Is there an interleukin 2 inhibitor in human serum?

Normal mouse serum has been shown to contain an inhibitor of interleukin 2 (IL-2). Here we report that a molecule with similar activity cannot be found in normal human serum (NHS). Although NHS inhibited the IL-2-dependent proliferation of mouse CTLL cells, as expected of an IL-2 inhibitor, it also had inhibitory activity on IL-3-dependent cells and was cytolytic to IL-2-independent mouse cells as measured by a ⁵¹Cr release assay, indicating a nonspecific effect. In addition, NHS had no effect on the IL-2-dependent proliferation of human peripheral blood T-cell blasts. Fractionation of NHS by size exclusion HPLC failed to separate cytolytic activity from any putative true IL-2 inhibitor activity. The cytolytic component was not related to immunoglobulin since it had a molecular weight of 50,000 to 60,000 and was not bound by protein-A-Sepharose. However, its molecular weight, heat lability, and trypsin sensitivity suggest it to be a protein.     

Why are multiple chains required for the interleukin 2 receptor?

The interleukin 2 receptor (IL-2R) is composed of at least two proteins, that is, a 55 kDa L chain (p55, α chain) and a 75 kDa H chain (p75, β chain). The high-affinity binding of IL-2 results in the formation of the ternary complex consisting of IL-2, the L chain and the H chain. Kinetic studies on the IL-2 binding to the high-affinity IL-2R have shown that the association of IL-2 to the L chain is the first step of the ternary complex formation and that expression of a larger number of L chains accelerates the association of IL-2 to the high-affinity IL-2R in agreement with the stepwise binding/affinity conversion model. This conclusion was supported by experiments using several monoclonal antibodies directed to either H or L chain and murine T cell lines which was transfected by the human L chain cDNA. Temperature-sensitive IL-2 binding to the high-affinity receptor is also consistent with the above conclusion. Signal transduction by the IL-2R appears to involve the activation of tyrosine protein kinase. IL-2 signal transduction seems to require the H chain and another yet unidentified molecule, which might have the kinase activity.     

Effects of recombinant human interleukin 1a and interleukin 1β on cell growth and alkaline phosphatase of the mouse osteoblastic cell line MC3T3-E1

Recombinant human interleukin 1 (rhIL-1)α and rhIL-1β were examined for their effects on DNA synthesis, cell growth and alkaline phosphatase activity of the mouse osteoblastic cell line MC3T3-E1. The relative activity of rhIL-1α and rhIL-1β was compared in terms of the units which induced half-maximal [³H]thymidine uptake into mouse thymocyte cultures exposed to IL-1. Both rhIL-1α and rhIL-1β significantly inhibited DNA synthesis and division of the cells in a concentration- and cultivation time-dependent fashion. In contrast, rhIL-lα and rhIL-1β markedly increased alkaline phosphatase activity, which is a marker of osteoblastic differentiation. This activity in cells treated with rhIL-1α and rhIL-1β increased about 2.0- and 1.7-fold, respectively, compared with that of control cultures. Inhibition of the DNA synthesis and stimulation of alkaline phosphatase activity by both types of rhIL-1 were completely neutralized by treatment with their respective polyclonal antisera. Also, inhibition of DNA synthesis was unaffected by the addition of cyclooxygenase and lipoxygenase inhibitors, and stimulation of alkaline phosphatase activity was unaffected by the addition of indomethacin. These results indicate that both rhIL-1α and rhIL-1β have qualitatively similar biological effects on osteoblastic cells. They also suggest that IL-1 is an important modulator of the growth and differentiation of osteoblasts.     

Influence of culture conditions on the production of heterologous interleukin 1β by Kluyveromyces lactis

Summary Under the control of the repressible PHO5 promoter, the expression of gene encoding interleukin 1 (Il1) was derepressed when the medium was depleted of free inorganic phosphate (Pi). Maximum heterologous protein synthesis was obtained in the presence of 75 mg KH₂PO₄/1 (for 20 g glucose/l). The successful heterologous protein production greatly depends on nutritional culture conditions as Il1 production efficiency was increased by 83% through optimization of the growth medium. Comparison of different phosphate-limited cultivation strategies led to the development of a batch culture procedure with nutrient pulses to delay induced oxido-fermentative glucose metabolism and increase the Il1 production to 135 mg/l.     

Decreased interleukin 27 expression is associated with active uveitis in Beh?et’s disease

Instruction

Interleukin 27 (IL-27) is an important regulator of the proinflammatory T-cell response. In this study, we investigated its role in the pathogenesis of Behçet’s disease (BD).

Methods

IL-27 mRNA in peripheral blood mononuclear cells (PBMCs) was examined by performing RT-PCRs. Cytokine levels in sera or supernatants of PBMCs, naïve CD4⁺ T cells, dendritic cells (DCs) and DC/T cells were determined by enzyme-linked immunosorbent assay. We used RNA interference in naïve CD4⁺ T cells to study the role of interferon regulatory factor 8 (IRF8) in the inhibitory effect of IL-27 on Th17 cell differentiation. Flow cytometry was used to evaluate the frequency of IL-17- and interferon γ–producing T cells.

Results

The expression of IL-27p28 mRNA by PBMCs and IL-27 in the sera and supernatants of cultured PBMCs were markedly decreased in patients with active BD. A higher frequency of IL-17-producing CD4⁺ T (Th17) cells and increased IL-17 production under Th17 polarizing conditions were observed in patients with active BD. IL-27 significantly inhibited Th17 cell differentiation. Downregulation of IRF8 by RNA interference abrogated the suppressive effect of IL-27 on Th17 differentiation. IL-27 inhibited the production of IL-1β, IL-6 and IL-23, but promoted IL-10 production, by DCs. IL-27-treated DCs inhibited both the Th1 and Th17 cell responses.

Conclusions

The results of the present study suggest that a decreased IL-27 expression is associated with disease activity in BD patients. Low IL-27 expression may result in a higher Th1 and Th17 cell response and thereby promote the autoinflammatory reaction observed in BD. Manipulation of IL-27 may offer a new treatment modality for this disease.     

Distribution of immune cells and expression of interleukin receptors in ileal Peyer’s patches of calves

Newborn calves lack a mature immune system. The immune system develops with age, but the role of the expression of cytokine receptors in the development of immune cells of Peyer’s patches (PPs) in the intestines of calves in the first 2 months has not yet been elucidated. In this study, the distribution of immune cells and the expression of interleukin (IL) receptors (R) in the ileal PPs of newborn and 2-month-old calves were investigated immunohistochemically with monoclonal antibodies against bovine CD4, CD8, IgM, γδTCR, T19, WC3, WC5, and WC6 antigens. The expression of ILRs was examined with antibodies against CD25 (IL-2Rα), IL-2Rγ, IL-4R, IL-6R, IL-10R, and IL-13R antigens. CD4⁺, CD8⁺, γδTCR⁺, T19⁺, and WC6⁺ cells were found to be more widely distributed in the ileal PPs of 2-month-old calves than in those of newborn calves. Moreover, the expression of CD25 (IL-2Rα), IL-4R, and IL-13R in the ileal PPs of 2-month-old calves was more prominent than that in newborn calves. These data suggest that the immune system of calves at 2 months of age is developed by reactions to foreign antigens and aging.     

Interaction of the conceptus and endometrium to establish pregnancy in mammals: role of interleukin 1β

Implantation and the establishment of pregnancy in mammals involves an intricate interplay of hormones, cytokines, growth factors, proteins, lipids, ions and the extracellular matrix between the uterine epithelium, stroma, immune cells and the conceptus trophectoderm. The divergent nature of implantation in the mouse, human and pig provides not only an interesting contrast in the establishment of pregnancy and early embryonic development but also intriguing similarities with regard to early endometrial-conceptus signaling. An interesting pro-inflammatory cytokine expressed in a number of mammalian species during the period of implantation is interleukin-1β (IL1B). The presence of IL1B might be involved with immunotolerance at the maternal-placental interface and has been proposed as one of the mediators in placental viviparity. The production of IL1B and other proinflammatory cytokines might play a role in establishing pregnancy through modulation of the nuclear factor kappa-B (NFKB) system in a number of species. A model for the regulation of cellular progesterone receptor expression and NFKB activation for endometrial receptivity and conceptus attachment is continuing to evolve and is discussed in the present review.     

The effect of tumor necrosis factor α (TNFα), interleukin 1β (IL1β) and interleukin 6 (IL6) on endometrial PGF2α synthesis, metabolism and release in early-pregnant pigs

Cytokines produced by the porcine uterus and embryos may be involved in the regulation of endometrial prostaglandin synthesis, metabolism, and release. We studied the effect of tumor necrosis factor α (TNFα), interleukin 1β (IL1β) and interleukin 6 (IL6) on: 1) endometrial release of prostaglandin F₂α (PGF₂α), 2) expression of the terminal enzyme of PGF₂α synthesis - PGF synthase mRNA (PGFS mRNA), 3) secretion of PGF₂α metabolite - 13,14-dihydro-15-keto PGF₂α (PGFM) by the endometrium and 4) presence and activity of endometrial NAD-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH). The effects of cytokines were determined on days 10-11 and days 12-13, e.g., before and during maternal recognition of pregnancy, and on days 15-16, e.g., during the peri-implantation period and compared with its effect in cyclic gilts on corresponding days of the estrous cycle. TNFα did not affect endometrial release of PGF₂α in pregnant and cyclic pigs. IL1β enhanced endometrial PGF₂α release on days 12-13 and 15-16 in pregnant and cyclic pigs, respectively. IL6 increased PGF₂α release mainly on days 15-16 of pregnancy. Expression of PGFS mRNA was decreased by IL1β on days 12-13 of pregnancy (P < 0.05) and increased in response to IL1β, TNFα and IL6 on 12-13 (P < 0.05) and 15-16 (P < 0.01) of the estrous cycle. IL1β increased release of PGFM in gravid pigs on days 12-13, 15-16 and in non-gravid pigs 10-11 and 15-16 of the cycle. On days 15-16 of pregnancy TNFα and IL6 increased endometrial secretion of PGFM. We determined that in porcine endometrium NAD-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH) is present. In gravid pigs, the highest expression of endometrial 15-PGDH occurred during days 12-13 of pregnancy, while in non-gravid pigs during days 10-11 of the estrous cycle. These data provide new evidence that TNFα, IL1β, IL6 are involved in the regulation of endometrial synthesis, release and metabolism of PGF₂α to protect CL during early pregnancy or to facilitate its regression in cyclic females.     

Combined effects of interferon α and interleukin 2 on the induction of a vascular leak syndrome in mice

Summary Immunotherapy with interleukin 2 (IL-2) alone or in combination with lymphokine-activated killer cells can mediate tumor regression in mice and in man. Further dose escalation of IL-2 along with lymphokine-activated killer cells has been prevented by the development of a vascular leak syndrome produced by IL-2. Because we have found that interferon (IFN-) or tumor necrosis factor (TNF-) has synergistic antitumor effects when administered together with IL-2, we have tested the vascular leakage induced by these lymphokine combinations. We used a murine model to quantify vascular leakage by measuring the extravasation of ¹²⁵I-albumin from the intravascular space as well as the wet and dry lung weights after treatment with different cytokines. Cytokines (or Hanks balanced salt solution) were administered to C57BL/6 mice and 4 h after the last injection the vascular leak was quantified. IFN- alone did not cause extravasation of radiolabel or increase in wet lung weights, though when given in combination with IL-2, significantly greater extravasation (P<0.01) as well as increase in lung water weights (P<0.05) was observed compared to the response in mice treated with IL-2 alone. IFN- in combination with IL-2 induced significant vascular leakage earlier than the response induced by IL-2 alone. For example treatment with IFN- and IL-2 induced accumulation of 14674±605 cpm in the lungs at day 1 while IL-2 alone induced 12340±251 cpm. The degree of vascular leakage was highly related to the dose of IFN- administered along with IL-2 and increased vascular leak syndrome was evident even at low doses (5000 units) of IFN-. Immunosuppression of mice by pretreatment irradiation (500 rad) markedly decreased the development of vascular leak syndrome induced by IL-2 and IFN-. Interestingly IFN- and TNF- did not induce vascular leakage in the lungs when given alone, and did not add or synergize with IL-2 in causing the syndrome. Thus the administration of IFN- in combination with IL-2 produces a dose-limiting vascular leakage that is more severe than that caused by IL-2 alone, and may be mediated, directly or indirectly by host radiosensitive cells. Abbreviations used: LAK, lymphokine-activated killer; IFN, interferon; TNF, tumor necrosis factor; IL-2, interleukin-2     

1H, 13C and 15N backbone and side chain resonance assignments of human interleukin 1α

As part of our NMR structure determination of the human Interleukin-1α, we report nearly complete NMR chemical shift assignments for the ¹H, ¹³C and ¹⁵N nuclei.     

Human blood monocyte activation byNocardia rubra cell wall skeleton for productions of interleukin 1 and tumor necrosis factor-α

Human blood monocytes were obtained from peripheral blood of healthy donors by counter-flow centrifugal elutriation. Functional integrity of monocytes for production of interleukin 1 (IL-1) and tumor necrosis factor (TNF-) in response toNocardia rubra cell wall skeleton (N-CWS) was examined by bioassay and enzyme immunoassay. Monocytes treated with N-CWS at more than 0.5 g/ml produced IL-1 and TNF- extracellularly. Extracellular TNF activity appeared within 4 h, and maximally, 16 h after N-CWS stimulation, whereas longer time was needed for IL-1 activity to appear, the peak production being at 24 h. The neutralizing experiment also showed that anti TNF- antibody did not affect IL-1 production by the monocytes treated with N-CWS, suggesting independen cy of IL-1 production of TNF-.These results suggest that the therapeutic antitumor effect of N-CWS is due, in part at least, to the augmented production of these monokines.     

Anti-inflammatory and antiarthritic effects of piperine in human interleukin 1β-stimulated fibroblast-like synoviocytes and in rat arthritis models

Introduction  

The objective of this study was to determine the anti-inflammatory, nociceptive, and antiarthritic effects of piperine, the active phenolic component in black pepper extract.     

Interleukin 8 production in whole blood assays: Is interleukin 10 responsible for the downregulation observed in sepsis?

Reduced cytokine production in ex vivo cultures has been regularly reported in patients suffering from sepsis syndrome. Using whole blood assays, we have now demonstrated that in sepsis patients, normal production of IL-8 was achieved with the higher concentration of lipopolysaccharide (LPS; 1 microg/ml) and with heat-killed streptococci, whereas the IL-8 production induced by lower LPS concentration (0.1 microg/ml) was significantly reduced as compared to healthy controls. In contrast, in patients undergoing cardiac surgery associated with cardio-pulmonary bypass, a group of patients with inflammation in the absence of infectious insult, none of the studied IL-8 productions were affected. Among the various anti-inflammatory cytokines known to regulate IL-8 production which we tested (i.e. IL-4, IL-10, IL-13, TGF-beta), IL-10 was the most active inhibitory cytokine in whole blood assays performed with blood samples from healthy subjects. However, its activity was not influenced by the amounts of LPS used. In addition, IL-10 also inhibited the heat-killed streptococci-induced IL-8 production and was the only cytokine to inhibit the release of IL-8 when TNF was added to LPS. It is worth noting that IL-13 which also inhibited the heat-killed streptococci-induced IL-8 production, failed to do so when the TNF production was analysed. Together, these data suggest that while circulating IL-10 in septic patients may be responsible for the hyporeactivity of circulating leukocytes, its presence is not sufficient to explain the observed dysregulation which occurs in septic patients.     

Differential glycosylation of interleukin 2, the molecular basis for the NOD Idd3 type 1 diabetes gene?

The insulin-dependent diabetes (Idd) gene, Idd3, has been localised to a 0.35 cM region of chromosome 3 containing the structural gene for the cytokine interleukin 2 (IL-2). While variation of the N-terminal amino acid sequence of IL-2 has been shown to correlate with Idd3 allelic variation, differences in induction of proliferation by IL-2 allotypes have not been detected. In the current study, we examined the electrophoretic migration of IL-2 allotypes and have found two distinct patterns, consistent with differences in glycosylation, that correlate with diabetes-resistance and susceptibility. These findings strongly suggest that IL-2 variants may be functionally distinct.     

Common variants within the interleukin 4 receptor α gene (IL4R) are associated with susceptibility to osteoarthritis

Primary osteoarthritis (OA) is a common late-onset arthritis that demonstrates a complex mode of transmittance with both joint-site and gender-specific heterogeneity. We have previously linkage-mapped an OA susceptibility locus to a 12-cM interval at chromosome 16p12.3-p12.1 in a cohort of 146 affected female sibling-pair families ascertained by total hip replacement (female-THR families), with a maximum multipoint LOD score of 1.7. Despite the low LOD score, we were encouraged to investigate this interval further following the report of a linkage to the same interval in an Icelandic pedigree with an early-onset form of hip OA. Using public databases, we searched the interval for plausible candidates and concluded that the gene encoding the interleukin 4 receptor chain (IL4R) was a particularly strong candidate based on its known role in cartilage homeostasis. We genotyped nine common single nucleotide polymorphisms (SNPs) from within IL4R, including six non-synonymous SNPs, in the 146 probands from our female-THR families (stage 1) and in an independent cohort of 310 female-THR cases (stage 2). We compared allele frequencies with those of 399 age-matched female controls. All individuals were UK Caucasians. The minor alleles of two SNPs demonstrated association in both stages, with the most significant association having a P-value of 0.004 with an odds ratio (OR) of 2.1. These two SNPs defined two associated SNP groups. Inheriting a minor SNP allele from both groups was a particular risk factor (OR=2.4, P=0.0008). Our data suggest that functional variants within the IL4R gene predispose to hip OA in Caucasian females.