Pimecrolimus and tacrolimus differ in their inhibition of lymphocyte activation during the sensitization phase of contact hypersensitivity.

BACKGROUND: As reported previously, oral administration of the calcineurin inhibitors (CNI) pimecrolimus and tacrolimus resulted in equipotent inhibition of the elicitation phase of contact hypersensitivity (CHS) in mice. The sensitization phase was inhibited by tacrolimus but was unaffected by pimecrolimus, even at higher doses. OBJECTIVE: The kinetics of lymph node hyperplasia and up-regulation of T and B cell activation antigens were analyzed to obtain a better understanding of the divergent CNI profile in CHS. METHODS: Lymph node (LN) cells of CNI-untreated and treated mice were examined with flow cytometry at various time points after sensitization with oxazolone. LN hyperplasia and drug levels were also determined. RESULTS: Sensitization induced a higher portion of LN cells expressing the activation antigens CD25, CD69 and CD134 and an increase in activated B cells (B220(+)/CD40(+)) compared to naïve mice. Up-regulation of these markers was completely or profoundly blocked with tacrolimus, whereas pimecrolimus at the three-fold higher dose caused significantly less inhibition. Tacrolimus also completely blocked the sensitization-associated increase of CD11c(+) antigen presenting cells (APC) in LN, whereas pimecrolimus showed significantly less inhibition. In contrast to tacrolimus, LN weight and cellularity were not affected by pimecrolimus at any time point after sensitization. Concentration of tacrolimus in blood and in the draining LN substantially exceeded that of pimecrolimus by factors 6.7-14 and 5.6-5.8, respectively, at the same dose levels. CONCLUSION: In contrast to tacrolimus, systemic treatment of mice with pimecrolimus only weakly interferes with lymphocyte activation and does not affect hyperplasia of the draining lymph nodes during sensitization.

Pimecrolimus does not affect the differentiation, maturation and function of human monocyte-derived dendritic cells, in contrast to corticosteroids.

Clinically, corticosteroids (CS) are among the first line drugs in the therapy of autoimmune and allergic diseases and potently inhibit the activation of immune cells. However, due to their pleiotropic mode of action, the prolonged use of CS is generally associated with a range of undesirable side-effects. In this study, we compared the activity of pimecrolimus, a novel immunomodulatory drug for the treatment of inflammatory skin disorders, and the CS dexamethasone (Dex) and beta-methasone-valerate (beta-MSV) in different in vitro assays addressing the cytokine-induced differentiation and maturation of monocyte-derived dendritic cells (M-DC), the susceptibility of M-DC to drug-induced apoptosis and the potency of differentiated M-DC to induce primary T cell activation. In contrast to pimecrolimus, Dex and beta-MSV strongly induced apoptosis of M-DC precursors if added at the start of the DC differentiation culture. Flow cytometric analysis of surviving cells on day 6 of culture showed that the expression of several DC-specific antigens such as CD1a, CD40 and CD80 was inhibited by 50% to 80% at concentrations between 1 nm and 10 nm of either Dex or beta-MSV. Furthermore, the presence of CS during the final maturation of M-DC inhibited the synthesis of IL-12p70, the expression of critical DC costimulatory molecules, such as CD83 and CD86 and impaired their ability to activate primary CD4+ T cell proliferation. In contrast, pimecrolimus did not inhibit the LPS-induced secretion of IL-12, surface expression of costimulatory molecules or the maturation of M-DC into potent stimulators of T cells. Taken together, these data indicate that pimecrolimus does not interfere with the differentiation and viability of dendritic cells and their precursors or with the function of mature M-DC to prime naïve T lymphocytes, and thus may have a lower potential than CS to interfere with DC-mediated immunosurveillance.

Pimecrolimus inhibits up-regulation of OX40 and synthesis of inflammatory cytokines upon secondary T cell activation by allogeneic dendritic cells.

Pimecrolimus is a new non-steroidal inhibitor of T cell and mast cell activation. In the present study, we compared the potency of pimecrolimus and cyclosporin A (CyA) to inhibit cytokine synthesis of alloantigen-primed T cells and the expression of CD134 (OX40), an inducible co-receptor molecule thought to be critical for the survival and expansion of inflammation-mediating T cells. To mimic the physiological situation of recurrent antigenic stimulation, we have used dendritic cells (DC) as stimulators of purified CD4+ T cells in the primary and secondary allogeneic mixed lymphocyte culture (allo-MLC). Pimecrolimus inhibited surface expression of OX40 and prevented the up-regulation of CD25 and CD54 with a 10-fold higher potency compared to CyA. Similarly, 50% inhibition of allo-DC-mediated T cell proliferation by pimecrolimus was obtained at 0.55 nm, compared to about 12 nm for CyA. Furthermore, pimecrolimus blocked the increase of OX40 on primed T cells restimulated on day 10 in secondary allo-MLC. Allo-DC-primed T cells showed a restricted cytokine profile characterized by the production of TNF-alpha, IFN-gamma and IL-2 but low to undetectable levels of IL-4 and IL-10. The synthesis of TNF-alpha and IFN-gamma and the up-regulation of OX40 on T cells after secondary allogeneic stimulation were almost entirely blocked by 10 nm pimecrolimus. Taken together, pimecrolimus inhibits T cell proliferation and Th1 cytokine synthesis and also prevents the up-regulation of the OX40 co-receptor on primed T cells indicating its potential in the therapy of chronic inflammation and autoimmunity.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) has opposing effects on the capacity of monocytes versus monocyte-derived dendritic cells to stimulate the antigen-specific proliferation of a human T cell clone.

GM-CSF is widely used in combination with IL-4 to differentiate monocytes into potent T cell stimulatory cells, referred to as monocyte-derived dendritic cells (MoDC). These cytokines further increased the stimulatory function of MoDC when present during their incubation with antigen, as determined by the proliferative response of an allergen-specific T cell clone. Conversely, the incubation of freshly isolated monocytes with antigen in the presence of GM-CSF or GM-CSF and IL-4 strongly inhibited the specific stimulation of the T cells, compared with monocytes pulsed in the absence of cytokines. This suppression was partly due to the secretion of prostaglandin E2 (PGE2) and IL-10 by GM-CSF-treated monocytes, since the combined use of indomethacin and anti-IL-10 antibodies during GM-CSF incubation and antigen pulsing restored T cell growth to about 65% of control levels. As confirmed by culture supernatant transfer experiments, maximal inhibition of T cell stimulation was also dependent on the direct contact between the T cells and GM-CSF-treated monocytes during antigen presentation. Collectively, these results imply that GM-CSF can either inhibit or enhance the re-stimulation of primed T cells by antigen-presenting monocytes or MoDC, respectively.

Function of the human interleukin 4 receptor (IL-4R)-derived acidic motif revealed by cytoplasmic domain chimeras of the IL-4R alpha chain and the IL-2R beta chain.

Interleukin 2 (IL-2)- and IL-4-mediated stimulation of survival and growth, reflected by the induction of bcl2 and c-myc, respectively, depends on the integrity of the membrane-proximal region (S-region) in the IL-2 receptor beta-chain (IL-2R beta) and the haematopoietin homology box1-containing region of the IL-4 receptor alpha-chain (IL-4R alpha). In contrast to IL-4, IL-2 induces the expression of c-fos and c-jun family genes, mediated by the acidic region (A-region) within the cytoplasmic domain of IL-2R beta. A highly acidic motif is also present in IL-4R alpha, and evidence in favour and against its importance has been published. The authors have constructed chimeric receptors between IL-2R beta and IL-4R alpha by substitution of either the S-region or the A-region of IL-2R beta with sequences derived from IL-4R alpha. These chimeras were stably transfected into BA/F3 cells and assayed for the capacity to restore functions of IL-2 beta, such as growth mediation by IL-2 and the induction of proto-oncogenes (c-myc, c-junB and c-fos). Replacement of both the S- and A-region of IL-2R beta with IL-4R alpha derived regions of similar size and cytoplasmic location supported growth-stimulation by IL-2 as well as proto-oncogene induction. In contrast, all IL-2R functions were lost by exchange of the S-region with the corresponding part of IL-4R alpha. Induction of c-junB and c-fos RNA as an indicator of A-region function, however, was maintained in an IL-2R beta chimera containing the acidic box-bearing region of IL-4R alpha. These data indicate a functional role of the acidic region in the IL-4R alpha-chain.

Interleukin-13 is a potent activator of JAK3 and STAT6 in cells expressing interleukin-2 receptor-gamma and interleukin-4 receptor-alpha.

The lymphocyte growth factors interleukin-2 (IL2), IL4, IL7, IL9 and IL15 use the common IL2 receptor-gamma (IL2R gamma) and activate the IL2R gamma-associated tyrosine kinase JAK3 (Janus kinase 3). IL13 is structurally related to IL4, competes with IL4 for binding to cell surface receptors and exhibits many similar biological effects. The molecular basis for this functional overlap between IL4 and IL13 has been attributed mainly to a shared use of the 140 kDa IL4R alpha, since these cytokines appear to be uniquely different in that, according to several recent reports, IL13 does not recruit the IL2R gamma or JAK3. This notion has been supported by the identification of a novel 70 kDa IL13 receptor in certain IL13-responsive cell lines that lack IL2R gamma. The present study sheds new light on the issue of functional overlap between IL13 and IL4, by demonstrating for the first time that, in cells that express both IL2R gamma and IL4R alpha, IL13 can mimic IL4-induced heterodimerization of IL2R gamma and IL4R alpha, with consequent marked activation of JAK3 and the transcription factor STAT6 (IL4-STAT). Reconstitution experiments in BA/F3 cells showed that both cytokines require the simultaneous presence of IL4R alpha and IL2R gamma to mediate JAK3 and proliferative responses, and analysis of 12 IL4R alpha variants showed that IL4 and IL13 signals were equally affected by mutations of the cytoplasmic domain. We conclude that IL13 activates the IL2R gamma-associated JAK3 tyrosine kinase in appropriate cell types, and propose that IL13 is capable of interacting with multiple receptor subunits in a cell-dependent and combinatorial manner. Consequently, we predict that partial disruption of IL13 signal transduction also contributes to the severe combined immuno-deficiency syndromes associated with inactivation of the IL2R gamma or JAK3 genes.

IL-4-dependent proliferation of BA/F3 cells expressing a growth-negative mutant of the human IL-4 receptor is restored by enforced expression of Bcl-2.

We have studied the regulation of growth and apoptosis in murine BA/F3 cells stably expressing cytoplasmic deletion mutants of the human interleukin-4 receptor (hIL-4R). Previously, we showed that BA/F3 cell transfectants expressing a cytoplasmic deletion mutant of the hIL-4R that lacks the region between Thr(462) and Ala(580), referred to as delta R3, fails to proliferate in the presence of hIL-4. Here we report that supertransfection of delta R3-expressing cells with a constitutively active murine bcl-2 gene results in prolonged survival of the delta R3/bel-2 double transfectants in the absence of cytokines. More importantly, however, the constitutive expression of Bcl-2 restored their capacity to grow permanently with hIL-4. This may provide an explanation for the discrepancy with previous reports showing growth mediation by hIL-4R truncated at position 367.

Activation of JAK3, but not JAK1, is critical to interleukin-4 (IL4) stimulated proliferation and requires a membrane-proximal region of IL4 receptor alpha.

The tyrosine kinases JAK1 and JAK3 have been shown to undergo tyrosine phosphorylation in response to interleukin-2 (IL), IL4, IL7, and IL9, cytokines which share the common IL2 receptor gamma-chain (IL2R gamma), and evidence has been found for a preferential coupling of JAK3 to IL2R gamma and JAK1 to IL2R beta. Here we show, using human premyeloid TF-1 cells, that IL4 stimulates JAK3 to a larger extent than JAK1, based upon three different evaluation criteria. These include a more vigorous tyrosine phosphorylation of JAK3 as measured by anti-phosphotyrosine immunoblotting, a more marked activation of JAK3 as determined by in vitro tyrosine kinase assays and a more manifest presence of JAK3 in activated IL4-receptor complexes. These observations suggest that IL4 receptor signal transduction does not depend on equimolar heterodimerization of JAK1 and JAK3 following IL4-induced heterodimerization of IL4R alpha and IL2R gamma. Indeed, when human IL4R alpha was stably expressed in mouse BA/F3 cells, robust IL4-induced proliferation and JAK3 activation occurred without detectable involvement of JAK1, JAK2, or TYK2. The present study suggests that JAK1 plays a subordinate role in IL4 receptor signaling, and that in certain cells exclusive JAK3 activation may mediate IL4-induced cell growth. Moreover, mutational analysis of human IL4R alpha showed that a membrane-proximal cytoplasmic region was critical for JAK3 activation, while the I4R motif was not, which is compatible with a role of JAK3 upstream of the recruitment of the insulin receptor substrate-1/4PS signaling proteins by IL4 receptors.

Distinct sequence motifs within the cytoplasmic domain of the human IL-4 receptor differentially regulate apoptosis inhibition and cell growth.

Hematopoietin receptors generally function as multimeric complexes composed of a unique ligand-binding chain and a second component often shared between several members of this receptor family. To better understand the signal transduction of the human IL-4 receptor (hIL4R), we analyzed the functionality of targeted mutations in two cytoplasmic regions of the ligand-binding hIL4R chain that we previously identified to be necessary for growth mediation in factor-dependent murine Ba/F3 cells. Here, we provide evidence that transient inhibition of apoptotic death of Ba/F3 cells and the competence to proliferate indefinitely depend on separated and distinct sequence motifs of the hIL4R. In particular, hIL4R constructs with a truncation of the recently described gp130 box1 from P242 to K264, or a deletion of the acidic region between S330 and S365, fail to stimulate growth or to mediate the inhibition of apoptosis. hIL4R bearing a point mutation within the gp130 box1 (P242S) is defective for growth stimulation but still signals the transient inhibition of apoptotic cell death and the induction of c-myc RNA. A third region required for IL4-mediated cell growth is localized between T462 and S476 and includes the sequence NPAY previously described to serve as interaction motif in signaling of epidermal growth factor and insulin receptors. Conversion of Y472 into F472 within the latter hIL4R motif affects the competence of stably transfected BA/F3 cells to proliferate indefinitely in the presence of hIL4. Sequences C-terminal of S476 are not essential for growth stimulation of BA/F3 transfectants.

Transforming growth factor-beta inhibits IL-4 and IFN-gamma production by stimulated human T cells.

The present study investigates the effect of transforming growth factor (TGF)-beta on the production of IL-4 and IFN-gamma by the leukemia Th0 type cell line HUT78, by freshly isolated human T cells, and by antigen specific human T cell clones. We found that IL-4 and IFN-gamma, but not IL-2, production by stimulated HUT78 cells was inhibited by TGF-beta 1. TGF-beta 1 also reduced the accumulation of IL-4 and IFN-gamma specific mRNA in stimulated HUT78 cells. However, IL-2 and IL-7 co-stimulated IL-4 and IFN-gamma production, whereas IL-1, IL-3, IL-5, IL-6, IL-8, tumor necrosis factor-alpha or granulocyte macrophage colony stimulating factor had no effect. Because IL-2 is an important helper cytokine for the production of IL-4 and IFN-gamma, we investigated whether signal transduction through the IL-2 receptor is impaired by TGF-beta 1. We found that tyrosine phosphorylation in response to IL-2 in HUT78 cells was strongly inhibited by a short preincubation with TGF-beta 1. Evidence for an antagonistic role for TGF-beta 1 and IL-2 comes from the finding that high doses of IL-2 could partially overcome TGF-beta 1 mediated inhibition of IL-4 and IFN-gamma production. Similar to its effect on HUT78 cells, TGF-beta 1 also inhibited IL-4 and IFN-gamma production by freshly isolated T cells as well as by human T cell clones. Taken together, our experiments show that the IL-2 dependent cytokines IL-4 and IFN-gamma are both negatively controlled by TGF-beta under conditions where IL-2 production is unaffected by a mechanism which partially involves an inhibition of IL-2/IL-2R signal transduction. These data identify TGF-beta and IL-2 as mutual antagonists in the regulation of IL-4 and IFN-gamma production.

Human interleukin-4 receptor signaling requires sequences contained within two cytoplasmic regions.

The signaling pathway used by the murine or human interleukin-4 receptor (hIL-4R) has not been elucidated so far. As an approach to mapping the cytoplasmic regions of the hIL-4R that are essential for mediating the biological response upon IL-4 binding we have cloned and expressed the wild-type hIL-4R and several cytoplasmic deletion mutants in the murine IL-3-dependent pro-B cell line BA/F3. Transfection of the wild-type hIL-4R conferred the ability on BA/F3 cells to proliferate in a dose-dependent way when treated with human IL-4. The analysis of six deletion mutants indicated that the signaling function of the hIL-4R depends on sequences within two discontinuous regions that are located between amino acid IIe233 and Ser365 and Thr462 and Ala580 of the intracytoplasmic domain. The deletion of either of these regions totally abrogated IL-4-inducible growth. The relevance of our data is discussed in relation to the results obtained from similar studies with other members of the hematopoietin receptor superfamily.

Interleukin-4 production by the malignant cell line HUT78.

Interleukin-4 (IL-4) is a cytokine difficult to induce in large quantities in freshly isolated cells. No malignant cell line has been described to date to produce IL-4 either constitutively or following activation. Here we report that HUT78 cells can be induced to produce IL-4 either by CD3 stimulation under cross-linking conditions or by soluble CD3 mAbs in the presence of PMA. IL-4-specific mRNA could be demonstrated readily under such conditions. Using this experimental model we investigated the role of IL-2 for IL-4 production. The addition of IL-2 strongly costimulated CD3-induced IL-4 production, whereas blockade of the IL-2/IL-2R pathway reduced IL-4 production by approximately 50%. IL-2 alone, however, could not induce IL-4 production. These results indicate that IL-2 is a helper of cytokine necessary, but not sufficient, for IL-4 production in HUT78 cells.

Regulation of interleukin-4 production in human mononuclear cells.

Interleukin (IL)-4 is a cytokine with a broad range of effects on immune cells, however, little is known regarding the regulation of its production in freshly isolated human peripheral blood mononuclear cells (PBMC). Here we report the production of IL-4 in such cells following stimulation with monoclonal antibodies (mAb) directed against different cell surface antigens. We show that triggering via CD2 is more efficient for IL-4 production than triggering via the CD3 complex. The addition of a CD28 mAb enhances IL-4 production approximately threefold. Cell depletion experiments show that among CD2 plus CD28-stimulated PBMC the production of IL-4 is restricted to the CD8-CD45RA-T cell subpopulation. mAb interfering with the binding of IL-2 to its receptor can inhibit the production of IL-4 in CD2 plus CD28-stimulated PBMC. As IL-2 induces cell proliferation and production of interferon-gamma, but not production of IL-4, it follows that IL-2 is necessary but not sufficient for IL-4 production.

Skin-derived aeroallergen-specific T-cell clones of Th2 phenotype in patients with atopic dermatitis.

T-cell lines have been derived from aeroallergen-induced eczematous patch test sites of patients with atopic dermatitis (AD). Biopsy specimens were obtained 24 hours after allergen application to the skin, and only in vivo-activated T cells were propagated. From one patient, the T-cell lines were subcloned at 0.3 cells per well. With allergen-induced interleukin (IL) production, all clones tested (n = 13) were found to be specific for the allergen, producing IL-4 and granulocyte-macrophage-colony-stimulating factor. No IL-2 or interferon-gamma was found. The allergen-specific proliferative response of these clones, although the response was dependent on exogenous IL-2 or IL-4, also proved that all clones were allergen specific. Under optimal stimulation (aCD3 plus phorbol myristate acetate), 15% of the clones appeared to be of Th0 phenotype and 70% of Th2 phenotype. In 15% of the clones, IL-4 was produced in the absence of IL-2, IL-5, or interferon-gamma. Supernatants of all clones tested induced IgE production by B cells from normal non-atopic donors. The T-cell lines of the other patient demonstrated similar results; allergen-specific proliferation was dependent on exogenous IL-2 or IL-4 and stimulation with aCD3 plus phorbol myristate acetate demonstrated that the T cells in these lines were of the Th2 phenotype. In conclusion, our data reveal that, in AD, percutaneous sensitization to aeroallergens may occur and indicate that allergen-specific Th2 type T cells may be responsible for the high levels of (specific) IgE found in 80% of patients with AD.

Differential induction of cytokine-specific mRNA in human PBL after in vitro culture with either IL2 or IL4.

The present study was designed to investigate whether human equivalents of murine T helper cell subsets can be demonstrated by propagation of peripheral blood lymphocytes (PBL) with either recombinant human (rh) interleukin (IL) 2 or rhIL4 in the presence of neutralising antibodies. Cells of both cultures, termed T-IL2 or T-IL4, respectively, were challenged on day 8 using a combination of phorbol-12-myristate-13-acetate (PMA) and the Ca2(+)-ionophore A23187 (Io). Total cellular RNA was isolated at different time points after PMA/Io-stimulation and the expression of 7 distinct cytokine genes was assessed by Northern analysis. Whereas maximal accumulation of mRNA species for IL2, GM-CSF, TNF alpha and TNF beta did not reveal major differences between cells of T-IL2 and T-IL4 cultures, substantial differences emerged for the induction of IFN gamma and IL3 messages. Accumulation of IFN gamma-mRNA consistently was 2- to 13-fold higher in T-IL2 than in T-IL4 cells, depending on the time point of RNA harvest. In contrast, IL3-specific mRNA levels induced in T-IL4 cells were 2-5 times greater than those in T-IL2. If PBL cultured with IL2 for 7-8 days were subsequently shifted to IL4 and further propagated until day 14, the mRNA induction pattern seen for IFN gamma and IL3 was similar to that obtained if cells had continuously been propagated with IL2. Collectively, these results indicate a selective outgrowth of distinct responder phenotypes by IL2 or IL4 rather than a direct modulation of cytokine expression by these factors.