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C M Hilkens

Publications and source records attributed to C M Hilkens.

At least 19 recordsLinked to original sources
PubMed2001

A region encompassing the FERM domain of Jak1 is necessary for binding to the cytokine receptor gp130.

The terminal portion of the Janus kinases (Jaks) contains a divergent FERM (Four-point-one, Ezrin, Radixin, Moesin) homology domain comprising 19 conserved hydrophobic regions. To determine the role of this domain in governing recruitment of Jak1, but not Jak3, to the gp130 subunit of the interleukin-6 family of cytokine receptors, the interaction of three Jak1/Jak3 chimeras with gp130 was investigated. Chimeras 1, 2 and 3 (Jak1 FERM regions 1-19, 1-18 and 1-8/Jak3, respectively) were all enzymically active. Chimeras 1 and 2 interacted with the cytoplasmic domain of gp130, although less efficiently than Jak1. Only chimera 2, however, restored gp130 signalling in Jak1-negative cells. The data are consistent with recruitment of Jak1 to gp130 through the Jak1 FERM domain, but also emphasise the likely requirement for precise Jak/receptor orientation to sustain function.

Amino Acid Sequence↗
PubMed2001

IL-12-induced reversal of human Th2 cells is accompanied by full restoration of IL-12 responsiveness and loss of GATA-3 expression.

IL-12 is a potent inducer of IFN-gamma production and drives the development of Th1 cells. Human polarized Th2 cells do not express the signaling beta2-subunit of the IL-12R and, therefore, do not signal in response to IL-12. The question was raised as to what extent the loss of the IL-12Rbeta2 chain in Th2 cells has bearing on the stability of the human Th2 phenotype. In the present report, we show that restimulation of human fully polarized Th2 cells in the presence of IL-12 primes for a shift towards Th0/Th1 phenotypes, accompanied by suppression of GATA-3 expression and induction of T-bet expression. These reversed cells are further characterized by a marked IL-12Rbeta2 chain expression and fully restored IL-12-inducible STAT4 activation. The IL-12-induced phenotypic shift proved to be stable as a subsequent restimulation in the presence of IL-4 and in the absence of IL-12 could not undo the accomplished changes. Identical results were obtained with cells from atopic patients, both with polyclonal Th2 cell lines and allergen-specific Th2 cell clones. These findings suggest the possibility of restoring IL-12 responsiveness in established Th2 cells of atopic patients by stimulation in the presence of IL-12, and that IL-12-promoting immunotherapy can be beneficial for Th2-mediated immune disorders, targeting both naive and memory effector T cells.

Allergens↗
PubMed2001

How to deal with polarized Th2 cells: exploring the Achilles' heel.

The central effector cells in the pathogenesis of atopic allergic diseases are type 2 T helper (Th2) cells, which display an aberrant cytokine profile dominated by type 2 cytokines. Initial reports from mouse studies indicated that established and committed Th2 cells are stable and unsusceptible to modulation. However, there is a growing awareness that in humans, established effector Th2 cells are more flexible and can be reverted to predominant Th1 phenotypes. In fact, the Th1-driving cytokine interleukin (IL)-12 is the crucial factor in this respect. IL-12 is mainly produced by dendritic cells (DC), which can be primed for high or low IL-12 production, depending on inflammatory and/or microbial signals they encounter during their residence in the peripheral tissues. Accordingly, both the regulation of and the priming for IL-12 production in DC form ideal targets for therapeutic intervention. The development of new therapies for atopic allergy now focuses on local IL-12-promoting substances to target both the development of new Th2 cells and the persistent population of established allergen-specific Th2 cells.

Animals↗
PubMed1999

Final maturation of dendritic cells is associated with impaired responsiveness to IFN-gamma and to bacterial IL-12 inducers: decreased ability of mature dendritic cells to produce IL-12 during the interaction with Th cells.

Activation of immature CD83- dendritic cells (DC) in peripheral tissues induces their maturation and migration to lymph nodes. Activated DC become potent stimulators of Th cells and efficient inducers of Th1- and Th2-type cytokine production. This study analyzes the ability of human monocyte-derived CD1a+ DC at different stages of IL-1 beta and TNF-alpha-induced maturation to produce the major Th1-driving factor IL-12. DC at the early stages of maturation (2 and 4 h) produced elevated amounts of IL-12 p70 during interaction with CD40 ligand-bearing Th cells or, after stimulation with the T cell-replacing factors, soluble CD40 ligand and IFN-gamma. The ability to produce IL-12 was strongly down-regulated at later time points, 12 h after the induction of DC maturation, and in fully mature CD83+ cells, at 48 h. In contrast, the ability of mature DC to produce IL-6 was preserved or even enhanced, indicating their intact responsiveness to CD40 triggering. A reduced IL-12-producing capacity of mature DC resulted mainly from their impaired responsiveness to IFN-gamma, a cofactor in CD40-induced IL-12 p70 production. This correlated with reduced expression of IFN-gamma R (CD119) by mature DC. In addition, while immature DC produced IL-12 and IL-6 after stimulation with LPS or Staphylococcus aureus Cowan I strain, mature DC became unresponsive to these bacterial stimuli. Together with the previously described ability of IL-10 and PGE2 to stably down-regulate the ability to produce IL-12 in maturing, but not in fully mature, DC, the current data indicate a general resistance of mature DC to IL-12-modulating factors.

Antigens, Bacterial↗
PubMed1999

The paradigm of type 1 and type 2 antigen-presenting cells. Implications for atopic allergy.

Optimal clearance of the various pathogen types encountered by the human body requires the selective activation of particular cellular and/or humoral immune responses. The orchestration of the types of effector responses is directed by Th cells through the production of type 1 (Th1 cell-associated) and type 2 (Th2 cell-associated) cytokines. The way in which the Th cell cytokine profile is matched to the type of invading pathogen, and why these profiles sometimes derail and lead to disease, is not well understood. Here, we will discuss the concept that antigen-presenting cells (APC) provide Th cells not only with antigen and costimulatory signals, but also with a polarizing signal (signal 3). This signal can be mediated by many APC-derived factors, but IL-12 and PGE2 seem to be of major importance. The Th2-biased responses in atopic allergy appeared to be associated with monocytes with a decreased IL-12/PGE2 ratio and, consequently, with the down-regulation of type 1 cytokine production in Th cells. As for Th cells, APC can be functionialy polarized. In vitro experiments with monocyte-derived dendritic cells (DC) showed that the presence of IFN-gamma during activation of immature DC primes for mature DC with the ability of high IL-12 production and, consequently, a Th1-driving capacity (APC1 or DC1). In contrast, PGE2 primes for a low IL-12 production ability and a Th2-driving capacity (APC2 or DC2). These findings suggest that pathogens provoke either Th1- or Th2-cell development by inducing the production of a certain pattern of inflammatory DC-polarizing mediators (e.g. IFN-gamma and PGE2) at the site of infection. The type of immune polarization will not only depend on the type of pathogen, but also varies with the type of infected tissue, i.e. that different tissues produce different mediators in response to the same pathogen. In the case of atopic allergy, this concept implies that the Th2-cell bias may be related to low levels of cross-regulatory infections, to Th1 cell-inducing pathogens, or to an aberrant function of stromal cells in peripheral tissues.

Animals↗
PubMed1998

Prostaglandin E2 induces the final maturation of IL-12-deficient CD1a+CD83+ dendritic cells: the levels of IL-12 are determined during the final dendritic cell maturation and are resistant to further modulation.

Activation of immature dendritic cells (DC) in peripheral tissues induces their migration to lymph nodes and their maturation into CD83+ DC, which are able to prime naive T cells. The inflammatory cytokines IL-1beta and TNF-alpha induce mature DC, which can secrete IL-12 and promote the development of Th0/Th1-biased cells. DC maturation factors with a Th2-promoting function have not been described. Here we show that PGE2, although it does not induce final DC maturation by itself, synergizes with IL-1beta and TNF-alpha, and allows their effectiveness at 100-fold lower concentrations. While being phenotypically identical with the DC matured in the presence of high concentrations of IL-1beta and TNF-alpha alone, DC matured in the additional presence of PGE2 show impaired IL-12 production and bias naive Th cell development toward the Th2. The ability of DC to produce IL-12 is also suppressed by IL-10, which in contrast to PGE2, inhibits their maturation. The differences in the ability to produce IL-12, established during the final DC maturation, are stable after the removal of modulatory factors. Importantly, fully mature DC become unsusceptible to PGE2 and IL-10. This indicates that the levels of IL-12 production in vivo, in mature DC interacting with Th cells within the lymph nodes, are mainly predetermined at the stage of immature DC in peripheral tissues. These data imply that the character of pathogen-induced local inflammatory reaction can "instruct" local DC to initiate Th1 or Th2-biased responses.

Adjuvants, Immunologic↗
PubMed1998

The role of antigen-presenting cells in the regulation of allergen-specific T cell responses.

Allergic reactions in atopic patients follow from a generalized enhanced polarization of Th cells, predominantly imposed by factors derived from antigen-presenting cells from a pathogen-stressed tissue; these sample information not only on antigen structures but also on the nature of the stress. Antigen-presenting cells of atopic individuals show aberrant characteristics which, through a highly interactive communication network, play an active role in aberrant Th-cell polarization. This generalized bias may follow from intrinsic abnormalities of antigen-presenting cells and also from a low degree of cross-regulation by micro-organisms.

Allergens↗
PubMed1998

High-level IL-12 production by human dendritic cells requires two signals.

IL-12 is a key cytokine in the development of Th1 responses. IL-12 production by antigen-presenting cells (APC) can be induced by the interaction between CD40 on the APC and CD40 ligand (CD40L) expressed on T cells after activation. Our previous study indicated that in dendritic cells (DC), the only APC that can activate naive T(h) cells efficiently, the mere CD40 engagement is insufficient to induce IL-12 production. The aim of the present study was to dissect the conditions for efficient IL-12 production by DC further. Using populations of naive and memory Th cells, recombinant CD40L, neutralizing and blocking antibodies, and by determining IFN-gamma production and CD40L expression levels, we here show that T cell-induced IL-12 production by DC results from the action of two signals, mediated by CD40L and IFN-gamma, and that the inability of naive T(h) cells to induce IL-12 production resides in their inability to produce IFN-(gamma). Other factors than CD40L and IFN-gamma can provide the required signals for IL-12 production by DC, as either factor could be replaced by lipopolysaccharide (LPS). The two-signal requirement proved unique for the production of IL-12, since either CD40 engagement or LPS was sufficient for the efficient production of tumor necrosis factor-alpha, IL-8 and the p40 subunit of IL-12, and may be considered as a safety mechanism for optimal control of potentially harmful T(h)1 responses.

CD40 Antigens↗
PubMed1997

Human dendritic cells require exogenous interleukin-12-inducing factors to direct the development of naive T-helper cells toward the Th1 phenotype.

Dendritic cells (DC) are important initiators of specific primary immune responses because they are the only APC that can efficiently activate naive Th cells. DC have the capacity to produce interleukin-12 (IL-12), a cytokine that plays a pivotal role in the development of Th1-mediated cellular immune responses. The present study focuses on the conditions under which human DC produce bioactive IL-12 p70 and, consequently, direct the development of naive T helper (Th) cells toward the Th1 phenotype. Bacteria or bacterial compounds such as Staphylococcus aureus Cowan strain I (SAC) or lipopolysaccharide (LPS) induced substantial IL-12 levels in DC, which could be further upregulated by interferon-gamma (IFN-gamma), whereas induction of IL-12 production via CD40 ligation required IFN-gamma as an obligatory, complementary signal. Also, activated naive Th cells were poor inducers of IL-12 production, unless exogenous IFN-gamma was present, whereas activated memory Th cells were effective inducers of IL-12 production and did not require exogenous IFN-gamma. Next, the cytokine profiles of matured Th cells that were primed by DC under different conditions were examined. DC promoted the development of naive Th cells into memory Th0 cells that produced both the type 1 cytokine IFN-gamma and the type 2 cytokine IL-4. In contrast, after activation with SAC, DC efficiently directed the development of Th1 cells through the release of IL-12. An APC-independent Th cell maturation model, using either recombinant IL-12 or supernatants of SAC-activated DC and neutralizing anti-IL-12 antibodies, confirmed that DC-derived IL-12 was the major Th1 skewing factor. Together, these data indicate that the contact between DC and naive Th cells during the initiation of specific immune responses does not result in the efficient induction of IL-12 production and that, consequently, exogenous IL-12-inducing factors are required to promote primary Th1-mediated cellular immune responses.

Cell Differentiation↗
PubMed1997

IL-12-deficient dendritic cells, generated in the presence of prostaglandin E2, promote type 2 cytokine production in maturing human naive T helper cells.

We studied to what extent the presence of an inflammatory mediator PGE2, during the development of dendritic cells (DC) affects their subsequent ability to induce Th1- and Th2-type cytokines in maturing naive Th cells. PGE2 (10(-9)-10(-6) M) did not alter the morphology or the expression of class II MHC and costimulatory molecules on DC obtained from monocytes in the presence of granulocyte-macrophage CSF and IL-4, although at concentrations above 10(-8) M, PGE2 prevented the acquisition of CD1a marker. Both control DC and DC maturing in the presence of PGE2 (PGE2-DC) were potent stimulators of naive Th cells. In contrast to control DC, which produced high amounts of IL-12 and trace amounts of IL-10, PGE2-DC produced no IL-12 and high amounts of IL-10 when stimulated in the absence of PGE2. This distinct cytokine profile of PGE2-DC was stable for at least 48 h of additional culture in the absence of PGE2. Control DC induced the development of Th0-like cells from superantigen-activated naive Th cells, whereas PGE2-DC promoted the development of Th cells that produced high amounts of IL-4 and IL-5. Experiments using IL-12-neutralizing Abs or rIL-12 indicated a crucial role of IL-12 deficiency in the induction of type 2 cytokine profiles. These findings suggest that elevated levels of PGE2 promote type 2 Th responses by stably impairing the ability of maturing DC to produce IL-12. Since type 2 Th responses are protective in several Th1-related autoimmune disorders, PGE2-DC may be considered for use in immunotherapy.

Cell Communication↗
PubMed1997

Dendritic cells, obtained from peripheral blood precursors in the presence of PGE2, promote Th2 responses.

In order to investigate the impact of an inflammatory mediator PGE2 on the functions of maturing DC we used an in vitro model of DC generation from peripheral blood monocytes. Addition of PGE2 (10(-9) M-10(-6) M) to the cultures performed in the presence of GM-CSF and IL-4 did not alter the morphology nor high levels of expression of class II MHC and co-stimulatory molecules on arising DC, although at concentrations above 10(-8) M, the acquisition of CD1a was selectively prevented. Control DC and the DC maturing in the presence of PGE2 (PGE2-DC) induced a similar proliferation of naive Th cells. Control DC produced high amounts of IL-12, and only trace amounts of IL-10, whereas PGE2-DC produced no IL-12 and high levels of IL-10, when stimulated after the removal of PGE2. The deficient IL-12 production by PGE2-DC was observed after stimulation both in the absence and in the presence of IFN gamma, and was not compensated during further 48 h culture in the absence of PGE2. Compared to control DC, PGE2-DC induced development of Th cells secreting elevated amounts of IL-4 and IL-5, from naive precursors. These data indicate that elevated tissue levels of PGE2 may promote type 2 Th responses by impairing the ability of locally maturing DC to produce IL-12. Since Th2 responses mediate protection in Th1-related autoimmune disorders, the use of PGE2-DC in immunotherapy of such disorders may be considered.

Cell Communication↗
PubMed1996

Lack of IL-12 signaling in human allergen-specific Th2 cells.

IL-12 is a powerful skewer of CD4+ T cell responses toward the Th1 phenotype by inducing IFN-gamma production in naive Th cells. In the present study we addressed the question of whether IL-12 can reverse established Th2 responses into Th1/Th0 responses by inducing IFN-gamma production in memory Th2 cells. To this aim, allergen-specific CD4+ T cell clones (TCC) were generated from the peripheral blood of three atopic patients, and their cytokine profiles were analyzed. The majority of these TCC exhibited a strongly polarized Th2 cytokine profile, and the production of IFN-gamma could not be induced by exogenous IL-12. Only those TCC with low IFN-gamma levels in the absence of IL-12 responded to IL-12 by additional enhancement of IFN-gamma production. The IL-12 nonresponsiveness of the Th2 clones was further evident by the total lack of IL-12-induced phosphorylation of STAT4 (signal transducer and activator of transcription-4), a transcription factor that is typically involved in IL-12 signaling. Consequently, IL-12 also failed to induce the DNA-binding activity of STAT4-containing complexes in the nuclei of these Th2 clones. All TCC expressed equal levels of the low-affinity IL-12R beta1 subunit. Our results indicate that human allergen-specific Th cells with strongly polarized Th2 cytokine profiles do not respond to IL-12 and, therefore, cannot be induced to produce IFN-gamma. The apparent high frequency of IL-12-nonresponsive Th cells within the allergen-specific populations in atopic patients predicts a limited skewing potential of IL-12 in the case of established Th2 responses, but only affecting newly recruited naive Th cells.

Allergens↗
PubMed1996

Reduced IL-12 production by monocytes upon ultraviolet-B irradiation selectively limits activation of T helper-1 cells.

The capacity of APC to stimulate the proliferation of human peripheral blood T cells decreases upon ultraviolet-B (UVB) irradiation. The aim of this study was to investigate whether all T cell subsets are equally sensitive to this reduced APC function. Established human Th1, Th2, and Th0 clones were stimulated with monocytes in a soluble CD3 mAb-mediated assay that is dependent on the presence of APC. Monocytes were exposed to low nonlethal doses of UVB radiation before coculture with T cells. UVB irradiation inhibited the capacity of monocytes to stimulate the proliferation and IFN-gamma production of Th1 cells in a dose-related fashion. In contrast, UVB-treated monocytes induced normal proliferation and IL-4 production in Th2 cells. Stimulation of Th0 cell proliferation by UVB-irradiated monocytes was normal, but a preferential suppression of IFN-gamma production was observed, thus leading to a more Th2-like cytokine response. The loss of Th1 proliferation upon stimulation with UVB-irradiated monocytes could be overcome by rIL-2; however, IFN-gamma production remained suppressed. IFN-gamma production could be completely restored by rIL-12, whereas the addition of IL-1 beta, TNF-alpha, or indomethacin had no such effect, nor did the addition of mAb to CD28, added to compensate for the reduced B7 expression of UVB-irradiated monocytes. Monocytes exposed to UVB radiation exhibited reduced expression of mRNA for the IL-1 2 subunits p35 and p40 and suppressed production of the IL-12 p70 protein. Our results thus indicate that UVB irradiation of APC selectively impairs Th1-like responses, a phenomenon caused by the UVB-induced suppression of monocyte IL-12 production.

Cytokines↗
PubMed1996

Accessory cell-derived IL-12 and prostaglandin E2 determine the IFN-gamma level of activated human CD4+ T cells.

IL-12 and PGE2 are two immunomodulators produced by accessory cells (AC) in response to various stimuli. IL- 12 enhances IFN-gamma production by activated CD4+ T cells, whereas PGE2 inhibits the secretion of this cytokine. Because these AC-derived factors exert clearly opposite modulatory effects on IFN-gamma production, we examined 1) the net-IFN-gamma production of CD4+ T cells, stimulated in the presence of both IL-12 and PGE2, 2) the susceptibility of activated CD4+ T cells in time by adding these modulators at different timepoints after stimulation, and 3) the relative contributions of AC-derived IL-12 and PGE2 to IFN-gamma levels by stimulating CD4+ T cells in the presence of LPS-activated monocytes and inhibitors of PGE2 or IL-12. Here, we demonstrate that 1) IL-12 and PGE2 do not abrogate the modulatory action of each other and that the net-IFN-gamma production is determined by their concentration ratio, 2) T cells become insensitive to PGE2, whereas susceptibility to IL-12 is retained after activation, and 3) activated monocytes potently modulate IFN-gamma levels of stimulated CD4+ T cells via release of IL-12 and PGE2. The relative contributions of these AC-derived factors shift in time, due to different production kinetics, from a dominant IL-12 effect to a mixed IL-12/PGE2 effect. Because the net IFN-gamma production of CD4+ T cells is largely determined by the ratio of IL-12 and PGE2 at the timepoint of T cell activation, an imbalance in the production of these immunomodulators may, therefore, lead to immunologic dysfunction.

Adjuvants, Immunologic↗
PubMed1996

CD30 expression does not discriminate between human Th1- and Th2-type T cells.

CD30 is a member of the TNF receptor superfamily that is commonly used as a marker for Hodgkin and Reed-Sternberg cells in Hodgkin's disease. More recently, it has been proposed that CD30 is preferentially up-regulated on Th2-type human T cells. We analyzed regulation of CD30 expression on both peripheral blood T cells and T cell clones. In short-term culture, CD30 expression could be induced on T cells by Ags that elicit Th2-type responses (Schistosoma haematobium, adult worm Ag, and Toxocaria canis, excretory/secretory Ag) and Th0-type responses (tetanus toxoid), as well as Th1-type responses (tuberculin purified protein derivative). Moreover, simultaneous measurement of membrane phenotype and cytokine production showed that CD30-expressing cells can produce IFN-gamma. Finally, within panels of randomly generated as well as Ag-specific T cell clones, CD30 expression was found on Th0-, Th2-, and Th1-type clones. We conclude that induction of CD30 on activated T cells is not related to differentiation in Th0-, Th1-, or Th2-type cells.

Cell Differentiation↗
PubMed1996

Regulation of bioactive IL-12 production in lipopolysaccharide-stimulated human monocytes is determined by the expression of the p35 subunit.

IL-12 enhances IFN-gamma production by NK cells and T lymphocytes and plays a pivotal role in the development of Th1 cells. Bioactive IL-12 (p70) is composed of two subunits, p35, which is only secreted as part of the p70 dimer, and p40, which can also be secreted by itself. The IL-12 subunits are encoded by two separate genes. Therefore, we studied to what extent the expression of each of the subunits contributes to the regulation of IL-12 production. We found that in LPS-stimulated whole blood and purified monocytes, p70 and p40 production are enhanced by IFN-gamma and inhibited by IL-10 and IL-4. However, IFN-gamma and IL-10 had stronger effects on p70 production than on p40 production, and IL-4 affected p40 production more strongly. Concomitantly, in all experimental conditions tested, p40 production greatly exceeded p70 production, suggesting that p35 expression was limiting. Analysis of p35 and p40 mRNA expression by PCR confirmed this notion. Resting purified monocytes expressed neither p40 nor p35 mRNA. The effects of IFN-gamma, IL-10, and IL-4 on the p35 mRNA expression in LPS-stimulated purified monocytes were similar to the effects of these cytokines on p70 protein production, and the p40 mRNA expression corresponded to p40 protein production. Our results imply that production of the two IL-12 subunits is differently regulated for each subunit, mainly at the level of mRNA expression, and that the level of bioactive IL-12 production in monocytes in response to LPS and cytokines is determined by the level of p35 expression.

Antibodies, Monoclonal↗