Biomedical subjects

R Debets

Publications and source records attributed to R Debets.

At least 19 recordsLinked to original sources

PubMed2006

Phoenix-ampho outperforms PG13 as retroviral packaging cells to transduce human T cells with tumor-specific receptors: implications for clinical immunogene therapy of cancer.

We have designed a transgene that encodes a scFv(G250) chimeric receptor, which is specific for carboxyanhydrase IX (G250-ligand, G250L), a molecule overexpressed by renal cell cancer (RCC). Retroviral transduction of this transgene into primary human T lymphocytes confers these cells with specific functional responses towards G250L-positive RCC cells. In preparation of a clinical phase (I/II) study in RCC patients, we set up a protocol for gene transduction and expansion of primary human T cells. For this purpose, we directly compared two packaging cell lines, that is, the GALV-pseudotyped MLV producing cell line PG13, and the MLV-A-producing cell line Phi-NX-Ampho (a.k.a. Phoenix-A). We generated and characterized stable scFv(G250)-positive clones of both PG13 and Phoenix cells and optimized the retrovirus production conditions. Transductions of primary human T cells yielded 30-60% scFv(G250)+ T cells using PG13-derived retrovirus versus up to 90% scFv(G250)+ T cells using Phoenix-derived retrovirus. The median number of transgene integrations per scFv(G250)+ T cell differed only 1.5-fold as determined by real-time PCR (mean number of integrations per T cell 2.6 and 3.7 for PG13 and Phoenix-based transductions, respectively). In addition, T cells transduced with Phoenix-derived retrovirus showed, on a per cell basis, 10-30% higher levels of scFv(G250)-mediated TNFalpha production and cytolysis of G250L+ RCC cells than T cells transduced with PG13-derived retrovirus. The improved functional transduction efficiency together with a limited increase in the number of integrations per recipient cell, made us select Phoenix clone 58 for our clinical immunogene therapy study.

Antigens, Neoplasm↗

PubMed2005

Redirecting human CD4+ T lymphocytes to the MHC class I-restricted melanoma antigen MAGE-A1 by TCR alphabeta gene transfer requires CD8alpha.

Adoptive immunotherapy involving the transfer of autologous tumor or virus-reactive T lymphocytes has demonstrated its effectiveness in the eradication of cancer and virally infected cells. Clinical trails and in vitro studies have focused on CD8+ cytotoxic T-cell receptor (TCR) alphabeta lymphocytes since these cells directly kill virally infected- and tumor cells after antigen-specific recognition via their TCR alphabeta. However, increasing evidence suggests that induction of sustained immunity against cancer and viral infections depends on the presence of tumor- or virus-specific CD4+ T lymphocytes, which are restricted by MHC class II. Here, we show that these MHC class II-restricted CD4+ T lymphocytes can efficiently be redirected to MHC class I-restricted tumor cells by retroviral introduction of an HLA-A1/MAGE-A1-specific chimeric two-chain TCR ValphaCalphazeta/VbetaCbetazeta (tcTCR/zeta). However, TCR-transduced CD4+ T lymphocytes were only able to specifically bind to HLA-A1/MAGE-A1 complexes and respond to HLA-A1+/MAGE-A1+ melanoma cells when the CD8alpha gene was cointroduced. These CD4+/CD8alpha+/TCR(POS) T lymphocytes produce IFN-gamma, TNFalpha and IL-2 when specifically stimulated via the introduced TCR with immobilized HLA-A1/MAGE-A1 complexes or HLA-A1+/MAGE-A1+ melanoma cells. Furthermore, introduction of the CD8alpha gene into TCR(POS) T lymphocytes rendered these T lymphocytes cytotoxic for HLA-A1+/MAGE-A1+ melanoma cells. These results demonstrate that human CD4+ T lymphocytes when genetically grafted with an HLA-A1/MAGE-A1-specific TCR and CD8alpha are induced to kill and produce cytokines upon specific interaction with the relevant melanoma cells. Hence, CD4+ T lymphocytes, in addition to CD8+ T lymphocytes, may be critical effector cells for adoptive immuno-gene therapy to generate a sustained tumor-specific immune response in cancer patients.

Antigens, Neoplasm↗

PubMed2005

Parallel detection of transduced T lymphocytes after immunogene therapy of renal cell cancer by flow cytometry and real-time polymerase chain reaction: implications for loss of transgene expression.

We have started a phase I/II immunogene therapy study of metastatic renal cell cancer (RCC), using autologous T lymphocytes transduced ex vivo with a gene encoding a single-chain receptor based on the monoclonal antibody (mAb) G250 [scFv(G250)]. G250 recognizes carbonic anhydrase IX, which is overexpressed by RCC cells. We have developed and validated flow cytometric and real-time polymerase chain reaction (PCR) assays to quantitatively detect transduced T cells in patient blood. The flow assay was based on staining with the anti-G250 idiotype mAb NuH82 and showed a sensitivity of 0.06% scFv(G250)(1) cells within CD3(1) T cells. The real-time PCR method showed a sensitivity of 14 copies of scFv(G250) DNA per 100 ng of total DNA, which enabled detection of 0.008% scFv(G250)(1) T cells within leukocytes. Both assays were further validated for their specificity and reproducibility. When applied to blood samples from three RCC patients treated with intravenous infusions of scFv(G250)(1) T cells, the kinetics of scFv(G250)(1) T cell counts as detected by flow cytometry were similar to those detected by real-time PCR, although PCR allowed detection of transduced T cells over a longer period of time (i.e., for patient 3, 7 versus 32 days, respectively). Interestingly, follow-up studies of patient 3 demonstrated that the number of circulating scFv(G250)(1) T cells remained fairly constant during the first 7 days posttreatment, whereas the number of gene copies increased during the same period of time. These results suggest loss of scFv(G250) membrane expression on adoptive transfer, which would have important implications for the antitumor efficacy of this form of immunogene therapy.

Antibodies, Monoclonal↗

PubMed2001

A critical role for interleukin 18 in primary and memory effector responses to Listeria monocytogenes that extends beyond its effects on Interferon gamma production.

The stimulation of interferon (IFN)-gamma by interleukin (IL)-12 has been shown to provide protection from intracellular pathogens such as Listeria monocytogenes. Tumor necrosis factor (TNF) is also a major player in the resolution of Listeria infections and is suggested to have more global effects than can be explained by the induction of IFN-gamma alone. Since IL-18 synergizes with IL-12 to induce IFN-gamma production by natural killer and T helper (Th)1 cells, we determined its role in responses to Listeria. IL-18 appeared to be even more potent than either IL-12 or IFN-gamma for protection against this pathogen and IL-18 enhanced bacterial clearance in the complete absence of IFN-gamma. Indeed IL-18 was comparable to TNF in its ability to resolve the infection and showed a lowered protective capacity in the absence of TNF. Moreover, IL-18 induced macrophages to secrete both TNF and nitric oxide after a Listeria infection. IL-18 was also essential for optimal IFN-gamma production by antigen-specific T cells. Therefore, IL-18 operates via its effects on both the innate immune response, including macrophages, as well as on Th1 cells, to protect against Listeria.

Animals↗

PubMed2001

Two novel IL-1 family members, IL-1 delta and IL-1 epsilon, function as an antagonist and agonist of NF-kappa B activation through the orphan IL-1 receptor-related protein 2.

IL-1 is of utmost importance in the host response to immunological challenges. We identified and functionally characterized two novel IL-1 ligands termed IL-1delta and IL-1epsilon. Northern blot analyses show that these IL-1s are highly abundant in embryonic tissue and tissues containing epithelial cells (i.e., skin, lung, and stomach). In extension, quantitative real-time PCR revealed that of human skin-derived cells, only keratinocytes but not fibroblasts, endothelial cells, or melanocytes express IL-1delta and epsilon. Levels of keratinocyte IL-1delta are approximately 10-fold higher than those of IL-1epsilon. In vitro stimulation of keratinocytes with IL-1beta/TNF-alpha significantly up-regulates the expression of IL-1epsilon mRNA, and to a lesser extent of IL-1delta mRNA. In NF-kappaB-luciferase reporter assays, we demonstrated that IL-1delta and epsilon proteins do not initiate a functional response via classical IL-1R pairs, which confer responsiveness to IL-1alpha and beta or IL-18. However, IL-1epsilon activates NF-kappaB through the orphan IL-1R-related protein 2 (IL-1Rrp2), whereas IL-1delta, which shows striking homology to IL-1 receptor antagonist, specifically and potently inhibits this IL-1epsilon response. In lesional psoriasis skin, characterized by chronic cutaneous inflammation, the mRNA expression of both IL-1 ligands as well as IL-1Rrp2 are increased relative to normal healthy skin. In total, IL-1delta and epsilon and IL-1Rrp2 may constitute an independent signaling system, analogous to IL-1alphabeta/receptor agonist and IL-1R1, that is present in epithelial barriers of our body and takes part in local inflammatory responses.

Amino Acid Sequence↗

PubMed2001

A phage display selected fab fragment with MHC class I-restricted specificity for MAGE-A1 allows for retargeting of primary human T lymphocytes.

The clinical benefit of adoptive transfer of MHC-restricted cytotoxic T lymphocytes(CTL) for the treatment of cancer is hampered by the low success rate to generate antitumor CTLs. To bypass the need for tumor-specific CTL, we developed a strategy that allows for grafting of human T lymphocytes with MHC-restricted antigen specificity using in vitro selected human Fab fragments fused to the Fc(epsilon)RI-gamma signaling molecule. Retroviral introduction of a Fab-based chimeric receptor specific for MAGE-A1/HLA-A1 into primary human T lymphocytes resulted in binding of relevant peptide/MHC complexes. Transduced T lymphocytes responded to native MAGE-A1/HLA-A1POS target cells by specific cytokine production and cytolysis. Therefore, peptide/MHC-specific Fab fragments represent new alternatives to TCR to confer human T lymphocytes with tumor specificity, which provides a promising rationale for developing immunogene therapies.

Antigens, Neoplasm↗

PubMed2001

Cytokine networking in lungs of immunocompetent mice in response to inhaled Aspergillus fumigatus.

Cytokine networking in the lung in response to inhaled Aspergillus fumigatus was assessed using a murine model of primary pulmonary aspergillosis in immunocompetent Crl:CF-1 mice. Inhalation of virulent A. fumigatus (6 x 10(6) CFU) resulted in the induction of interleukin 18 (IL-18), tumor necrosis factor alpha (TNF-alpha), IL-12, and gamma interferon (IFN-gamma) protein in bronchoalveolar lavage fluid and/or lung tissue. Induction of immunoreactive IL-18 preceded induction of TNF-alpha protein, which preceded induction of immunoreactive IL-12 and IFN-gamma. Real-time reverse transcriptase (RT) PCR analysis of infected lung tissue demonstrated that induction of IL-18 protein also preceded induction of pulmonary TNF-alpha, IL-12, and IFN-gamma mRNAs. Mice were subsequently treated with cytokine-specific neutralizing monoclonal antibodies (MAbs) to the IL-18 receptor (anti-IL-18R MAb), TNF-alpha (anti-TNF-alpha MAb), IL-12 (anti-IL-12 MAb), and/or IFN-gamma (anti-IFN-gamma MAb), and effects on intrapulmonary cytokine activity and growth of A. fumigatus were assessed in infected lung homogenates. Simultaneous neutralization of IL-12 and IL-18 resulted in decreased levels of immunoreactive TNF-alpha, while neutralization of IL-18, TNF-alpha, or IL-12 alone or of IL-18 and IL-12 together resulted in decreased levels of immunoreactive IFN-gamma. Simultaneous neutralization of IL-12 and IL-18 or neutralization of TNF-alpha alone or in combination with IL-12, IL-18, or IFN-gamma also resulted in a significant increase in A. fumigatus CFU in lung tissue. Taken together, these results demonstrate that endogenous IL-18, IL-12, and TNF-alpha, through their modulatory effects on both intrapulmonary cytokine activity and growth of A. fumigatus, play key roles in host defense against primary pulmonary aspergillosis.

Adjuvants, Immunologic↗

PubMed2000

IL-18 receptors, their role in ligand binding and function: anti-IL-1RAcPL antibody, a potent antagonist of IL-18.

IL-18 is critical in eliciting IFN-gamma production from Th1 cells both in vitro and in vivo. Th1 cells have been implicated in the pathogenesis of autoimmune disorders, making antagonists of IL-18 promising therapeutics. However, specificity and binding characteristics of IL-18R components have only been superficially explored. In this study, we show that IL-1R related protein 1 (IL-1Rrp1) and IL-1R accessory protein-like (IL-1RAcPL) confer responsiveness to IL-18 in a highly specific (no response to other IL-1 ligands) and unique manner (no functional pairing with other IL-1Rs and IL-1R-like molecules). Cotransfection with both receptor components resulted in expression of both low and high affinity binding sites for IL-18 (K:(d) of 11 and 0.4 nM, respectively). We prepared anti-IL-1RAcPL mAb TC30-28E3, which, in contrast to soluble R proteins, effectively inhibited the IL-18-induced activation of NF-kappaB. Quantitative PCR showed that Th1 but not Th2 cells are unique in that they coexpress IL-1Rrp1 and IL-1RAcPL. mAb TC30-28E3 inhibited IL-18-induced production of IFN-gamma by Th1 cells, being at least 10-fold more potent than anti-IL-18 ligand mAb. This study shows that IL-1RAcPL is highly specific to IL-18, is required for high affinity binding of IL-18, and that the anti-IL-1RAcPL mAb TC30-28E3 potently antagonizes IL-18 responses in vitro, providing a rationale for the use of anti-IL-1RAcPL Abs to inhibit Th1-mediated inflammatory pathologies.

Animals↗

PubMed2000

Computational identification, cloning, and characterization of IL-1R9, a novel interleukin-1 receptor-like gene encoded over an unusually large interval of human chromosome Xq22.2-q22.3.

The Interleukin-1 receptor (IL-1R) and Toll signaling pathways share the evolutionarily conserved Toll homology domain (THD), which is a critical component in the signaling cascade of the host defense responses to infection and inflammation. Our initial genomic database searches uncovered a novel THD signature sequence between DNA markers DXS87 and DXS366. The feasibility of subsequently applying a coordinated computational approach, including various exon-finding programs, homology-based searches, and receptor profile searches, in revealing the exons encoding this novel IL-1R family member is described. IL-1R9 shows restricted expression in fetal brain and is highly homologous to IL1RAPL (A. Carrie et al., 1999 Nat. Genet. 23: 25-31), which is reportedly involved in nonsyndromic X-linked mental retardation. These genes are scattered over separate genomic intervals in excess of 1.0 Mb and encode receptors with extended C-terminal tails. In our functional NF-kappaB reporter assays, IL1RAPL, IL-1R9, or versions lacking the extended C-terminal sequences failed in responding either to IL-1 directly or to IL-18 when various permutations of IL-18R ectodomain chimeras were fused to their cytoplasmic domains. Evolutionary sequence analyses reinforce our conclusion that these novel orphan receptors probably form a functionally distinct subset of the IL-1R superfamily.

Amino Acid Motifs↗

PubMed2000

Immunomodulatory role of endogenous interleukin-18 in gamma interferon-mediated resolution of replicative Legionella pneumophila lung infection.

The in vivo role of endogenous interleukin-18 (IL-18) in modulating gamma interferon (IFN-gamma)-mediated resolution of replicative Legionella pneumophila lung infection was assessed using a murine model of Legionnaires' disease. Intratracheal inoculation of A/J mice with virulent bacteria (10(6) L. pneumophila organisms per mouse) resulted in induction of IL-18 protein in bronchoalveolar lavage fluid (BALF) and intrapulmonary expression of IL-18 mRNA. Real-time quantitative RT-PCR analysis of infected lung tissue demonstrated that induction of IL-18 in BALF preceded induction of IL-12 and IFN-gamma mRNAs in the lung. Blocking intrapulmonary IL-18 activity by administration of a monoclonal antibody (MAb) to the IL-18 receptor (anti-IL-18R MAb) prior to L. pneumophila infection inhibited induction of intrapulmonary IFN-gamma production but did not significantly alter resolution of replicative L. pneumophila lung infection. In contrast, blocking endogenous IL-12 activity by administration of anti-IL-12 MAb) alone or in combination with anti-IL-18R MAb inhibited induction of intrapulmonary IFN-gamma and resulted in enhanced intrapulmonary growth of the bacteria within 5 days postinfection. Taken together, these results demonstrate that IL-18 plays a key role in modulating induction of IFN-gamma in the lung in response to L. pneumophila and that together with IL-12, IL-18 regulates intrapulmonary growth of the bacteria.

Animals↗

PubMed2000

Human keratinocytes are major producers of IL-18: predominant expression of the unprocessed form.

The cytokine network in the skin is a tightly regulated system in which IL-1 isoforms, as well as their receptors and antagonists have a central role. The recently discovered IL-1 isoform IL-18 (also known as interferon gamma-inducing factor (IGIF) or IL-1gamma), promotes IFN-gamma expression by T cells in concert with IL-12. Because IFN-gamma plays an important role in many inflammatory skin diseases by facilitating the development of Th1 cells, it is important to elucidate the role of mediators which regulate the production of this cytokine. We demonstrate that human keratinocytes constitutively express IL-18 at the mRNA as well as at the protein level. The protein was mainly expressed intracellularly in the 24 kD unprocessed pro-form, but was also secreted. Histochemistry revealed a diffuse staining of IL-18 in the epidermis of normal skin, which is in line with our in vitro data. Furthermore, we show that the level of IL-18 expressed in freshly isolated normal human epidermal cells, whether or not containing HLA-DR+ cells, significantly exceeded the expression levels of other cell types such as monocytes and bronchial epithelial cells. Finally, our results show that stimulation of the keratinocyte cell line HaCaT with PMA LPS or IL-1beta, does not significantly affect intracellular or released (pro) IL-18 levels. These experiments show for the first time that human keratinocytes relative to monocytes, PBMC or leukocytes produce a considerably larger amount of pro-IL-18, which is also readily released. High constitutive levels of IL-18 may contribute to the skewing towards a Th1-like environment, which is apparent in many human inflammatory skin diseases.

Cell Line↗

PubMed1999

IL-1 beta and IFN-gamma induce the regenerative epidermal phenotype of psoriasis in the transwell skin organ culture system. IFN-gamma up-regulates the expression of keratin 17 and keratinocyte transglutaminase via endogenous IL-1 production.

Skin biopsies from healthy human skin and non-lesional skin from patients with psoriasis were cultured for 24 h and stimulated with interleukin-1 beta (IL-1 beta) and interferon-gamma (IFN-gamma) in a skin organ culture model and the induction of the psoriasiform regenerative epidermal phenotype was analysed using immunostaining. In the presence of IL-1 beta, the psoriasiform regenerative epidermal phenotype was clearly induced. This involved strong up-regulation of the expression of keratin 16, keratin 17, and keratinocyte transglutaminase (TGk) in the suprabasal layers, strong up-regulation and a shift of the expression of keratin 5 and integrin beta 1 from the basal to suprabasal keratinocytes, and induction of the expression of ICAM-1 and HLA-DR on basal keratinocytes. The effects of IL-1 beta in the organ cultures of normal skin could be completely neutralized by anti-IL-1 polyclonal antibodies. The effects of IFN-gamma in healthy and non-lesional psoriatic skin were qualitatively similar to those of IL-1 beta. The IFN-gamma-induced epidermal expression of keratin 17 and TGk could be completely blocked by culturing the biopsies in the presence of IL-1ra or anti-IL-1 antibodies, while the induction of HLA-DR and ICAM-1 was not inhibited. The induction of the psoriasiform regenerative epidermal phenotype by IFN-gamma is partially mediated via endogenous epidermal IL-1.

Adult↗

PubMed1998

Interleukin 4 receptors on human bronchial epithelial cells. An in vivo and in vitro analysis of expression and function.

Asthma is considered a Th2-like disease, characterized by locally increased levels of interleukin (IL) 4. The bronchial epithelium plays an important role in the initiation and perpetuation of inflammatory reactions within the airways. However, little is known about the presence of IL-4 receptors on human bronchial epithelial cells, or the effects of IL-4 on these cells. In this report, definitive evidence of IL-4 receptor expression on human bronchial epithelial cells using several methods is presented. IL-4 receptor expression on human bronchial epithelial cells in vivo was demonstrated using in situ hybridization and immunohistochemistry. No difference in IL-4 receptor protein expression was observed between bronchial biopsies of healthy subjects compared to allergic asthmatics. Cultured human bronchial epithelial cells also expressed IL-4 receptor mRNA and protein (as determined by RT-PCR analysis and flow cytometry, respectively). IL-4 receptor protein expression by bronchial epithelial cells could be increased by stimulation with PMA+calcium ionophore, whereas IL-1beta and IL-6 decreased IL-4 receptor expression. A cyclic AMP analogue and IL-4 had no effect. Finally, it is shown that the IL-4 receptor is functionally active as IL-4 stimulates the release of IL-8, monocyte chemoattractant protein 1, and particularly IL-1 receptor antagonist by human bronchial epithelial cells. It is concluded that human bronchial epithelial cells express IL-4 receptors both in vivo and in vitro. Stimulation of human bronchial epithelial cells by IL-4 may result in the release of both pro- and anti-inflammatory mediators known to be upregulated in asthmatic airways.

Asthma↗

PubMed1997

The IL-1 system in psoriatic skin: IL-1 antagonist sphere of influence in lesional psoriatic epidermis.

The epidermal expression of IL-1 in psoriasis is clearly altered, but data are still incomplete and poorly understood. To thoroughly study the IL-1 system in psoriasis, we semiquantitatively analyzed the expression of all currently characterized IL-1 isoforms and their receptors in parallel in both lesional (PP) and nonlesional psoriatic (PN) epidermis. Immunostaining of skin sections showed that IL-1alpha, located in the basal keratinocytes of normal control (NN) and PN epidermis, was significantly decreased to negligible levels in PP epidermis. IL-1 receptor antagonist (IL-1ra) and IL-1R type II (IL-1RII) were both significantly overexpressed in mutually exclusive compartments of PP epidermis, the suprabasal and basal compartment, respectively. A significant inverse correlation was found between the expressions of IL-1alpha and these two IL-1 antagonists, which may be inherent to the accelerated terminal differentiation of the psoriatic keratinocyte. In situ hybridization of IL-1(R) mRNAs confirmed the staining results. Levels of IL-1ra mRNA, however, were not increased in PP epidermis, suggesting that the overexpression of IL-1ra protein may be explained at the level of translation. The more sensitive PCR demonstrated a clearly increased expression of IL-1beta mRNA in PP epidermal cells (EC), which may be related to the inflammatory response in psoriasis. IL-1RI mRNA was clearly present in both PP and NN EC. The mRNA levels of the secreted IL-1ra isoform, but not intracellular IL-1raI and II, and IL-1RII were elevated in PP EC and paralleled those of IL-1beta. In summary, this study provides a defined phenotype of the complete epidermal IL-1 system in psoriasis; it shows that the expressions of IL-1(R) isoforms are coordinately altered, resulting in a predominance of IL-1 antagonists, which may represent a negative feedback response to IL-1 agonists, leading to a decreased IL-1 responsiveness.

Epidermis↗

PubMed1997

Heterogeneity of pituitary folliculo-stellate cells: implications for interleukin-6 production and accessory function in vitro.

The population of folliculo-stellate (FS) cells of the rat anterior pituitary has been shown to be ultrastructurally and immunohistochemically heterogeneous. Based on the overlap of ultrastructural characteristics, the localization in the anterior pituitary and the co-expression within the same cel of the S-100 protein (a marker for FS cells) and MHC-class II determinants (an immune marker) we concluded that a partial overlap exists between the population of FS cells and the monocyte-derived dendritic cells (DC). In this report we describe that interleukin-6 (IL-6) immunoreactivity was found in situ in stellate cells of the rat, mouse and human anterior pituitary at a very low density (< 1% of the cells); the topography was reminiscent of the distribution of FS cells. In the present study we also analyse three different pituitary cell separation methods, in order to study the functional heterogeneity of the FS cells in vitro, and to verify whether functionally distinct subpopulations exist within the FS cell group. Production of bioactive IL-6 was measured in conditioned media of rat anterior pituitary cells separated by (i) bovine serum albumin (BSA) gradient sedimentation at 1 g, (ii) Nycodenz gradient and (iii) a magnetic cell separation (MACS) technique. Production of bioactive IL-6 by cell cultures of 1 to 4 days was correlated with the proportional number of S100 immunoreactive and S100 producing cells, but was not correlated with the proportional number of MHC-class II expressing (OX6-positive) dendritic cells (DC). The distribution pattern of OX6-positive DC was found to partly overlap with the distribution pattern of S100-positive cells in the BSA gradient. Co-sedimentation of S100-positive FS cells and MHC-class II-expressing DC was not restricted to the top fractions of the BSA gradient, but was also found in the low density Nycodenz fraction. MACS separation of the rat anterior pituitary cells resulted into a population enriched in OX6 and OX62 positive DC and a population devoid of such cells, while S100+ cells were equally divided into these two subpopulations. Although there was a significantly decreased production of IL-6 as compared to that of an original pituitary cell population, both MACS separated populations were equal in IL-6 production. The diminution in IL-6 production in both populations may be the result of an impediment of paracrine communication due to the MACS separation into these two populations. Our data also show that a subpopulation of FS cells was capable of stimulating T cell proliferation in vitro. Concomitantly with the distribution pattern of S100- and OX6-immunoreactive cells in the BSA and Nycodenz gradient fractions, we found a similar pattern of stimulation of T cell proliferation. Unlike the IL-6 production pattern, the T cell stimulating capacity was present in the MHC-class II-enriched cell population but absent in the MHC-class II-depleted cell population. These findings-together with earlier in situ histochemical data-suggest that there is an OX6+ S100- subpopulation of FS cells in the anterior pituitary that in itself is capable of stimulating T cell proliferation in vitro, and acts as lymphoid DC. There is also an S100+ OX6- population that is unable to stimulate T cell proliferation in vitro. Both populations are able to produce IL-6, but probably need stimuli from other subpopulations of pituitary cells (or exogeneous stimuli) to produce maximal amounts of IL-6.

Animals↗

PubMed1997

Inflammatory properties of peptidoglycan are decreased after degradation by human N-acetylmuramyl-L-alanine amidase.

Human N-acetylmuramyl-L-alanine amidase (EC 3.5.1.28) degrades peptidoglycan, a major component of bacterial cell walls with potent pro-inflammatory cytokine-inducing properties. We postulate that degradation of peptidoglycan by N-acetylmuramyl-L-alanine amidase is important for the inactivation of inflammatory peptidoglycan products in human tissues. The inflammatory activities of peptidoglycan digested by lysozyme and/or amidase were investigated using two properties of peptidoglycan: its capacity to induce the release of the inflammatory cytokines IL-1, IL-6 and TNF-alpha in vivo and in vitro and its capacity to induce arthritis in Lewis rats. The results show that after subsequent treatment with both lysozyme and amidase, the peptidoglycan products were unable to induce arthritis in Lewis rats. The production of pro-inflammatory cytokines in mice after intravenous injection of cell wall fragments was lower after in vitro degradation of the cell wall fragments by amidase. These in vivo results were confirmed with whole blood assays in which the production of pro-inflammatory cytokines was measured after stimulation with lysozyme- and amidase-treated peptidoglycan. The results show that human N-acetylmuramyl-L-alanine amidase possesses an enzymatic activity capable of inactivating inflammatory peptidoglycan by lowering its cytokine-inducing properties.

Animals↗

PubMed1996

Expression of cytokines and their receptors by psoriatic fibroblast. I. Altered IL-6 synthesis.

Interleukin(IL-)6 is overproduced in psoriatic lesions. We investigated the contribution of dermal fibroblasts to the local IL-6 production. Fibroblasts (passageno. 2 to 6) derived from lesional psoriatic (PP) and normal human (NN) skin were used to analyse the secretion of IL-6, and the related cytokines IL-1, IL-8 and TNF-alpha, and the expression of their corresponding mRNA by bioassay, ELISA and Northern hybridization, respectively. PP fibroblasts cultured under serum-free conditions produced increased amounts of bioactive IL-6 when compared to NN fibroblasts. Differences were partially restored in the presence of growth factors or serum. The serum-induced IL-6 production reached a maximum within 24 h after seeding and remained unchanged in PP fibroblasts, whereas comparable amounts of IL-6 were produced only 6 days later in NN fibroblasts. There was a clear expression of IL-6 mRNA in both types of fibroblasts under serum-free conditions. Unexpectedly, fetal calf serum, inactivated fetal calf serum as well as human serum completely inhibited the expression of IL-6 mRNA in all the PP fibroblast cultures investigated. NN fibroblasts were clearly less sensitive to this inhibiting effect of serum. Furthermore, medium supplemented with serum-free component or calcium also repressed IL-6 mRNA expression in PP fibroblasts in contrast to NN fibroblasts. Cycloheximide fully restored the repressing effect of serum indicating that serum induced a labile repressor protein. PP and NN fibroblasts produced negligible amounts of IL-1 and TNF-alpha, but the production of IL-8, however, was comparable to that of IL-6. Our results show a differently regulated IL-6 synthesis in PP fibroblasts in vitro, suggesting an active contribution of dermal fibroblasts to the local IL-6 production in psoriasis.

Blotting, Northern↗

PubMed1996

Expression of cytokines and their receptors by psoriatic fibroblasts. II. decreased TNF receptor expression.

Psoriatic fibroblasts produce enhanced amounts of IL-6 in vitro. This state of activation may reflect an altered expression of cytokine receptors, involved in auto/paracrine induction of IL-6. Cultures of dermal fibroblasts derived from lesional psoriatic (PP) and normal control (NN) skin were therefore analysed for their ability to bind biotinylated recombinant human cytokines using flow cytometry. PP and NN fibroblasts bound negligible amounts of IL-1 alpha and IL-1 beta, but clearly bound IL-4, IL-6 and TNF-alpha. Serum upregulated the number of NN fibroblasts which bound TNF-alpha, and to a lesser extent IL-6, but not the number of binding sites per cell. In contrast, this upregulation was significantly less in PP fibroblasts. This was not a result of differences in growth characteristics, receptor occupancy or an inability of stimulated PP fibroblasts to bind TNF-alpha. Immunocytochemistry of cells grown on slides showed that the TNF receptor type 1 (TNFR1, p55) was the predominant receptor in NN fibroblasts and was localized to the nucleus of cytoplasma. The expression of TNFR1 was clearly decreased in PP fibroblasts, which confirmed the binding studies. A slow and serum-induced shedding of TNFR1 was observed, but not of the TNFR2 (p75), in both types of fibroblasts. Confluent multi-passaged PP fibroblasts display both a decreased TNFR expression as well as an enhanced IL-6 production under serum conditions. These inherent abnormalities of PP fibroblasts imply the involvement of dermal fibroblasts in the maintenance of chronic inflammation in psoriasis.

Calcium↗