Prostaglandin-E2 is a potent inhibitor of human interleukin 12 production.

During human immunodeficiency virus infection and allergic diseases, characterized by a dominant T helper (Th) 2 response, overproduction of prostaglandin E2 (PGE2) is observed. In this paper we studied the effect of PGE2 on interleukin (IL)-12 synthesis, because this cytokine has been described to be essential in induction of Th1 responses. IL-12 synthesis was induced in monocytes that were stimulated with Neisseria meningitidis-derived lipopolysaccharide in whole blood cultures. PGE2 almost completely inhibited lipopolysaccharide induced IL-12 production, whereas IL-6 production was only partially inhibited by PGE2. In contrast, the production of IL-10 was approximately twofold enhanced at these conditions. The effects of PGE2 were due to its cAMP-inducing capacity, since they could be mimicked by other cAMP inducers. Recombinant human IL-10 also inhibited IL-12 and IL-6 production. However, the inhibitory effect of PGE2 on IL-12 production was independent of IL-10 since neutralizing anti-IL-10 antibodies were unable to reverse this inhibition. These results suggest that the capacity of an antigen to induce PGE2 synthesis may play a crucial role in the development of either a Th1 or Th2 response.

On the interaction of IgG subclasses with the low affinity Fc gamma RIIa (CD32) on human monocytes, neutrophils, and platelets. Analysis of a functional polymorphism to human IgG2.

An allotypic form of the low affinity IgG Fc receptor Fc gamma RIIa (CD32), termed low responder (LR) because of its weak reactivity with mouse (m) IgG1, interacts efficiently with human (h) IgG2. Fc gamma RIIaLR is the first known human FcR that binds this IgG subclass. In this study, we analyzed the role of Fc gamma RIIa in binding of stable hIgG-subclass dimers, and in induction of T cell mitogenesis using chimeric anti-CD3 mAb. We demonstrate that the functional polymorphism to hIgG2 is expressed on the majority of Fc gamma R-bearing peripheral blood cells: monocytes, neutrophils, and platelets. We were able to assess Fc gamma RII-mediated IgG-binding without interference of other Fc gamma R-classes, by blockade of Fc gamma RI on monocytes, and by using neutrophils of an individual deficient for the Fc gamma RIIIB gene. This study indicates as subclass specificity: hIgG3 >hIgG1,hIgG2 >> hIgG4 for Fc gamma RIIaLR and hIgG3,hIgG1 >> hIgG2 > hIgG4 for Fc gamma RIIaHR. Comparing the serum hIgG levels of individuals homozygous for the two fc gamma RIIa allotypic forms, we observed significantly lower hIgG2 serum levels in individuals expressing the hIgG2-binding LR allotypic form. This observation may implicate that Fc gamma RIIa regulates hIgG subclass production or turnover in man.

Histamine inhibits the production of interleukin-12 through interaction with H2 receptors.

IL-12 is essential for T helper 1 (Th1) development and inhibits the induction of Th2 responses. Atopic diseases, which are characterized by Th2 responses, are associated with the overproduction of histamine. Here we present evidence that histamine, at physiological concentrations, strongly inhibits human IL-12 p40 and p70 mRNA and protein production by human monocytes. The use of specific histamine receptor antagonists reveals that this inhibition is mediated via the H2 receptor and induction of intracellular cAMP. The inhibition of IL-12 production is independent of IL-10 and IFN-gamma. The observation that histamine strongly reduces the production of the Th1-inducing cytokine IL-12 implies a positive feedback mechanism for the development of Th2 responses in atopic patients.

Sensitive ELISA for interleukin-6. Detection of IL-6 in biological fluids: synovial fluids and sera.

A monoclonal antibody and an affinity purified polyclonal antibody, both raised against recombinant human IL-6, have been employed in an ELISA procedure to quantitate human IL-6. Both antibodies were very potent in neutralizing the biological activity of recombinant as well as natural human IL-6. The monoclonal antibody was used as the capture antibody whilst the polyclonal antibody, in biotinylated form, was used as the detecting antibody in combination with a streptavidin horseradish peroxidase conjugate and a signal amplification system. The detection limit for natural as well as recombinant IL-6 was 1 pg/ml. A good correlation was found between the ELISA and the B9 biological assay when IL-6 was measured in crude culture supernatants, in synovial fluids of rheumatoid arthritis patients and in the sera of patients with diverse diseases. Immunoprecipitation of IL-6, produced by different cell types, such as monocytes, endothelial cells and smooth muscle cells or derived from biological fluids, such as the serum of a patient with septic shock or the synovial fluid of a rheumatoid arthritis patient, revealed in every case only molecules in the molecular weight range of 21,000-26,000.

Differential induction of interleukin-6 production in monocytes, endothelial cells and smooth muscle cells.

Studying the production of IL-6 (interleukin-6) by monocytes, endothelial cells and smooth muscle cells we observed that cytokine inducers like IL-1, TNF alpha (tumor necrosis factor alpha), LPS (lipopolysaccharide), SAC (Staphylococcus Aureus Cowan 1) and PMA could be divided roughly into two categories. Bacterial products such as LPS or SAC have a potent IL-6 inducing effect on monocytes and minor or no effect on endothelial- and smooth muscle cells. The other category comprising IL-1, TNF alpha and PMA induces IL-6 production in endothelial- and smooth muscle cells. Only IL-1 induces IL-6 production in monocytes as well as in endothelial cells and smooth muscle cells. In addition to IL-6, also IL-1 and TNF alpha are produced by monocytes however with different kinetics. None of the stimuli had any inhibitory effect on IL-6 production with the exception of PMA. Whereas PMA induced IL-6 production in endothelial cells and it potentiated the induction of IL-6 by IL-1 in these cells, it inhibited LPS-stimulated IL-6 production in monocytes. In line with the effects of PMA, staurosporin induced IL-6 production in monocytes and it inhibited IL-1 driven IL-6 production by endothelial cells.

Differential induction of interleukin-6 production by monocytes, endothelial cells and smooth muscle cells.

Studying the production of IL-6 by monocytes, endothelial cells and smooth muscle cells we observed that cytokine inducers like IL-1, TNF alpha, LPS, SAC and PMA could be divided roughly into two categories. One, that consisted of LPS and SAC, which had a potent IL-6 inducing effect on monocytes and minor or no effect on endothelial- and smooth muscle cells. The other category, consisting of IL-1, TNF alpha and PMA, induced IL-6 production in endothelial- and smooth muscle cells and of which IL-1 also induced IL-6 in monocytes. Only IL-1 induced IL-6 production in monocytes as well as in endothelial cells and smooth muscle cells. Besides IL-6, also IL-1 and TNF alpha were produced by monocytes however with different kinetics. None of the stimuli had any inhibitory effect on IL-6 production with the exception of PMA. Whereas PMA induced IL-6 production in endothelial cells and it potentiated the induction of IL-6 by IL-1 in these cells, it inhibited LPS-stimulated IL-6 production in monocytes. In line with the effects of PMA, staurosporin induced IL-6 production in monocytes and it inhibited IL-1 driven IL-6 production by endothelial cells.

Functional discrimination between interleukin 6 and interleukin 1.

In this study we have investigated the specificity of bioassays in which interleukin (IL) 1 and/or IL 6 are active. The thymocyte assay cannot be used to discriminate between IL 1 and IL 6; both monokines are active in this assay. Moreover the detection limit for both IL 1 and IL 6 is around 100 pg/ml. IL 6 activity can be measured with a murine hybridoma cell line (B9). The detection limit for human as well as murine IL 6 is about 0.5 pg/ml. The assay is specific for IL 6 and is not influenced by a variety of other cytokines except for murine IL 4 which shows some activity in this variety of other cytokines except for murine IL 4 which shows some activity in this assay. IL 1 can be measured specifically with D10 cells. The detection limit for IL 1 alpha and IL 1 beta is around 1 pg/ml whereas IL 6 is not active in this assay at all. Upon stimulation by IL 1 and/or IL 2 D10 cells produce IL 6. However, this IL 6 does not seem to be involved in the proliferation of these cells.

IL-6 is an intermediate in IL-1-induced thymocyte proliferation.

Both IL-1 and IL-6 have been shown to be comitogenic for lectin-stimulated thymocytes. Thymocytes cultured in the presence of IL-1 produce IL-6 themselves. This IL-6 production is caused by a cell population with low buoyant density. After removal of these cells, IL-6 or IL-2 are still co-mitogenic for thymocytes whereas IL-1 is not. Addition of IL-1 to such thymocytes renders them about 100-fold more sensitive to IL-6. At all conditions proliferation is inhibitable with antibodies to IL-2 and to the IL-2R. Our experiments show that IL-1-driven proliferation of thymocytes is dependent on endogenous IL-6 production and that in the classical thymocyte assay IL-1 has a dual role: it induces IL-6 production and it greatly increases the sensitivity for IL-6.

Functional analysis of synovial fluid and peripheral blood T cells from patients with rheumatoid arthritis.

Rheumatoid arthritis is a T cell-mediated autoimmune disease. The lack of knowledge of the involved target antigens severely hampers research on relevant T cells in patients. Here we describe the functional analysis of freshly isolated T cells from the peripheral blood and the site of the lesion (synovial fluid or synovial membrane) of patients with rheumatoid arthritis. Healthy donors and osteoarthritis patients served as controls. Using various polyclonal stimuli, we analyzed CD4+ T cells with respect to proliferation and their ability to produce lymphokines. Our data show that lesion-derived CD4+ T cells of patients with rheumatoid arthritis are severely defective in proliferation and lymphokine (interleukin-2, interleukin-4, tumor necrosis factor-alpha, interferon-gamma) production. This activation defect was most pronounced at lower cell densities and was present in both synovial fluid derived and synovial membrane derived CD4+ T cells of all patients tested. No difference was found between responses of synovial fluid derived CD4+ T cells from osteoarthritis patients and those observed with peripheral blood derived T cells from all groups. The observed defect in lesion-derived CD4+ T cells from rheumatoid arthritis patients was not due to the effect of inflammatory factors in the synovial fluid because preincubation with synovial fluid could not induce a similar defect in control T cells. Together, our data show a rheumatoid arthritis specific, general defect in the activation of lesion-derived CD4+ T cells.

Growth inhibitory effects of 5-aza-2'-deoxycytidine in HL-60 promyelocytic leukemia cells resistant to differentiation induction.

In the original HL-60 cells (HL-60-S) and an HL-60 subline (HL-60-R) respectively susceptible and resistant to induction of differentiation by retinoic acid or dimethyl sulfoxide, 5-aza-2'-deoxycytidine inhibited growth equally but induced differentiation to a greater extent in HL-60-S. Flow cytometry showed that 5-aza-2'-deoxycytidine produced in both HL-60 lines an increased proportion of cells in G2+M rather than G0/G1 as with retinoic acid. 5-aza-2'-deoxycytidine may have a differentiation-inducing effect in HL-60 provided cells have the competence to differentiate, indicating the importance of an alternate mechanism of action.

Induction of T-cell proliferation by recombinant mouse and chimeric mouse/human anti-CD3 monoclonal antibodies.

The isotype of anti-CD3 mAb has a dramatic effect on anti-CD3 induced T-cell activation, as was previously reported for switch variants (IgG2b to IgA) of a high-avidity IgG1 anti-CD3 mAb (CLB-T3/4.1). In order to study and compare the isotype dependency of T-cell activation with anti-CD3 mAb of various mouse and human subclasses, we now prepared recombinant anti-CD3 mAb. The variable region of the anti-CD3 Ig heavy chain was cloned, joined with genes for the heavy chain constant region and expressed in a cell line only secreting autologous mouse chi light chains. Thus we obtained cell lines that produced mouse (m) IgM, mIgG3 and chimaeric mouse/human (h) IgM, hlgG1, hlgG2, hlgG3, hlgG4, hlgE and hlgA2 anti-CD3. The matched set of mouse and mouse/human chimaeric anti-CD3 isotypes switch variants was then used to study activation of T cells in an accessory cell-dependent system. hlgG1, hlgG4, hlgE, mlgG2a and mlgE induced T-cell proliferation in PBMC of all donors tested, whereas PBMC from a subset of donors were unresponsive to stimulation with hlgG2, hlgG3, hlgA2, mlgG1 and mlgG2b anti-CD3 mAb. hlgM, mlgM and mlgA were only able to induce T-cell mitogenesis in combination with PMA. Our panel of anti-CD3 mAb variants may prove a powerful tool to study mouse and human isotype-dependent effector functions and their influence on T-cell activation requirements in detail.

Characterization of IgG FcR-mediated proliferation of human T cells induced by mouse and human anti-CD3 monoclonal antibodies. Identification of a functional polymorphism to human IgG2 anti-CD3.

T cell activation induced by mouse anti-CD3 mAb has shown to be dependent on the Ig isotype of these antibodies. A study of isotype dependency of human antibodies, however, seems more relevant to human effector systems, especially in view of the availability of humanized antibodies for clinical applications. We constructed a panel of mouse and mouse/human chimeric anti-CD3 mAb, which differ only in their CH region and hence have identical binding sites and affinity. By using these antibodies, we now studied their ability to induce T cell proliferation in human PBMC and analyzed the classes of IgG FcR involved in these responses. The human (h)IgG1, hIgG3, and hIgG4, as well as mouse (m)IgG2a and mIgG3 anti-CD3 mAb induced an Fc gamma RI (CD64)-dependent T cell proliferation in all donors. Activation with hIgG2 and mIgG1 anti-CD3 mAb was observed to be mediated via the low affinity Fc gamma RII (CD32). It was found that leukocytes in a normal donor population display a functional polymorphism with respect to hIgG2 anti-CD3 responsiveness. This polymorphism was found to be inversely related to the previously defined Fc gamma RII-polymorphism to mIgG1 anti-CD3 mAb. Monocytes expressing the Fc gamma RII mIgG1 low responder (LR) allele support hIgG2 anti-CD3 induced T cell proliferation efficiently, whereas cells homozygous for the Fc gamma RII mIgG1 high responder (HR) allele do not. This observation could be confirmed in T cell activation studies using hFc gamma RIIa-transfected mouse fibroblasts, expressing either the mIgG1 anti-CD3 HR or LR Fc gamma RII-encoding cDNA.

The role of IL-13 in IgE synthesis by allergic asthma patients.

IgE antibodies play a crucial role in allergic type I reactions. Only IL-4 and IL-13 are able to induce an immunoglobulin isotype switch to IgE in B cells. A major question is to what extent these cytokines contribute to the production of IgE in allergic patients. To address this question we used an in vitro culture system in which the production of IgE is dependent on endogenously produced IL-4 and IL-13. In cultures of purified T and B cells from allergic asthma patients and non-atopic controls, T cells were polyclonally stimulated to obtain IL-4, IL-13 and subsequently IgE secretion. The absolute amount of IgE produced was not significantly different between patients and controls. When neutralizing IL-4 antibodies were included during culture, the production of IgE was dramatically inhibited in both patients and controls (production of IgE was reduced to 12%). However, neutralization of IL-13 led to a significantly stronger inhibition of IgE production in the patient group: production of IgE was reduced to 23 +/- 3% versus 50 +/- 10% in the control group. Corresponding with these results, we also observed a higher production of IL-13 by the patients, while the production of IL-4 was not significantly different. A more detailed analysis of the production of IL-13 revealed that patients' T cells were less sensitive to a negative signal controlling IL-13 production. Our results indicate that, at least in vitro, IgE production in allergic asthma patients is more dependent on IL-13 than in non-atopics, due to enhanced IL-13 production and to enhanced IgE production in response to IL-13.

Patients with systemic lupus erythematosus with high plasma levels of sFas risk relapse.

OBJECTIVE: We related soluble Fas (sFas) levels to the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) in a longitudinal series of plasma samples of patients with SLE to evaluate the relation between excessive production of sFas and disease activity. METHODS: We generated 21 monoclonal antibodies against Fas. Two of these were used to develop and validate a sensitive sandwich ELISA for the longitudinal analysis of sFas levels in plasma of 30 patients and 25 controls. RESULTS: At the start of followup, a significant elevation (p<0.0001) was found in sFas levels in SLE (1167+/-347 pg/ml sFas) compared to controls (618+/-98 pg/ml sFas). Also, at the start of the followup a significant difference (p = 0.0028) existed between patients who were going to have a relapse (1236+/-402 pg/ml sFas) during followup and patients who were not (809+/-276 pg/ml sFas). While sFas did not fluctuate with disease activity in individual patients, we found a strong correlation (r = 0.75, p<0.0001) between sFas and SLEDAI, but only at the time of relapse, when we analyzed the patients as a group. CONCLUSION: In individual patients with SLE, sFas does not fluctuate with disease activity. However, patients with high plasma levels of sFas are at risk of relapse.

Human IL-13 production is negatively influenced by CD3 engagement. Enhancement of IL-13 production by cyclosporin A.

IL-13, a T cell-derived cytokine, shares many of its biologic activities with the Th2 cytokine IL-4, including induction of a class switch to IgE and anti-inflammatory properties. Its potential impact on development of Th2 responses makes it interesting to determine how the production of IL-13 is regulated and which cell types produce IL-13. In this work, we show that IL-13 is produced optimally by T cells when stimulated with a combination of anti-CD28 and PMA. Unexpectedly, additional ligation of the TCR complex with Abs to CD3 caused an approximately fivefold inhibition of IL-13 production. Moreover, this inhibition could be reversed by cyclosporin A (CsA). The effect of CsA did not depend on the presence of PMA; upon CD3 and CD28 stimulation, CsA equally enhanced IL-13 production. Both naive and memory CD4+ T cells and CD8+ T cells produced IL-13, and production in all cell types could be enhanced by CsA. In contrast to IL-13, IL-4 production was observed mainly in CD4+ memory cells, required costimulation through CD3, and was inhibited by CsA. The unusual regulation and relative abundance of IL-13 make it an important candidate to be controlled tightly by dose and type of TCR ligands. CsA is used widely to inhibit T cell function. The finding that IL-13 production is enhanced instead of diminished in the presence of CsA may explain the Th2-inducing effects of CsA in vivo.

High IL-13 production by human neonatal T cells: neonate immune system regulator?

Neonates are highly susceptible to diseases and display biased type 2 immune responses, although no skewing to type 2 cytokines has been reported. In view of the emerging importance of IL-13 in type 2 inflammatory responses and clinical allergy, we analyzed IL-13 production by neonatal T cells. We found that, mainly CD8 T cells produced high levels of IL-13, while producing low levels of IL-4, IL-10 and IFN-gamma, upon primary and secondary stimulation. Our results point towards a possible immunoregulatory role of CD8 T cells in neonate responses. Moreover, they suggest that the abundance of IL-13 in the neonate immune system might account for the type 2 bias in neonates, providing a basis for the high disease susceptibility of newborns, for instance to allergic diseases.

On the interaction between agalactosyl IgG and Fc gamma receptors.

One of the serum abnormalities observed in autoimmune diseases such as rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) is the occurrence of IgG that lacks the terminal galactose on asparagine-linked biantennary complex type oligosaccharides [Gal(0)-IgG] located in the CH2 domain. Additionally, IgG without glycosylation is known to be defective in several effector functions due to a reduced ability to bind to its specific receptors (Fc gamma R). It has thus been speculated that, by analogy with unglycosylated IgG, Gal(0)-IgG may also be functionally impaired or exert altered effector mechanisms. If this were true, Gal(0)-IgG could contribute to the phenotype of above-mentioned autoimmune diseases, like impaired immune complex clearance and defective down-regulation of activated B cells. Here, we show by three different methods that the interaction of Gal(0)-IgG and normally glycosylated IgG with the low-affinity Fc gamma RII (CD32) is indistinguishable with respect both to binding and receptor-mediated signalling. These data argue against a prominent role for Fc gamma R-dependent Gal(0)-IgG interactions in the etiology or pathogenesis of autoimmune diseases.

Reduced production of IL-12 and IL-12-dependent IFN-gamma release in patients with allergic asthma.

In atopic patients, allergen-specific T cells have acquired the Th2 phenotype, which is considered to be responsible for the class switch to IgE Ab formation. Because IL-12 is a key cytokine for the induction of Th1 responses, a reduced capacity to produce this cytokine could lead to aberrant Th2 development. Therefore, we examined the production of IL-12 in whole blood cultures from patients with allergic asthma (n = 15) in comparison with nonatopic control subjects (n = 15) to different stimuli. After stimulation with Staphylococcus aureus Cowan I strain (SAC) we observed a 2.6-fold reduction of IL-12 p70 production in the patient group (p < 0.005). This was not due to a general failure of monocytes from these patients to produce cytokines, because the production of IL-6 was normal. SAC also induced the production of IFN-gamma, which was blocked by neutralization of IL-12. In line with the reduced levels of IL-12 secretion, the patient group showed a 3-fold reduction of IL-12-dependent IFN-gamma production (p < 0.005). The amounts of IL-12 and IFN-gamma were positively correlated in both the patient (R = 0.51 at 0.05% SAC and R = 0.64 at 0.01% SAC) and the control groups (R = 0.64 at 0.05% SAC and R = 0.70 at 0.01% SAC). The IFN-gamma:IL-12 ratio was not different between patients and control subjects, indicating a normal response to IL-12. Diminished production of IL-12 and IFN-gamma could not be explained by an increased production of IL-10, because in SAC-stimulated cultures IL-10 was hardly induced in both groups. Furthermore, after stimulation with Escherichia coli, the production of IL-10 was similar in patients and control subjects.

An IL-13 promoter polymorphism associated with increased risk of allergic asthma.

IL-13 plays a crucial role in the development of allergic asthma by several mechanisms, including induction of IgE antibodies, airway eosinophilia and hyper-reactivity. We previously established a deregulated production of IL-13 by T cells from allergic asthma patients. In this report we describe the identification of a novel IL-13 promoter polymorphism (C to T exchange) at position -1055. The IL-13 -1055 TT genotype is associated with allergic asthma (P = 0.002), altered regulation of IL-13 production (P < 0.002), and increased binding of nuclear proteins to this region. We postulate that the presence of this polymorphism predisposes to the development of allergic asthma.