Characterization of tumour necrosis factor-alpha genetic variants and mRNA expression in patients with severe sepsis.

Tumour necrosis factor-alpha (TNFalpha) has been implicated in the pathogenicity of severe sepsis by both genetic association studies and animal models. Conflicting functional data have emerged in relation to genetic variants and TNFalpha protein production. Therefore, we assessed the functionality of TNFalpha genetic variants in terms of mRNA production and their potential influence on outcome in the setting of severe sepsis. Sixty-two Irish Caucasian patients presenting with severe sepsis were recruited and TNFalpha mRNA and protein levels were quantified. Patient DNA was analysed for the presence of common promoter polymorphisms and haplotypes were inferred. An A allele at position -863 was associated with more TNFalpha mRNA on day 1 compared to C homozygotes (P = 0.037). There was a trend for G homozygotes at position -308 to produce more TNFalpha mRNA on day 1 than those carrying an A allele (P = 0.059). The presence of an A allele at -863 was associated with greater levels of TNFalpha mRNA in comparison with patients carrying the A allele at -308 on day 1 (P = 0.02). Patients homozygous for the A allele at position -308 had a higher mortality than those carrying the G allele (P = 0.01). Our data are consistent with recent reports suggesting that a deficient proinflammatory response may be harmful in human sepsis. This deficient inflammatory response may be mediated in part by polymorphisms in the TNFalpha promoter.

A rapid test of alloreactivity based on interleukin-2 mRNA expression might identify liver transplant recipients with donor-specific hyporesponsiveness.

BACKGROUND: Immunosuppression withdrawal is feasible in some liver transplant (OLT) recipients but may lead to severe rejection in others, underlying the need for reliable biomarkers to identify patients with tolerant profile in whose weaning/withdrawal could be safely proposed. We evaluated the value of real-time polymerase chain reaction (PCR)-based measurement of interleukin (IL)-2 mRNA in mixed lymphocyte reaction (MLR) to monitor in vitro anti-donor reactivity in OLT patients. METHODS: MLR were performed in three patients undergoing living donor OLT using a tolerogenic protocol including donor stem cells. IL-2 mRNA production in MLR was measured by PCR at several intervals after OLT. RESULTS: In the early posttransplant period, three patients presented with global immunodeficiency, as indicated by low IL-2 mRNA production against both donor and third-party antigens. In the two patients who has immunosuppression successfully withdrawn, donor-specific hyporesponsiveness was observed thereafter: IL-2 mRNA production against donor cells remained low, while IL-2 mRNA production against a third-party antigen-presenting cells progressively recovered. No such modulation of the anti-donor response was observed in the patient in whom withdrawal led to rapid rejection. CONCLUSION: Measurement of IL-2 mRNA production in MLR might prefer a tool to monitor anti-donor reactivity after OLT for decisions to minimize or withdraw immunosuppression in patients displaying donor-specific hyporesponsiveness.

[The immunology-hematology-transfusion department]. / Le Service d'Immunologie-Hématologie-Transfusion.

New immunotherapies derived from biotechnology offer fascinating perspectives in different fields of medicine including anti-infectious vaccines, cancer, organ transplantation and autoimmune diseases. In this paper, we illustrate how the Department of Immunology can contribute to the development of these new treatments within a academic hospital such as the Erasme Hospital at the Université Libre de Bruxelles.

Examination of Escherichia coli from poultry for selected adhesin genes important in disease caused by mammalian pathogenic E. coli.

A collection of 1601 extraintestinal and intestinal Escherichia coli isolated from chickens, turkeys and ducks, in Belgium, France and Spain, was hybridised with gene probes specific for fimbrial and afimbrial adhesins (F17, F18, S , Bfp, Afa, Cs31A, Intimin , Aida-1) of intestinal, urinary and invasive E. coli of mammals and with a probe specific for the P (Pap/Prs) fimbrial adhesin of urinary and invasive E. coli of mammals and birds. Three hundred and eighty-three strains (23.9%) were P-positive, 76 strains (4.8%) were Afa-positive, 75 strains (4.7%) were F17-positive, 67 strains (4.2%) were S-positive, 23 (1.4%) were Intimin-positive, and all were F18-, Cs31A-, Aida1- and Bfp-negative. The 75 F17-positive strains harboured different major subunit A-encoding gene variants, but the f17Ac variant was the most frequent (52 strains, 69.3%) and seven strains (9.3%) were not typeable. The f17G gene variant coding for the GII adhesin was the most frequent (56 strains, 75.0%), whereas the f17GI gene variant was present in four strains (5%) and 15 strains (20.0%) were not typeable. All Afa-positive strains harboured the afa-8 variant. The 23 Intimin-positive E. coli tested positive for the beta-variant (16 strains; 69.6%) or for the gamma-variant (seven strains; 30.4%) of the eae gene. Chicken and turkey E. coli were more frequently probe-positive (43.6 and 43.1%, respectively) than duck E. coli (31.5%) and extraintestinal E. coli were also more frequently probe-positive (48.4%) than intestinal strains (18.5%). Different combinations of probe positive hybridisation results were observed in 72 of the 540 probe-positive E. coli (13.3%). The most frequent combinations were between AfaE-8 and F17 probes (47 strains; 8.7%) and between P and S probes (13 strains; 2.4%). Although f17- and afa-8-related DNA sequences can be plasmid-located in mammalian E. coli, they were not in avian E. coli. Besides the P fimbrial adhesins, F17 and S fimbrial and Afa-VIII and Intimin afimbrial adhesins may thus represent colonisation factors of avian pathogenic E. coli.

Cultured Kaposi's sarcoma tumor cells exhibit a chemokine receptor repertoire that does not allow infection by HIV-1.

BACKGROUND: HIV-1 is known to play a critical role in the pathogenesis of AIDS-associated Kaposi's sarcoma (KS). However, it remains controversial whether KS cells are target cells for HIV infection. The aim of this study was to investigate the expression of chemokine receptors in KS cell cultures and to determine whether these cells can be infected by HIV-1. MATERIAL AND METHODS: KS-derived cells and KS-Y1 cells were investigated using RT-PCR for the expression of CD4, CCR3, CCR5, CCR8 and CXCR4 mRNA. HIV infectivity of these cells was determined by p24 antigen and HIV-1 RNA production, as well as by HIV-1 DNA integration. RESULTS AND DISCUSSION: With the exception of CCR8 which is expressed by KS-derived spindle cell cultures but not by KS-Y1 cells, unstimulated KS cells express no significant levels of CD4, CCR3, CCR5 or CXCR4 mRNA. HIV infectivity assays showed that KS cells were unpermissive to HTLVIIIB and JRFL strains. Although the expression of CXCR4 mRNA could be upregulated by interleukin-1beta, stimulation of KS cells by this cytokine did not allow infection by HIV-1. CONCLUSIONS: This shows that KS cells exhibit a chemokine receptor repertoire that does not allow infection by HIV-1. Other cell types making up KS lesions, such as inflammatory cells, are likely to represent the source of HIV-1 products cooperating to promote KS development and progression.

Transforming growth factor-beta inhibits interleukin-10 synthesis by human monocytic cells.

Transforming growth factor-beta (TGF-beta1) enhances interleukin-10 (IL-10) synthesis by mouse monocytes/macrophages, suggesting a potential role of IL-10 in mediating some of the anti-inflammatory properties of TGF-beta1. Since differences exist between the transcriptional regulation of human and mouse IL-10, the studies reported here examined whether TGF-beta1 up-regulated IL-10 production by human monocytes/macrophages as well. Exposure of PMA-differentiated U-937 promonocytic cells to TGF-beta1 resulted in an unexpected, dose-dependent decrease in IL-10 production as assessed by specific ELISA. TGF-beta1 was effective when added at the time of the PMA stimulus or 6 hours after. In addition, TGF-beta1 suppressed induction of IL-10 by three different stimuli other than PMA. TGF-beta1 inhibition of IL-10 protein release was associated with proportional changes in IL-10 mRNA accumulation as assessed by quantitative kinetic ELISA PCR. This would result from a decrease in IL-10 gene transcription as TGF-beta1 did not affect IL-10 mRNA stability, and TGF-beta1 limited the luciferase activity in cells transfected with reporter gene constructs containing 1,308 bp of the 5' non-coding sequence of human IL-10 gene. Blocking tumour necrosis factor-alpha (TNF-alpha) with neutralizing anti-TNF-alpha antibody did not modify the response to TGF-beta1, indicating the involvement of TNF-alpha-independent mechanisms in the overall process. Thus, the present study provides the first evidence that TGF-beta1 prevents IL-10 production by human monocytic cells at a transcriptional level.

Clonal Th2 cells associated with chronic hypereosinophilia: TARC-induced CCR4 down-regulation in vivo.

We analyzed the expression of chemokine receptors on clonal Th2-type CD4(+)CD3(- )lymphocytes isolated from blood of two patients with chronic hypereosinophilia. First, we observed that these Th2 cells express membrane CCR5 and CXCR4 but neither CCR3 nor CCR4 when analyzed immediately after purification. However, CCR4 appeared following culture in human serum-free medium, suggesting that it was down-regulated in vivo. Indeed, patient's serum, but not control human serum, strongly down-regulated CCR4 expression on cultured Th2 cells. As high levels of TARC, a CCR4 ligand, were detected in the serum of four hypereosinophilic patients with CD3(-)CD4(+) clonal Th2 cells, we evaluated the effect of TARC neutralization in this system. Addition of a neutralizing anti-TARC mAb inhibited CCR4 down-regulation by patient's serum, indicating that circulating TARC contributed to CCR4 down-regulation on Th2 cells in vivo. Clonal Th2 cells did not secrete high levels of TARC themselves but induced a sustained production of TARC by monocyte-derived dendritic cells, a phenomenon that was inhibited by addition of blocking mAb against IL-4 receptor. We conclude that high circulating levels of TARC in serum of patients with chronic hypereosinophilia, most likely derived from antigen-presenting cells stimulated by Th2-type cytokines, induce down-regulation of CCR4 on Th2 cells in vivo.

Iron induces Bcl-2 expression in human dermal microvascular endothelial cells.

Iron is suspected to be involved in the induction and/or progression of various human tumors. The present study was designed to investigate the effects of iron on endothelial cells, keeping in mind that the homeostasis of microvessels plays a critical role in neo-angiogenesis. Applying a model of human dermal microvascular endothelial cell terminal differentiation and death induced by serum deprivation, we found that iron salts (iron chloride and ferric nitrilotriacetate) provided a survival advantage to endothelial cells. Using immunohistochemistry and Western Blot analysis, we found that the extended cellular life span induced by iron was paralleled by an increase of Bcl-2 protein expression. Taken together, these observations suggest that iron may give a survival advantage to endothelial cells and represent a novel mechanism through which iron may contribute to tumorigenesis.

Interferon alpha prevents spontaneous apoptosis of clonal Th2 cells associated with chronic hypereosinophilia.

A recent study identified a clonal expansion of CD3(-)CD4(+)cells secreting Th2-type cytokines in 4 patients with chronic hypereosinophilia. Because interferon alpha (IFN-alpha) is used in the therapy of the idiopathic hypereosinophilic syndrome, the effects of this cytokine on the survival of clonal Th2 cells isolated from the blood of 2 patients were determined. First, these cells displayed a high rate of spontaneous apoptosis on culture in cytokine-free medium and were also sensitive to Fas-mediated apoptosis induced by soluble Fas ligand. Addition of IFN-alpha or interleukin-2 (IL-2) to culture medium resulted in significant protection against spontaneous but not Fas-induced apoptosis. Although spontaneous apoptosis of the clonal Th2 cells was clearly associated with down-regulation of both bcl-2 and bcl-x(L) levels, IFN-alpha had no significant effect on the expression of these antiapoptotic proteins, whereas addition of IL-2 resulted in higher levels of bcl-2. On the other hand, IFN-alpha decreased the numbers of cells with disrupted mitochondrial transmembrane potential both during spontaneous apoptosis and after exposure to protoporphyrin IX. Thus, IFN-alpha might promote the survival of clonal Th2 cells, an effect that could be relevant to the therapeutic approach for patients with chronic hypereosinophilia caused by clonal expansion of Th2-type cells. (Blood. 2000;96:4285-4292)

Early inflammatory response after elective abdominal aortic aneurysm repair: a comparison between endovascular procedure and conventional surgery.

OBJECTIVE: To determine the nature of and to compare the inflammatory responses induced by (1) endovascular and (2) conventional abdominal aortic aneurysm (AAA) repair. MATERIAL AND METHODS: Twelve consecutive patients undergoing elective infrarenal AAA repair were prospectively studied. Seven patients were selected for endovascular procedures (the EAAA group); five patients underwent open surgery (the OAAA group). Three control patients undergoing carotid thromboendarterectomy were also included. Serial peripheral venous blood samples were collected preoperatively, immediately after declamping or placement of the endograft, and at hours 1, 3, 6, 12, 24, 48, and 72. Acute phase response expression of peripheral T lymphocyte and monocyte activation markers and adhesion molecules (flow cytometry), soluble levels of cell adhesion molecules (enzyme-linked immunosorbent assay), cytokine (tumor necrosis factor alpha, interleukin-6, and interleukin-8) release (enzyme-linked immunosorbent assay), and liberation of complement products (nephelometry) were measured. RESULTS: Regarding acute phase response, the EAAA and OAAA groups showed significant increases in C-reactive protein (P <.001 and P =.001), body temperature (P =.035 and P =.048), and leukocyte count (P <.001 and P <.001). Similar time course patterns were observed with respect to body temperature (P =.372). Statistically significant different patterns were demonstrated for C-reactive protein (P =.032) and leukocyte count (P =.002). Regarding leukocyte activation, a significant upregulation of peripheral T lymphocyte CD38 expression was observed in the OAAA group only (P =.001). Analysis of markers such as CD69, CD40L, CD25, and CD54 revealed no perioperative fluctuations in any group. Regarding circulating cell adhesion molecules, the EAAA and OAAA groups displayed significant increases in soluble intercellular adhesion molecule-1 (P =.003 and P =.001); there was no intergroup difference (P =.193). All groups demonstrated high soluble von Willebrand factor levels (P =.018, P =. 007, and P =.027), there being no differences in the patterns (P =. 772). Otherwise, soluble vascular cell adhesion molecule-1, soluble E-selectin, and soluble P-selectin did not appear to vary in any group. Regarding cytokine release, although a tendency toward high tumor necrosis factor alpha and interleukin-8 levels was noticed in the EAAA group, global time course effects failed to reach statistical significance (P =.543 and P =.080). In contrast, interleukin-6 showed elevations in all groups (P =.058, P <.001, and P =.004). Time course patterns did not differ between the EAAA and OAAA groups (P =.840). Regarding complement activation, the C3d/C3 ratio disclosed significant postoperative elevations in the EAAA and OAAA groups (P =.013 and P =.009). This complement product release was reduced in the EAAA group (P <.001). CONCLUSIONS: The current study indicated that both endovascular and coventional AAA repair induced significant inflammatory responses. Our findings showed that there were no large differences between the procedures with respect to circulating cell adhesion molecule and cytokine release. Moreover, the endoluminal approach produced a limited response in terms of acute phase reaction, T lymphocyte activation, and complement product liberation. This might support the concept that endovascular AAA repair represents an attractive alternative to open surgery. Given the relatively small sample size, further larger studies are required for confirmation of our observations.

Inhibition of the CD40 pathway of monocyte activation by triazolopyrimidine.

Blockade of the CD40/CD40L pathway of monocyte/macrophage activation represents a promising strategy for the treatment of several inflammatory disorders. So far, most pharmacological agents developed for that purpose target CD40L (CD154) expressed on activated T cells. Herein, we provide evidence that triazolopyrimidine, a chemical compound primarily developed for the prevention of arterial thrombosis, strongly inhibits the response of human monocytes to CD40 ligation. First, we found that triazolopyrimidine inhibits the production of IL-12, TNF-alpha, and IL-6 by monocytes activated by coculture with fibroblasts transfected with the CD40L gene as well as the induction of procoagulant activity at their membrane. This was related to a decreased expression of CD40 on monocytes exposed to triazolopyrimidine, an effect that was already apparent at the mRNA level. Furthermore, the addition of triazolopyrimidine to monocytes cultured with IL-4 and GM-CSF prevented their differentiation into fully competent dendritic cells (DC) as DC differentiated in the presence of triazolopyrimidine expressed less CD40 at their surface and were profoundly deficient in the production of IL-12 upon exposure to CD40L transfectants. We conclude that triazolopyrimidine strongly inhibits the CD40 pathway of monocyte activation at least in part by downregulating the gene expression of CD40.

Antiinflammatory properties of mycophenolate mofetil in murine endotoxemia: inhibition of TNF-alpha and upregulation of IL-10 release.

Mycophenolate mofetil (MMF) is a new immunosuppressive agent currently used in organ transplantation and under evaluation in immune-mediated inflammatory disorders such as rheumatoid arthritis. Although MMF was shown to inhibit purine nucleotide synthesis in lymphocytes, it is still unclear whether it might also exert direct antiinflammatory actions in vivo. To address this question, we evaluated the effects of MMF administration on the responses of mice to a single challenge with bacterial lipopolysaccharide (LPS). We observed that MMF treatment inhibits the release of tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO) upon LPS injection whereas it promotes IL-10 production. In parallel, MMF was found to protect mice from LPS-induced lethality. Inhibition of TNF-alpha release was also observed in IL-10-deficient mice indicating that it does not exclusively depend on the upregulation of IL-10 endogenous synthesis. In view of the differential effects of MMF on the LPS-induced production of TNF-alpha and NO on one hand and that of IL-10 on the other hand, we conclude that beside its immunosuppressive action at the lymphocyte level, MMF is also endowed with antiinflammatory properties.

CD40 engagement on endothelial cells promotes tissue factor-dependent procoagulant activity.

The CD40 molecule expressed on endothelial cells has been shown to transduce activation signals resulting in upregulation of adhesion molecules. Herein, we studied the impact of CD40 engagement on the induction of tissue factor (TF)-dependent procoagulant activity (PCA) at the surface of human umbilical vein endothelial cells (HUVECs). First, we found that co-incubation of HUVECs with 3T6 fibroblasts transfected with the CD40L gene (3T6-CD40L) resulted in a clear induction of PCA which was not observed with control untransfected fibroblasts. The specificity of this finding was established by inhibition experiments using monoclonal antibodies (mAbs) blocking CD40 or CD40L. PCA induced by CD40 ligation was TF-related as it was not observed in factor VII-deficient plasma and was associated with the accumulation of TF mRNA. To investigate the role of CD40/CD40L interactions in the induction of endothelial cell PCA by lymphocytes, interferon (IFN)-gamma-stimulated EC were incubated with T cells in the absence or presence of anti-CD40 or anti-CD40L mAb. The 60-70% inhibition of PCA induced by these mAbs but not their isotype-matched control indicated that the CD40 pathway is involved in the induction of PCA resulting from interactions between activated HUVECs and T cells. We conclude that activation signals elicited by CD40 engagement on endothelial cells result in the induction of TF-dependent PCA. The CD40/CD40L pathway might therefore be involved in the development of prothrombic states during diseases associated with endothelial cell and T cell activation.

ChemR23, a putative chemoattractant receptor, is expressed in monocyte-derived dendritic cells and macrophages and is a coreceptor for SIV and some primary HIV-1 strains.

Leukocyte chemoattractants act through a rapidly growing subfamily of G protein-coupled receptors. We report the cloning of a novel human gene encoding an orphan receptor (ChemR23) related to the C3a, C5a and formyl Met-Leu-Phe receptors, and more distantly to the subfamilies of chemokine receptors. ChemR23 transcripts were found to be abundant in monocyte-derived dendritic cells and macrophages, treated or not with LPS. Low expression could also be detected by reverse transcription-PCR in CD4+ T lymphocytes. The gene encoding ChemR23 was assigned by radiation hybrid mapping to the q21.2-21.3 region of human chromosome 12, outside the gene clusters identified so far for chemoattractant receptors. Given the increasing number of chemoattractant receptors used by HIV-1, HIV-2 and SIV as coreceptors, ChemR23 was tested in fusion assays for potential coreceptor activity by a range of viral strains. None of the tested HIV-2 strains made use of ChemR23 as a coreceptor, but several SIV strains (SIVmac316, SIVmac239, SIVmacl7E-Fr and SIVsm62A), as well as a primary HIV-1 strain (92UG024-2) used it efficiently. ChemR23 therefore appears as a coreceptor for immunodeficiency viruses that does not belong to the chemokine receptor family. It is also a putative chemoattractant receptor relatively specific for antigen-presenting cells, and it could play an important role in the recruitment or trafficking of these cell populations. Future work will be required to identify the ligand(s) of this new G protein-coupled receptor and to define its precise role in the physiology of dendritic cells and macrophages.

Role of reactive oxygen intermediates in interleukin 10 release after cold liver ischemia and reperfusion in mice.

BACKGROUND & AIMS: Reactive oxygen intermediates and cytokines are key effectors in reperfusion injury after liver ischemia. We hypothesized that reactive oxygen intermediates act as a signal for the release of tumor necrosis factor (TNF) and interleukin 10 (IL-10) after reperfusion of cold-preserved livers. METHODS: An endotoxin-free isolated perfused mouse liver system was designed. Harvested mouse livers were stored at 4 degrees C for 0-28 hours and reperfused for 90 minutes with a warm oxygenated Hank's balanced salt solution (alone or with additives). Cytokine messenger RNA (mRNA) from whole liver was measured by reverse-transcription polymerase chain reaction. Cytokine protein levels and liver injury assessed by alanine aminotransferase levels were evaluated in liver effluent during reperfusion. RESULTS: TNF and IL-10 mRNA and protein concentrations were increased after reperfusion of ischemic livers. N-Acetylcysteine and allopurinol dramatically decreased TNF (-64% and -62%) and IL-10 (-49% and -57%) levels in the effluents, as did an inhibitor of the transcription factor NF-kappaB mobilization (-73% and -76% for TNF and IL-10, respectively). Liver injury was decreased by -40%, -43%, and -54% for the three inhibitors, respectively. CONCLUSIONS: Reactive oxygen intermediates are involved in TNF and IL-10 release after reperfusion of cold-preserved livers.

Molecular cloning and chromosomal mapping of a novel human gene, ChemR1, expressed in T lymphocytes and polymorphonuclear cells and encoding a putative chemokine receptor.

We describe the cloning of a human gene, named ChemR1, encoding a new putative chemokine receptor sharing 48% identity with CC-chemokine receptor (CCR)4 and 44% identity with CCR1. It displays four extracellular cysteines that are conserved among all other chemokine receptors. ChemR1 transcripts were detected by Northern blotting in the T lymphoblastic cell lines Jurkat and MOLT-4, but not in the pre-B lymphoblastic cell line JM-1. ChemR1 receptor transcripts were also detected by reverse transcription and polymerase chain reaction analysis in unstimulated CD4+ and CD8+ T cells and polymorphonuclear cells prepared from peripheral blood. The chromosomal localization was performed by radiation hybrid mapping and testing of a panel of yeast artificial chromosome clones. This allowed the assignment of the ChemR1 receptor gene to the p21.3-24 region of human chromosome 3, in close proximity with the functionally characterized CCR. Future work is required to identify the ligand(s) of this new chemokine receptor and to define its role in the recruitment of white blood cell populations.

Adenosine enhances IL-10 secretion by human monocytes.

Adenosine is a potent endogenous antiinflammatory agent released by cells under metabolically unfavorable conditions. Its effects on the production of IL-10 by human monocytes were presently investigated. Pre-incubation with adenosine dose-dependently enhanced IL-10 release by TNF stimulated human monocytes (+29, +58, and +116% at 1, 10, and 100 muM, respectively.) Adenosine also significantly enhanced IL-10 production after hydrogen peroxide and LPS stimulation and dose-dependently inhibited TNF secretion. Pre-incubation was not mandatory to achieve these effects, since addition of adenosine at the time of or 30 min after the stimulus led to the same results. Blocking IL-10 with anti-IL-10 mAbs partially restored adenosine-induced TNF inhibition. The enhanced IL-10 production was not observed when cells were preincubated with adenosine A1 or A2 receptor agonists (R-phenylisopropyladenosine, 5'-N-ethylcarboxamido-adenosine, and 2-chloroadenosine) and was not affected by pretreatment with theophyllin, an antagonist of both A1 and A2 receptors, or with dipyridamole, an inhibitor of adenosine cellular uptake. In conclusion, adenosine, in the submillimolar concentration range, increases IL-10 secretion by stimulated monocytes. This phenomenon participates in TNF inhibition, a known property of adenosine, but is not mediated through the occupancy of A1 or A2 receptors. This may represent a novel antiinflammatory property of adenosine by which it could modulate inflammation and limit ischemia-reperfusion injury.

Recombinant interferon-alpha selectively inhibits the production of interleukin-5 by human CD4+ T cells.

The effects of recombinant IFN-alpha on the production of IL-5 by human CD4+ T cells were first analyzed on resting CD4+ T cells purified from normal PBMC and stimulated either with a combination of PMA and anti-CD28 mAb or anti-CD3 mAb cross-linked on B7-1/CD32-transfected mouse fibroblasts. We found that IFN-alpha profoundly inhibited in a dose-dependent manner IL-5 production by resting CD4+ T cells whereas IL-10 was upregulated in both systems. The addition of a neutralizing anti-IL-10 mAb to PMA and anti-CD28 mAb upregulated IL-5 production by resting CD4+ T cells but did not prevent IFN-alpha-induced IL-5 inhibition. We then analyzed the effect of IFN-alpha on the production of cytokines by differentiated type 2 helper (Th2) CD4+CD3- cells isolated from peripheral blood of two patients with the hypereosinophilic syndrome. In both cases, IFN-alpha markedly inhibited IL-5 production while it induced mild upregulation of IL-4 and IL-10. Finally, the inhibitory effect of IFN-alpha on IL-5 production was confirmed on a panel of Th2 and Th0 clones generated in vitro. In 2 out of 6 clones, IL-5 inhibition was associated with upregulation of IL-4 and IL-10. We conclude that IFN-alpha selectively downregulates IL-5 synthesis by human CD4+ T cells.

HLA-B SNA antigen: a BW6 associated B locus antigen belonging to the B5 CREG.

A new B locus antigen has been found in members of two unrelated families and in a patient originating from Morocco belonging to the Berber population. A complete analysis has been performed with antisera from the 9th and 10th IHWS defining the B5 CREG antigens. Serology, absorption experiments and family studies indicate that SNA antigen is a Bw6 associated subtype of B5 antigen closely related to but clearly distinct from each of the B5 CREG antigens. One-dimensional isoelectric focusing study (1D-IEF) confirms that the SNA antigen precipitates as a B locus gene product and has an isoelectric point identical to that of Bw52 and one of the charge variants of B51.

Thyroid-specific and cAMP-dependent hypersensitive regions in thyroglobulin gene chromatin.

Two regions hypersensitive to DNase I digestion were found in a 7-kb segment of thyroglobulin gene 5'-flanking sequences in the chromatin from bovine thyroid. The most upstream site (-2000 to -1600 bp relative to the transcriptional start) was found in thyroid chromatin only, but independently of actual expression of the gene. It therefore represents a tissue-specific characteristic which may be associated with the commitment of the thyroglobulin gene to transcriptional activity. The very 5' end of the gene and the proximal promoter sequences (-100 to +60 bp relative to transcriptional start), constitute the second site, the hypersensitive character of which could be directly correlated with transcriptional activity. The structural changes occurring in this region of the chromatin were dependent on cAMP stimulation of the thyroid cells.