L3T4 (CD4+) cells that mediate contact sensitivity to trinitrochlorobenzene express I-A determinants

The present study has further characterized the T cell-mediated inflammatory response of contact sensitivity (CS) to the hapten trinitrochlorobenzene (TNCB) in mice. A discernible CS response was found to be induced as early as 2 days after epicutaneous application of TNCB. The response peaked on Days 4 to 5 and it then declined to a nearly undetectable level by Days 10 to 11. Examination of the draining lymph nodes demonstrated that development of CS coincided with an increase in cellular proliferation and in the total number of cells present. Despite a severalfold increase in the cellular contents of the draining lymph nodes of sensitized mice, the relative percentages of most subsets of T cells remained unchanged. Flow cytometric studies revealed that the subpopulation of T cells characterized as Thy 1.2+ L3T4+ I-A+ increased substantially in comparison to its presence in unsensitized mice. Whether the Thy 1.2+ L3T4+ I-A+ cells that increased following sensitization represented the effector population that mediates CS was then examined. Four-day immune lymph node T cells or L3T4 cells positively selected from them were capable of adoptively transferring CS to normal mice. However, these cells, after treatment with anti-Ia antibody or anti-I-A monoclonal antibody and complement, were unable to transfer CS. These findings imply that expression of I-A determinants may indicate antigen-induced T cell activation in vivo and that L3T4 cells that mediate CS are I-A positive.     

Cell-mediated immunity: role of IL-3 and IL-6 in the regulation of contact sensitivity reaction

Delayed type hypersensitivity reaction (DTH) consists of a sequential cascade of steps depending on different types of T cells, as well as mast cells, endothelial cells and macrophages. Recently it has been shown that CD4+ TH1 lymphocytes ("inflammatory type") play a central role in DTH reaction. Activated TH1 cells produce a characteristic pattern of cytokines: IL-2, IL-3, TNF-beta, IFN-gamma. Using the contact sensitivity (CS) reaction on mice as a model system, the role of cytokines in the regulation of DTH is presented, particularly the significance of IL-3 and IL-6. The recent data can be interpreted to show that IL-6 released by activated macrophages (APC cells) in the induction phase of the CS reaction probably stimulate CD8+ T suppressor cells. These in turn inhibit the production of IL-2 and IL-3 by CD4+ TH1 cells followed by a state of unresponsiveness.     

Parallel regulation of IL-4 and IL-5 in human helminth infections.

To investigate the relationship between cytokine production and the increased levels of serum IgE and peripheral eosinophilia commonly accompanying human helminth infections, we studied the ability of PBMC of normal (N1) (n = 18) and eosinophilic individuals with helminth infections (H1) (n = 9) to produce IL-3, IL-4, IL-5, granulocyte-macrophage-CSF, and IFN-gamma in vitro after stimulation with PMA (50 ng/ml) and ionomycin (1 microgram/ml). The two groups differed in both the levels of serum IgE and eosinophilia. For mitogen-induced production of granulocyte-macrophage-CSF and IFN-gamma, there was no difference in cytokine production between the two groups. In marked contrast, supernatants from PBMC of infected individuals had significantly higher levels of IL-4 (mean = 213 pg/ml for N1 and 944 pg/ml for H1, p less than 0.02), IL-5 (mean = 180 pg/ml for N1 and 1118 pg/ml for HL, p less than 0.001), and IL-3 (mean = 13900 pg/ml for N1, 28029 pg/ml for H1, p less than 0.05). In addition, helminth-infected patients had approximately 5-fold greater numbers of T cells capable of producing IL-5 and 2.5-fold greater frequency of IL-4-secreting cells than did normal individuals; GM-CSF- and IFN-gamma-producing T cell numbers were not significantly different in the two groups. IL-3-producing cell frequencies could not be evaluated by this method. There was a direct correlation between IL-4 production and IL-5 production at the level of both protein production and frequency of T cells capable of producing these cytokines. These data indicate that individuals with reactive eosinophilia and elevated serum IgE have an expanded population of lymphocytes producing IL-4 and IL-5 and the association of the two suggests that the regulation of IL-4 and IL-5 may be linked.     

CD28 costimulation accelerates IL-4 receptor sensitivity and IL-4-mediated Th2 differentiation.

The development of Th1 and Th2 cells is determined by the type of antigenic stimulation involved in the initial cell activation step. Evidence indicates that costimulatory signals, such as those delivered by CD28, play an important role in Th2 development, but little is known about how CD28 costimulation contributes to Th2 development. In this study, TCR cross-linking was insufficient for Th2 development, while the addition of CD28 costimulation drastically increased Th2 generation through the IL-4-mediated pathway. Th2 generation following CD28 costimulation was not simply explained by the enhancement of IL-4 production in naive T cells. To generate Th2 cells after TCR cross-linking only, it was necessary to add a 20- to 200-fold excess of IL-4 generated after TCR and CD28 stimulation. TCR cross-linking increased the expression level and binding property of the IL-4R, but enhanced the sensitivity to IL-4 only slightly. In contrast, as evidenced by the enhanced phosphorylation of Jak3, the IL-4Ralpha-chain, and STAT6 following IL-4 stimulation, CD28 costimulation increased IL-4R sensitivity without affecting its expression and binding property. This evidence of the enhancement of IL-4R sensitivity increases our understanding of how CD28 costimulation accelerates Th2 development.     

IL-33 activates B1 cells and exacerbates contact sensitivity

B1 B cells produce natural IgM and play a critical role in the early defense against bacterial and viral infection. The polyreactive IgM also contributes to the clearance of apoptotic products and plays an important role in autoimmune pathogenesis. However, the mechanism of activation and proliferation of B1 cells remains obscure. In this study, we report that IL-33, a new member of IL-1 family, activates B1 cells, which express the IL-33 receptor α, ST2. IL-33 markedly activated B1 cell proliferation and enhanced IgM, IL-5, and IL-13 production in vitro and in vivo in a ST2-dependent manner. The IL-33-activated B1 cell functions could be largely abolished by IL-5 neutralization and partially reduced by T cell or mast cell deficiency in vivo. ST2-deficient mice developed less severe oxazolone-induced contact sensitivity (CS) than did wild-type (WT) mice. Furthermore, IL-33 treatment significantly exacerbated CS in WT mice with enhanced B1 cell proliferation and IgM and IL-5 production. Moreover, IL-33-activated B1 cells from WT mice could adoptively transfer enhanced CS in ST2(-/-) mice challenged with IL-33. Thus, we demonstrate, to the best of our knowledge, a hitherto unrecognized mechanism of B1 cell activation and IL-33 function, and suggest that IL-33 may play an important role in delayed-type hypersensitivity.     

Anti-inflammatory effects of IL-4 and dynamic compression in IL-1beta stimulated chondrocytes

Mechanical loading can counteract inflammatory pathways induced by IL-1beta by inhibiting *NO and PGE2, catabolic mediators known to be involved in cartilage degradation. The current study investigates the potential of dynamic compression, in combination with the anti-inflammatory cytokine, IL-4, to further abrogate the IL-1beta induced effects. The data presented demonstrate that IL-4 alone can inhibit nitrite release in the presence and absence of IL-1beta and partially reverse the IL-1beta induced PGE2 release. When provided in combination, IL-4 and dynamic compression could further abrogate the IL-1beta induced nitrite and PGE2 release. IL-1beta inhibited [3H]thymidine incorporation and this effect could be reversed by IL-4 or dynamic strain alone or both in combination. By contrast, 35SO4 incorporation was not influenced by IL-4 and/or dynamic strain in IL-1beta stimulated constructs. IL-4 and mechanical loading may therefore provide a potential protective mechanism for cartilage destruction as observed in OA.     

TLR-dependent IL-4 production by invariant Valpha14+Jalpha18+ NKT cells to initiate contact sensitivity in vivo

LPS stimulated B-1 cell polyclonal in vivo IgM responses depend on IL-4 release by invariant Valpha14+Jalpha18+ NKT (iNKT) cells. The IgM Abs can recruit effector T cells to mediate contact sensitivity. LPS activates the B-1 cell response just 1 day later, and depends on CD1d, iNKT cells, IL-4, TLR4, and MyD88. LPS in vivo and in vitro stimulates rapid preferential production of IL-4 in hepatic iNKT cells within 2 h. TLR4 were demonstrated in iNKT cells by flow cytometry and functional studies. Thus, innate microbial stimulation via TLR can activate iNKT cell and B-1 cell collaboration. The result is polyclonal IgM Ab responses capable of recruiting Ag-specific T cells into tissues. This may be involved in the promotion of autoimmunity by infectious agents.     

Role of the fourth complement component (C4) in the regulation of contact sensitivity. III. In vivo effect of purified C4

Lymph node cells obtained from CBA/J mice 4 days after painting with contact sensitizing agents such as picryl chloride or oxazolone ("4-day" cells), induce contact sensitivity into naive recipient mice by membrane-associated immunocomplexes. This immunizing capacity is abolished after incubation of the cells in serum from mice with high C4 levels (C4H), but not in serum from mice with low C4 levels (C4L), and the inhibitory activity of C4H serum is due to the activation of the early components of the classical complement pathway. The presence of 4-day cells depends on C4 levels: in fact, C4H mice lack these cells because they activate their own complement in vivo, whereas C4L mice fail to activate complement in vivo and possess 4-day cells. CBA/J (C4L) mice injected with purified C4 preparations from the C4H mice BALB/c, lose 4-day cells and show a short-term contact-sensitivity reaction, exactly as BALB/c mice, thus indicating that C4 levels play a role in the control of contact-sensitivity reaction to simple chemical haptens.     

Further studies on the regulation of lymphokine biosynthesis in contact sensitivity

Contact sensitivity (CS) results from a series of cellular interactions involving CD4+ T cells and macrophages. We have studied the production of lymphokines by T cells from mice immunized by contact sensitization and by a variety of stimuli leading to CS or tolerance to CS. Primed T cells from mice immunized for CS are predominantly of the TH1 type. Conversely, a failure in the activation of these antigen-specific TH1 cells is a key step in the induction of tolerance to CS. Spleen cells from tolerant mice can suppress the production of lymphokines from primed cells, both in an adoptive transfer system and when mixed with effector cells in vitro. The relative importance of lymphokine competition, prostaglandin production by adherent cells, and other mechanisms underlying this suppression is discussed.     

Cytoskeletal regulation of the sensitivity of brain synaptosomes to the depolarizing action of veratrine

Using a radioactive permeant cation 3H-tetraphenylphosphonium, the sensitivity of rat brain synaptosomes to depolarizing action of veratrine, which specifically opens the sodium channels, was compared before and after destruction of microtubules and microfilaments. Depolymerization of microtubules with colchicin and vinblastine decreased an apparent affinity of veratrine to its receptor in the channel, while destruction of microfilaments with cytochalasin B had the opposite effect. Colchicine did not change allosteric interactions between the receptor for veratrine and that for scorpion venom in the sodium channel evaluated by the ability of scorpion venom to facilitate veratrine-induced depolarization of synaptosomes. It is suggested that two main cytoskeleton subsystems control the state of sodium channels in the nerve ending.     

Anti-inflammatory response of IL-4, IL-10 and TGF-beta in patients with systemic inflammatory response syndrome

The systemic inflammatory response syndrome (SIRS) is an inflammatory process seen in association with a large number of clinical infective and non-infective conditions. The aim of this study was to investigate the role of anti-inflammatory cytokines such as interleukin-4 (IL-4), interleukin-10 (IL-10), and transforming growth factor-beta (TGF-beta). Serum levels of IL-4, IL-10 and TGF-beta were determined in 45 patients with SIRS: 38 patients had SIRS of infectious origin, whereas seven patients had non-infectious SIRS. Twenty healthy subjects were used as controls. Serum levels of IL-4, IL-10 and TGF-beta were determined by an immunoenzyme assay. A significant increase of IL-4 was observed in these patients at the time of diagnosis and 5 days later. In contrast, serum levels of IL-10 were not increased at the time of diagnosis, but a slight decrease was noted after 5 days. Serum levels of TGF-beta were not increased at time of diagnosis, and a slight increase was observed after 5 days. Serum levels of IL-4 were significantly higher in patients with infectious SIRS at the time of diagnosis, whereas no significant difference between infectious and non-infectious SIRS was noted for serum levels of IL-10 and TGF-beta at the time of diagnosis and 5 days later. During SIRS, serum levels of IL-4 were significantly increased with a significant correlation between IL-4 and mortality, and only levels of IL-4 were significantly increased in the SIRS caused by infectious stimuli.     

Role of the fourth complement component (C4) in the regulation of contact sensitivity. I. Analysis in mice with high and low C4 levels

Lymph node cells collected from CBA/J mice 4 days after painting the skin with picryl chloride are able to immunize naive recipients by hapten-IgM immuno complexes. These cells ("4-day" cells) activate the early components of the classical pathway of complement from mice of the H-2 Sd haplotype (high-C4), but fail to activate the classical pathway of complement from mice of the H-2 Sk haplotype (low-C4). Incubation of "4-day" cells in complement from mice with high-C4 levels abolishes the induction of contact sensitivity, probably as a consequence of the solubilization of membrane-bound immuno complexes caused by complement activation. The presence of "4-day" cells is determined by the levels of C4. In fact, using strains of mice which differ only at the S region of the H-2 complex, we found that mice of the H-2 Sd (and perhaps H-2 Sb) haplotype (high-C4 levels) lack "4-day" cells in their lymph nodes and this is due to the activation of the early components of the classical complement pathway which occurs in vivo in these mice during sensitization with picryl chloride. The finding that contact sensitivity reaction to picryl chloride in H-2 Sk mice lasts about 21 days, whereas H-2 Sd mice show a contact sensitivity reaction until 7 days after sensitization, strongly suggests that the S region, and in particular C4 levels, controls the persistence of "4-day" immunogenic cells, and so play a role in the duration of the contact sensitivity reaction to picryl chloride in the mouse.     

Sensitivity and resistance to regulation by IL-4 during Th17 maturation

Th17 cells are highly pathogenic in a variety of immune-mediated diseases, and a thorough understanding of the mechanisms of cytokine-mediated suppression of Th17 cells has great therapeutic potential. In this article, we characterize the regulation of both in vitro- and in vivo-derived Th17 cells by IL-4. We demonstrate that IL-4 suppresses reactivation of committed Th17 cells, even in the presence of TGF-β, IL-6, and IL-23. Downregulation of IL-17 by IL-4 is dependent on STAT6 and mediated by inhibition of STAT3 binding at the Il17a promoter. Although Th1 cytokines were shown to induce IFN-γ expression by Th17 cells, IL-4 does not induce a Th2 phenotype in Th17 cells. Suppression by IL-4 is stable and long-lived when applied to immature Th17 cells, but cells that have undergone multiple rounds of stimulation, either in vivo during a Th17-mediated inflammatory disease, or in vitro, become resistant to suppression by IL-4 and lose the ability to signal through IL-4R. Thus, although IL-4 is a potent suppressor of the Th17 genetic program at early stages after differentiation, prolonged stimulation renders Th17 cells impervious to regulatory cytokines.     

T cell-specific negative regulation of transcription of the human cytokine IL-4.

IL-4 secreted by activated T cells is a pleiotropic cytokine affecting growth and differentiation of diverse cell types such as T cells, B cells, and mast cells. We investigated the upstream regulatory elements of the human IL-4 promoter. A novel T cell-specific negative regulatory element (NRE) composed of two protein-binding sites were mapped in the 5' flanking region of the IL-4 gene: -311CTCCCTTCT-303 (NRE-I) and -288CTTTTTGCTT-TGC-300 (NRE-II). A T cell-specific protein Neg-1 and a ubiquitous protein Neg-2 binding to NRE-I and NRE-II, respectively, were identified. Furthermore, a positive regulatory element was found 45 bp downstream of the NRE. The enhancer activity of the PRE was completely suppressed when the NRE was present. These data suggest that IL-4 promoter activity is normally down-regulated by an NRE via repression of the enhancer positive regulatory element. These data may have implications for the stringent control of IL-4 expression in T cells.     

Role of the fourth complement component (C4) in the regulation of contact sensitivity. II. Qualitative differences between C4 molecules from high- and low-C4 mouse strains

Lymph node cells collected from CBA/J mice 4 days after painting with picryl chloride induce contact sensitivity in naive recipient mice by virtue of hapten IgM immuno complexes. The immunizing capacity of these cells ("4-day" cells) is abolished after incubation of the cells with a C4-deficient guinea pig serum reconstituted with plasma or purified C4 from mice with high C4 levels (C4h), but not with plasma or purified C4 from mice with low C4 levels (C41). The inhibition of the immunizing capacity of 4-day cells is due to the activation of the early components of the classical complement pathway which is likely to result in the solubilization of membrane-bound immunocomplexes. However, the same amounts of CBA/J and BALB/c C4 have a different effect in inhibiting the induction of contact sensitivity by 4-day cells. In fact, by dose-response experiments, we have found that the amount of C41 able to inhibit the induction of contact sensitivity is about threefold higher than that of C4h. Analysis of the covalent binding ability of C4h and C41 reveals that C4h is able to bind to the surface of 4-day cells more efficiently than C41 and this probably accounts for the difference of the two C4 molecules in inhibiting the immunizing capacity of 4-day cells. Results are discussed in terms of different reactivities of C4h and C41 with the surface of 4-day cells.     

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.     

Fine-specificity of the contact sensitivity to 4-hydroxy-3-nitrophenyl acetyl (NP)

Dorf and colleagues (1-4) found that the contact sensitivity (CS) primed with (4-hydroxy-3-nitrophenyl)acetyl (NP) could be elicited as easily with the iodoanalog (NIP) as with NP when studied in Igh-1b mice but could only be elicited with NP, not NIP, in Igh-1j mice. Since this fine-specificity was parallel to the fine-specificity of anti-NP antibodies in the two types of mice and since anti-NP antibodies of Igh-1b mice are controlled by gene Igh-NPb the authors concluded that CS also was controlled by the Igh-NPb gene. The aim of this study was to confirm their findings with a more quantitative method (5). We confirmed equality of NP and NIP as elicitors of NP-primed CS in Igh-1b mice when the priming antigen was given subcutaneously into non-cyclophosphamide-treated mice (their method). We also found that this priming induced an anti-NP antibody response detectable at the time of challenge. Most experiments were carried out with a method that does not induce a detectable antibody response (pretreatment of mice with 200 mg/kg of cyclophosphamide; application of the sensitizing compound on skin). Since the NP-primed (and NBrP-primed) CS reactions exhibited "expected specificities," the immunizing compound was clearly the most efficient elicitor (relative efficiencies of homologs varied from 2 to 4). The Igh-NPb gene appears not to have a role in "antibody-free" reactions.     

Negative regulation of IL-17 production by OX40/OX40L interaction

The T-cell cytokine IL-17 is implicated in multiple inflammatory diseases through its induction of several pro-inflammatory cytokines and chemokines in a broad range of cell targets. Production of IL-17 defines the Th17 subset of helper T-cells associated with protection against microorganisms, a profile best characterized in the murine system. Multiple regulators of Th17 cell differentiation and IL-17 production are reported, but the impact of OX40L is not described. OX40 ligand (OX40L) is an early-stage activator of T-cells through its interaction with CD134 (OX40) that is up-regulated on antigen challenged T-cells. Here, we show that OX40L suppresses IL-17 production by PHA-stimulated human PBMC and purified CD4 and CD8 cells. In agreement with prior reports, OX40L signaling through CD134 increased IFNgamma and IL-4, both of which are reported to inhibit the production of IL-17. OX40L suppression of IL-17 was completely reversed by a neutralizing IFNgamma antibody while there was no effect with a neutralizing IL-4 antibody. Moreover, OX40L also suppressed IL-17 in the presence of IL-23, an established inducer of IL-17 and differentiation factor for Th17 cells. Presuming mediation by IFNgamma, we evaluated expression of this cytokine in the presence of OX40L and IL-23. Surprisingly, IL-23 also induced IFNgamma by PHA-stimulated T-cells and this effect was enhanced in the presence of OX40L. Addition of the IFNgamma antibody not only reversed the OX40L suppression of IL-17 in the presence of IL-23, it markedly enhanced the level of IL-17. These results further establish IFNgamma as a primary modulator of IL-17 production in the human cells, much as in the murine system.     

Oral administration of the contact sensitizer trinitrochlorobenzene: initial sensitization and subsequent appearance of a suppressor population

Our earlier studies have demonstrated that intragastric administration of the hapten trinitrochlorobenzene (TNCB) 2 to 3 weeks prior to attempting sensitization with epidermally applied hapten can abrogate development of systemic contact sensitivity (CS). In this paper, we have examined whether onset of tolerance following intragastric administration of the hapten is preceded by development of hapten-specific CS. Indeed, CS was found to be present 5 days after feeding TNCB and in most experiments the response decreased significantly by Days 10 to 12. The kinetics of development of CS by the oral and epidermal routes were strikingly similar except that the magnitude of reactivity (up to 5 days) in orally sensitized mice was somewhat less than that of epidermally sensitized mice. With the exception of Peyer's patches (PP), effector cells of CS were recovered from such gut-associated lymphoid tissues as mesenteric lymph nodes (MLN), lamina propria, and lymphocytes that are present in the intraepithelial compartment of the intestinal wall. These cells as well as spleen cells of TNCB-fed mice were able to adoptively transfer CS to naive mice. The capacity of MLN and spleen cells of TNCB-fed mice to confer CS adoptively was abrogated after treating cells with anti-Thy 1.2 and anti-Lyt 1.1 antibodies plus complement thereby identifying them as T lymphocytes. Although CS decreased by 10-12 days after feeding TNCB, the decline was reversed by pretreating mice with cyclophosphamide (CY) 2 days before giving the hapten. Whereas spleen cells from animals fed hapten 5 days earlier transferred CS readily, those from mice fed hapten 12 days earlier did not. However, when 12-day spleen cells were depleted of Lyt 2+ cells their ability to adoptively transfer CS was restored. These observations indicate that feeding TNCB to mice initially produces CS, mediated by Thy 1.2+, Lyt 1.1+ lymphocytes. CS is subsequently down-regulated by activation of Lyt 2+ suppressor cells, precursors of which are sensitive to CY.