogenously produced interleukin (IL) 4 and IL-10 in the regulation of inflammatory and immune reactions in the skin. In these experiments, irritant and contact hypersensitivity (CH) responses were elicited in mice with targeted disruptions of the IL-4 (IL-4T) or IL-10 (IL-10T) gene. Our study showed that IL-4T and wild- type (wt) mice exhibited equivalent responses to the irritant croton oil. In contrast, the response of IL-10T mice challenged with croton oil was abnormally increased. When IL-10T mice were exposed to a higher dose of irritant, irreversible tissue damage occurred. By comparison, any treatment of wt mice with croton oil resulted in far less tissue damage and resolution of inflammation. Neutralizing antibody studies demonstrated that the necrosis that occurred in IL-10T mice was due to the overproduction of tumor necrosis factor. The anti-tumor necrosis factor antibody treatment of IL-10T mice did not significantly reduce the edema or the influx of inflammatory cells, suggesting that these changes were due to the uncontrolled production of other proinflammatory cytokines. T cell-dependent immune responses were also evaluated using the contact sensitizer oxazolone. The response of IL-4T mice did not differ from wt mice. In contrast, IL-10T mice mounted an exaggerated CH response, increased in both magnitude and duration as compared with wt mice. Based on these studies, we have concluded that IL-10, but not IL-4, is a natural suppressant of irritant responses and of CH, and it limits immunopathologic damage in the skin.

title: Inhibition of allergic contact dermatitis to DNCB but not to oxazolone in interleukin-4-deficient mice.
abstract: The role of interleukin-4 as a regulator of immune responses in the skin is investigated with regard to the outcome of contact hypersensitivity reaction in interleukin-4-deficient BALB/C mice. In previous studies conflicting results were obtained concerning the role of interleukin-4 in contact hypersensitivity reactions supporting either a proinflammatory or rather an inhibitory function of this cytokine. Interleukin-4 deficient BALB/C mice sensitized to 2,4-dinitrochlorobenzene showed after challenge a significant reduction in magnitude and duration of the contact hypersensitivity response in comparison with wild-type mice. This attenuation was accompanied by a significant reduction of edema and cellular infiltrates in the dermis and a lacking induction of IL-10 mRNA expression in skin. Also, adoptive transfer experiments revealed that BALB/C mice failed to exhibit contact hypersensitivity after injection of lymph node cells obtained from sensitized interleukin-4 deficient mice. To examine further the role of the contact allergen used to induce the contact hypersensitivity response, mice were also sensitized and challenged with Oxazolone. Here a similar magnitude and duration of contact hypersensitivity in both the interleukin-4 deficient mice and BALB/C control mice was observed. This indicates that the contact hypersensitivity response to 2,4-dinitrochlorobenzene and Oxazolone may partly evolve on different pathways being dependent and independent of interleukin-4. Our results clearly show that the complete loss of endogenous interleukin-4 expression in BALB/C mice is associated with an impaired manifestation of contact hypersensitivity response to 2,4-dinitrochlorobenzene, implying an important proinflammatory function of this cytokine.

title: Essential role of Stat6 in IL-4 signalling
abstract: INTERLEUKIN-4(IL-4) is a pleiotropic lymphokine which plays an important role in the immune system1. IL-4 activates two distinct signalling pathways through tyrosine phosphorylation of Stat6, a signal transducer and activator of transcription, and of a 170K protein called 4PS2–7. To investigate the functional role of Stat6 in IL-4 signalling, we generated mice deficient in Stat6 by gene targeting. We report here that in the mutant mice, expression of CD23 and major histocompatibility complex (MHC) class II in resting B cells was not enhanced in response to IL-4. IL-4-induced B-cell proliferation costimulated by anti-IgM antibody was abolished. The T-cell proliferative response was also notably reduced. Furthermore, production of Th2 cytokines from T cells as well as IgE and IgGl responses after nematode infection were profoundly reduced. These findings agreed with those obtained in IL-4-deficient mice8,9 or using antibodies to IL-410 and the IL-4 receptor11. We conclude that Stat6 plays a central role in exerting IL-4-mediated biological responses.

title: Impaired IL-13-mediated functions of macrophages in STAT6-deficient mice.
abstract: IL-13 shares many biologic responses with IL-4. In contrast to well-characterized IL-4 signaling pathways, which utilize STAT6 and 4PS/IRS2, IL-13 signaling pathways are poorly understood. Recent studies performed with STAT6-deficient mice have demonstrated that STAT6 plays an essential role in IL-4 signaling. In this study, the functions of peritoneal macrophages of STAT6-deficient mice in response to IL-13 were analyzed. In STAT6-deficient mice, neither morphologic changes nor augmentation of MHC class II expression in response to IL-13 was observed. In addition, IL-13 did not decrease the nitric oxide production by activated macrophages. Taken together, these results suggest that the macrophage functions in response to IL-13 were impaired in STAT6-deficient mice, indicating that IL-13 and IL-4 share the signaling pathway via STAT6.

title: Blockade of costimulatory molecules B7‐1 (CD80) and B7‐2 (CD86) down‐regulates induction of contact sensitivity by haptenated epidermal cells
abstract: The hapten, trinitrobenzene sulphonic acid, induced weak B7‐1 (CD80) and moderate B7‐2 (CD86) expression on Langerhans cells and mRNA expression of both molecules in organ‐cultured murine skin. The intradermal injection of hapten‐treated epidermal cells induced hapten‐specific contact sensitivity in synergic mice. Cells of the keratinocyte cell line, Pam 212, or epidermal cells treated with a mixture of anti‐Ia/thy 1·2/γδ antibody plus complement, did not show any sensitizing ability. When hapten‐treated epidermal cells were injected into mice after incubation with  (CD86) or B7‐1 (CD80) antibody the resultant contact sensitivity reaction was decreased to less than 50% of the control reaction, a reduction which was similar to that seen with the anti‐ICAM‐1 and anti‐LFA‐1 antibody‐induced inhibition of contact sensitivity. Anti‐B7‐1 (CD80) or  (CD86) antibody also inhibited hapten‐specific lymphocyte proliferation or the allogenic mixed lymphocyte and epidermal cell reaction in vitro, although the inhibitory effect of  antibody was not as significant as that of  antibody. These results indicate that costimulatory signals induced by a hapten on epidermal Langerhans cells play an important role in the induction of hapten‐specific contact sensitivity in mice.

title: Functional CD86 (B7-2/B70) on cultured human Langerhans cells.
abstract: CD86 (B70/B7-2) has recently been identified as an alternative CD28/CTLA-4 ligand on activated B cells. CD86 has also been demonstrated as possibly serving as a primary costimulatory molecule in the initial immune response. Since the human Langerhans cell is one of the most potent antigen-presenting cells, we examined whether CD86 expression and function are found on organ-cultured skin, freshly isolated Langerhans cells, and cultured Langerhans cells in normal human epidermis. Immunohistochemical study in situ revealed that CD86 was expressed on dendritic cells with CD1a antigen in organ-cultured but not fresh skin. Fluorescence-activated cell sorter analysis revealed that no staining for either CD80 or CD86 was observed in freshly isolated Langerhans cells but that both CD80 and CD86 were expressed on cultured Langerhans cells. The actual expression of CD86 on cultured Langerhans cells was further confirmed by the detection of 70-kDa glycoprotein on Western blot analysis. Analysis of polymerase chain reaction demonstrated that both CD80 and CD86 were specifically amplified from purified cultured and freshly isolated Langerhans cells but not from Langerhans cell-depleted epidermal cells, indicating that both CD80 and CD86 genes were expressed by Langerhans cells. The functional importance of CD86 on Langerhans cells was confirmed by the allogeneic CD4 T cell proliferative responses with enriched Langerhans cells. A monoclonal antibody against CD86 caused 81% inhibition in contrast with 29% inhibition produced by anti-CD80 monoclonal antibody. This inhibitory effect was enhanced to 85.3% inhibition when a combination of anti-CD86 and anti-CD80 was administered. These results indicate that CD86 is predominantly expressed on the surface of cultured Langerhans cells and may transduce a primordial costimulatory signal in the interaction of Langerhans cells and T cells.

title: Initiation of delayed-type hypersensitivity by low doses of monoclonal IgE antibody. Mediation by serotonin and inhibition by histamine.
abstract: Elicitation of delayed-type hypersensitivity (DTH) responses by DTH effector T cells requires a prior phase of DTH initiation. This consists of an immediate hypersensitivity-like response mediated by Ag-specific DTH-initiating factors that are analogous to IgE antibodies in that they sensitize tissue mast cells for release of the vasoactive amine serotonin (5-HT). Experiments were conducted to determine whether IgE mAb injected i.v., or 5-HT injected locally, could initiate DTH. It was found that small doses of IgE (1 microgram/mouse), or of 5-HT (50 to 500 ng locally), which mediated small immediate responses, were optimal for DTH initiation. Even lower doses of IgE (10 ng/mouse), or of 5-HT (5 ng locally), which did not mediate macroscopically measurable immediate responses, were capable of DTH initiation. Higher doses of IgE (10 to 100 micrograms/mouse), which mediated large immediate responses, were not able to initiate DTH. A similar dose response for DTH initiation was found with IgG1 mAb, which is another mast cell-sensitizing isotype of Ig. The inability of high doses of IgE or IgG1 to mediate DTH initiation was probably caused by local release of large inhibitory amounts of histamine, because systemic treatment with the histamine-2 receptor antagonist cimetidine allowed high doses of IgE to initiate DTH. Thus, IgE and IgG1 antibodies could initiate DTH via release of small amounts of 5-HT, but simultaneous release of large amounts of histamine were inhibitory, probably via an effect on histamine-2 receptors of recruited T cells. We concluded the following: 1) IgE or IgG1 antibodies can initiate DTH; 2) DTH initiation need not be associated with macroscopically detectable early responses; 3) mast cell release of 5-HT acts positively whereas release of histamine acts negatively in murine DTH; 4) Ag-specific factors are not the only mechanism of DTH initiation.

title: T cell populations primed by hapten sensitization in contact sensitivity are distinguished by polarized patterns of cytokine production: interferon gamma-producing (Tc1) effector CD8+ T cells and interleukin (Il) 4/Il-10-producing (Th2) negative regulatory CD4+ T cells
abstract: Contact hypersensitivity (CHS) is a T cell-mediated response to hapten sensitization of the epidermis. The roles of CD4+ and CD8+ T cells in CHS have remained unclear, however, as studies to define either subset as the T cells mediating CHS have provided conflicting results. The goal of this study was to correlate the in vivo function of CD4+ and CD8+ T cells in CHS with the cytokines produced by each T cell population. Antibody-mediated depletion of CD4+ T cells before sensitization of BALB/c mice with 2,4-dinitrofluorobenzene (DNFB) or oxazolone (Ox) resulted in increased and prolonged CHS responses, indicating CD4+ T cells as negative regulators of the response. Depletion of CD8+ T cells resulted in low or abrogated responses, indicating CD8+ T cells as the effector cells in CHS. Sensitization with DNFB or Ox induced lymph node cell populations of CD8+ T cells producing interferon (IFN)-gamma and no interleukin (Il) 4 or Il-10, and CD4+ T cells producing Il-4 and Il-10 and no or little detectable IFN-gamma. The polarized patterns of cytokine production were stimulated by culture of hapten-primed lymph node cells either on anti- T cell receptor antibody-coated wells or with semipurified Langerhans cells isolated from hapten-sensitized mice. Stimulation of cytokine production during culture of hapten-primed CD4+ or CD8+ T cells with Langerhans cells was hapten specific and restricted to class II or class I major histocompatibility complex, respectively. The induction of the CD4+ and CD8+ T cells producing the polarized patterns of cytokines was not restricted to BALB/c mice, as cells from Ox sensitized C57B1/6 and B10.D2 mice produced the same patterns. Collectively, these results expose the induction of two polarized and functionally opposing populations of T cells by hapten sensitization to induce CHS: IFN-gamma-producing effector CD8+ T cells and Il-4/Il-10- producing CD4+ T cells that negatively regulate the response.

title: Low zone tolerance to contact allergens in mice: a functional role for CD8+ T helper type 2 cells
abstract: Normal skin is permeable to low molecular hydrophobic substances, including allergenic chemicals. Whereas such foreign matter appears to enter the skin naturally, it rarely induces contact hypersensitivity. This suggests that immunological tolerance would be the normal state of affairs. In search of a suitable model, we painted picryl chloride or oxazolone once or repeatedly on normal skin of BALB/c or C57B1/6 mice and found subsensitizing doses to be tolerogenic. The most effective doses in inducing tolerance were doses between those at the point of inflection from no responses to threshold sensitivity. But even doses three orders of magnitude lower than these suppressed subsequent sensitization if applied repeatedly. C57B1/6 mice (low responders) were consistently easier to make tolerant than BALB/c mice (high responders). The tolerant state established by a single painting was found to be fully developed at 48 h after initiation and long-lasting (>14 d). It could be adoptively transferred by intravenous injection of total spleen cells (SC), lymph node cells (LNC), or purified T cells and shown to be hapten specific. Pretreatment with cyclophosphamide (Cy) prevented tolerization. The T cells capable of transferring suppressive activity were found to be generated irrespective of the dose applied. On day 2 after painting, tolerance could be transferred with LNC from both tolerant and sensitized animals. On day 5, however, only cells from tolerant donors transferred tolerance. But by action of Cy, suppression was shown to be part of every sensitization, although masked. Production of hapten-specific antibodies was suppressed as well. Through depletion by monoclonal antibody in vitro the T suppressor cells were shown to belong to the murine CD8+ subset (Lyt2+). Upon restimulation in vitro by haptenized and irradiated normal SC, LNC from tolerant donors produced predominantly interleukin (IL)-4, IL-5, and IL-10. In contrast, LNC from sensitized donors produced preferentially IL-2 and interferon-gamma. Thus we demonstrate that painting subsensitizing doses of contact sensitizers on normal murine skin generates CD8+ Th2-like cells that give rise to hapten- specific tolerance. The model may have broader significance and apply to other species, including humans.

title: Cultured epidermal Langerhans cells activate effector T cells for contact sensitivity.
abstract: To investigate whether Langerhans cells are capable of inducing contact sensitivity effector T cells, we incubated purified T cells from naive mice with syngeneic cultured trinitrophenyl-modified Langerhans cells for 4-5 d. The cells were then expanded in interleukin-2 and fresh medium for another 6-9 d and injected intravenously into naive syngeneic recipient mice. After the ears of recipient mice were painted with 1% trinitrochlorobenzene, we observed an ear-swelling response peaking at 24-48 h. The ear-swelling response was hapten specific. CD8- but not CD4-depleted T cells mediated strong contact sensitivity. Systemic adoptive transfer into nude mice also lead to a hapten-specific delayed ear-swelling response. However, this response was less protracted than in euthymic animals, suggesting the participation of the recipient (non-immunized) T cells in the ear-swelling response of the euthymic mice. Lymphokine analysis of in vitro primed and restimulated T cells revealed predominant production of interleukin-2 but little or no interleukin-4. These in vitro primed cells therefore resemble type 1 or inflammatory T helper cell clones.

title: Th2 cells mediate IL-4-dependent local tissue inflammation.
abstract: We investigated whether polyclonal murine Th1 and Th2 cells obtained after short term culture in vitro were capable of mediating tissue inflammation in vivo. Th cells were pulsed with mAb to the TCR/CD3 complex and injected into the footpads or ears of naive syngeneic recipient mice. Th1 induced delayed swelling that peaked at 24 to 48 h and lasted > or = 5 days. Th2-induced swelling peaked at 6 h and lasted < or = 48 h. Similar responses were also observed in athymic nude mice. Lesions with both Th1 and Th2 cells contained a predominant neutrophilic infiltrate at 6 h, and mainly mononuclear cells at 48 h. The inflammatory response with Th2 was blocked by cyclosporin A, by mAb to IL-4 or by soluble rIL-4R. The requirement for IL-4 was early and transient. Four alloreactive short term Th2 clones induced swelling in allogeneic recipients. mAb- and IL-4-dependent swelling responses were also observed with two long term Th2 clones. Our results demonstrate that Th2 cells mediate IL-4-dependent tissue inflammation, and strengthen the concept that Th2 cells play an important role in some T cell-dependent immune reactions and, possibly, in allergic disorders such as atopic dermatitis and allergic asthma.

title: IL-4 inhibits the synthesis of IFN-gamma and induces the synthesis of IgE in human mixed lymphocyte cultures.
abstract: The T cell-derived lymphokine IL-4 is essential for the induction of IgE synthesis by human lymphocytes. The IgE-inducing effect of IL-4 is antagonized by IFN-gamma. The secretion of IFN-gamma is vigorously triggered in MLC. Thus, IL-4-stimulated MLC represent a suitable model to characterize the functional antagonism between IL-4 and IFN-gamma. In this report, we show that rIL-4 consistently induced IgE synthesis when added to human primary MLC. IL-4-dependent IgE production required cognate T/B cell recognition, because it was inhibited by antibodies to CD3 and MHC class II (HlA-DR) Ag. A neutralizing anti-IFN-gamma mAb dramatically enhanced IL-4-dependent IgE synthesis by MLC, indicating that endogenous IFN-gamma is a major inhibitor of IgE production. More importantly, addition of rIL-4 markedly inhibited the release of IFN-gamma in supernatants of MLC and Con A-activated PBMC. The decrease in IFN-gamma protein was accompanied by a decreased expression of IFN-gamma mRNA transcripts. The downregulation of IFN-gamma by IL-4 is likely to play an important role in the IL-4-dependent induction of IgE synthesis.

title: The cellular infiltrate in contact hypersensitivity to picryl chloride in the mouse.
abstract: In the present work, the contact hypersensitivity skin test reaction to picryl chloride in CBA mice was examined. The test was performed by applying the contact allergen to the ear skin and making a series of histological analyses up to 24 hours after challenge. An increment in ear thickness, measured with an engineer's micrometer 24 hours after challenge, was obvious in a group of sensitized mice when compared with a nonsensitized control group, and the difference was found to be highly significant. One hour after challenge, mononuclear cells appeared in the dermis, increasing in numbers during the following 12 hours. At this time, neutrophil granulocytes were the dominant cells in the infiltrate and remained so up to 24 hours after challenge. On the basis of the experiments performed here we conclude that measuring of the ear swelling with a micrometer 24 hours after challenge is a useful and reliable test of contact sensitivity in the mouse.

title: Pharmacologic modulation of picryl chloride-induced contact dermatitis in the mouse.
abstract: A biphasic response of ear swelling was observed 2 h and 24 h after application of the antigen to picryl chloride-sensitized Balb/c mice. A platelet-activating factor (PAF) antagonist, BN 52063, or the anti-inflammatory drug, betamethasone, applied topically or injected subcutaneously, inhibited in a dose-dependent fashion the antigen-induced increase in ear thickness observed after 24 h. In addition, BN 52063 and betamethasone presented a synergistic effect when administered in vivo simultaneously and subcutaneously. Indomethacin administered subcutaneously at the time of the antigen challenge significantly potentiated the early swelling phase and inhibited the late one. In contrast, the inhibitors of histamine and serotonin, ketotifen and methysergide, respectively, modulated mostly the early, and to a lower extent the late phase when administered at the time of antigen challenge. In contrast, none of these drugs inhibited the late phase reaction when administered 4 h after the antigen. A significant eosinophil and mononuclear-cell ear infiltrate was observed following topical application of the antigen, a phenomenon that was markedly reduced by either BN 52063 or betamethasone. These results demonstrate the effectiveness of PAF antagonists, either alone or in association with glucocorticosteroids, in experimental CD, the modulation of the infiltration of eosinophils and mononuclear cells possibly explaining part of the inhibitory action of these drugs.

title: Signal Transducer and Activator of Transcription Factor 6 (Stat6)-deficient Mice Are Protected from Antigen-induced Airway Hyperresponsiveness and Mucus Production
abstract: The pleiotropic cytokine interleukin 4 (IL-4) has been shown to regulate many processes thought to be important in the allergic diathesis. To determine the mechanism(s) by which IL-4 mediates allergic airway responses to inhaled allergens, we compared the effects of antigen sensitization and challenge on the development of allergic airway responses in mice in which the gene for the signal transducer and activator of transcription factor 6 (Stat6) was disrupted to those of their wild-type littermates. Strikingly, Stat6-deficient mice failed to develop airway hyperresponsiveness (AHR), which was observed in their wild-type littermates after allergen provocation. Moreover, antigen-induced increases in mucus-containing cells were found to be completely Stat6 dependent. Consistent with the lack of Th2 cytokine responses in Stat6-deficient mice, no ovalbumin-specific immunoglobulin (Ig)E was detected in their serum. In contrast, Stat6 signaling only partially mediated antigen-induced eosinophilia with no role in vascular adhesion molecule 1 expression. These results indicate that Stat6 signal transduction is critical in the development of allergen-induced AHR and that agents that specifically inhibit this pathway may provide a novel strategy for the treatment of allergic disorders.

title: STAT6 deficiency in a mouse model of allergen‐induced airways inflammation abolishes eosinophilia but induces infiltration of CD8+ T cells
abstract: The TH2‐type cytokines have been reported to contribute to the asthmatic response. STAT6 has an essential role in IL‐4 signalling and in production of TH2 cytokines from T cells and is involved in IgE and IgG1 responses after nematode infections, indicating that STAT6 has an important role in allergic diseases.

title: The late phase of the immediate wheal and flare skin reaction. Its dependence upon IgE antibodies.
abstract: IgE antibodies are usually thought to induce only immediate skin reactions. We have shown that the intradermal injection of a number of different allergens can produce a prolonged inflammatory reaction after the immediate wheal and flare in most sensitive subjects. This late inflammatory response occurs 6-12 h after challenge and is characterized by diffuse edema, erythema, pruritus, and heat. Both immediate and late responses can also be seen after passive sensitization of skin sites in nonatopic subjects. That IgE is involved in inducing the reaction was shown by the abolition of both immediate and late responses by passive transfer tests in the following experiments: (a) heating atopic serum at 56degreesC for 4 h, (b) removing IgE from the atopic serum by a solid phase anti-IgE immunoabsorbent, and (c) competitively inhibiting the binding of IgE antibodies to cells by an IgE myeloma protein. In addition, both responses were induced by affinity chromatography-purified IgE antibody, followed by antigenic challenge. Very similar lesions could also be induced by intradermal injection of Compound 48/80, thus suggesting a central role in the reaction for the mast cell or basophil. Histologically, the late phase is characterized by edema and a mixed cellular infiltration, predominantly lymphocytic but also containing eosinophils, neutrophils and basophils. Direct immunofluorescent staining did not show deposition of immunoglobulins or complement components, except IgM in 2 of 15 and C3 in 1 of 15 patients. This finding indicates that the late phase does not depend on the deposition of immune complexes. The results of the study suggest that IgE-allergen interaction on the surfaces of mast cells or on infiltrating basophils causes both immediate and late cutaneous responses.

title: Identification of histamine releasing factor(s) in the late phase of cutaneous IgE-mediated reactions.
abstract: We have shown that fluids collected from antigen-challenged skin blisters during the late phase reaction cause the release of substantial amounts of histamine (means = 42%, n = 14) from human basophils in vitro. Control fluids collected either during the immediate phase or from an unchallenged blister released less than or equal to 10% histamine from both basophils and lung mast cells. Late phase blister fluids induced low levels of histamine release from human lung cells (means = 11%, n = 4) that were slightly but not significantly greater than levels induced by control blister fluids. The characteristics of basophil release were similar to IgE-mediated stimuli in dose dependence, calcium and temperature requirements, and kinetics. The IgE dependence of the late phase blister fluid was demonstrated by desensitization of the basophils to anti-IgE, which obviated the response to anti-IgE and blister fluid but did not affect a non-IgE-mediated stimulus. Removal of the cell surface IgE with lactic acid also abolished the response to both anti-IgE and late phase blister fluid. Incubation of the "stripped" cells with serum containing IgE myeloma restored the response to anti-IgE but failed to affect response to late phase blister fluid. The characteristics of release obtained with this factor closely resemble those of an IgE-dependent histamine releasing factor from cultured macrophages previously described by our group.

title: Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus interleukin 4 and downregulated by tumor necrosis factor alpha
abstract: Using granulocyte/macrophage colony-stimulating factor (GM-CSF) and interleukin 4 we have established dendritic cell (DC) lines from blood mononuclear cells that maintain the antigen capturing and processing capacity characteristic of immature dendritic cells in vivo. These cells have typical dendritic morphology, express high levels of major histocompatibility complex (MHC) class I and class II molecules, CD1, Fc gamma RII, CD40, B7, CD44, and ICAM-1, and lack CD14. Cultured DCs are highly stimulatory in mixed leukocyte reaction (MLR) and are also capable of triggering cord blood naive T cells. Most strikingly, these DCs are as efficient as antigen-specific B cells in presenting tetanus toxoid (TT) to specific T cell clones. Their efficiency of antigen presentation can be further enhanced by specific antibodies via FcR- mediated antigen uptake. Incubation of these cultured DCs with tumor necrosis factor alpha (TNF-alpha) or soluble CD40 ligand (CD40L) for 24 h results in an increased surface expression of MHC class I and class II molecules, B7, and ICAM-1 and in the appearance of the CD44 exon 9 splice variant (CD44-v9); by contrast, Fc gamma RII is markedly and sometimes completely downregulated. The functional consequences of the short contact with TNF-alpha are in increased T cell stimulatory capacity in MLR, but a 10-fold decrease in presentation of soluble TT and a 100-fold decrease in presentation of TT-immunoglobulin G complexes.

title: Differentiation of human dendritic cells from monocytes in vitro
abstract: Since either macrophages (Mϕ) or dendritic cells (DC) differentiate from monocytes (MO) depending on culture conditions, we investigated the relationship of the DC and Mϕ differentiation pathways. Culturing MO‐enriched blood mononuclear cells with Mϕ colony‐stimulating factor (M‐CSF) or with granulocyte/Mϕ (GM)‐CSF induced Mϕ with a different morphology and CD14/CD1a expression. In contrast, in cultures with GM‐CSF and interleukin (IL)‐4, cells rapidly became nonadherent and acquired DC morphology, ultrastructure, CD1a expression, and most DC markers; they lost membrane CD14 and CD64 and capacity of phagocytosis, displayed less CD68 than Mϕ, but retained nonspecific esterase activity. These DC directly developed from MO without proliferation inasmuch as only day 0 FACS‐sorted MO, but not small CD14− cells, differentiated into DC when cultured with GM‐CSF and IL‐4, or to Mϕ with M‐CSF. While overall cell numbers declined, DC numbers plateaued from culture day 2 onwards, indicating that most had differentiasted by then. This differentiation was radioresistant and occurred without [3H]thymidine incorporation. Commitment to differentiate into DC with GM‐CSF and IL‐4 was irreversible by day 2, since discontinuing IL‐4 at this point did not revert cells to Mϕ. Alternatively, cells rapidly converted to DC when IL‐4 was added from day 2 to cultures initiated with GM‐CSF only. If cultures were initiated with M‐CSF and switched to GM‐CSF and IL‐4 after 2 or 5 days, about half of the cells still converted to DC. Thus, the capacity of MO and even of Mϕ to differentiate into DC was conserved for at least this period. The increased capacity to stimulate the mixed leukocyte reaction correlated with the relative number of CD1a− cells at any time and under each condition tested, a confirmation that these cells functionally qualify as DC. Thus, MO and even Mϕ can be directed to differentiate into DC depending on the cytokine microenvironment.

title: The ikaros gene is required for the development of all lymphoid lineages
abstract: The Ikaros gene encodes a family of early hematopoietic- and lymphocyte-restricted transcription factors. Mice homozygous for a germline mutation in the Ikaros DNA-binding domain lack not only T and B lymphocytes and natural killer cells but also their earliest defined progenitors. In contrast, the erythroid and myeloid lineages were intact in these mutant mice. We propose that Ikaros promotes differentiation of pluripotential hematopoietic stem cell(s) into the lymphocyte pathways. In the absence of a functional Ikaros gene, these stem cells are exclusively diverted into the erythroid and myeloid lineages.