Expression and Purification of Tag-removed Human IL37 by Digestion on Beads in Escherichia coli.

BACKGROUND: Human Interleukin 37 (IL37), a unique anti-inflammatory cytokine of IL1 family member, plays critical roles in innate and adaptive immunity and inflammation. OBJECTIVE: Preparation of high purity and tag-removed recombinant IL37 protein (rIL37) is critical for its clinical application. METHOD: In this study, we constructed an N-terminal cleavable GST-fused IL37 expression vector for recombinant expression. RESULTS: Subsequent to transformation and optimization of the induction temperature, the soluble expression level of rIL37 was 306.5 mg/L of culture medium at 18 °C induction in Escherichia coli. Meanwhile, rIL37 was digested on beads by GST-HRV3C protease during GST affinity chromatography. After further purification, the purity of rIL37 was higher than 99 %. Finally, the antiinflammatory activity of tag-removed protein was verified by the results showing that rIL37 suppressed IL1ß production in PBMCs. CONCLUSION: This work presents a protocol to produce high purity and tag-removed rIL37 with antiinflammatory activity, which provides the firm basis for advancing clinical application in human IL37-related inflammatory diseases.

High-level expression and one-step purification of a soluble recombinant human interleukin-37b in Escherichia coli.

Interleukin (IL)-37 is a novel member of the IL-1 cytokine family. However, as a result of lacking efficient method to generate relatively large quantity of IL-37, little is known of its functions in man. In the present study, the recombinant human IL-37b containing a C-hexahistidine tag was expressed in Escherichia coli (E. coli). The expression level of IL-37b in E. coli was very high after induction with IPTG. Furthermore, IL-37b protein was largely found in the soluble fraction. The expressed protein was readily purified by one-step immobilized metal-ion affinity chromatography using Ni(2+)-nitrilotriacetic acid agarose. The purified IL-37b appeared as a single band on SDS-PAGE and the purity was more than 97%. The yield was 90mg IL-37b from 1l of bacterial culture. Western blotting and N-terminal sequencing confirmed the identity of the purified protein. The purified IL-37b inhibited significantly the release of tumor necrosis factor-α and IL-1ß in lipopolysaccharide-activated THP-1 cells. Thus, this method provides an efficient way to obtain an active IL-37 with high yield and high purity.

A tale of two interferon bioassays: how frustration with discrepant results from slightly dissimilar methods can engender discovery.

This introductory article describes an episode that took place in the mid-1980s when the first wave of cytokine discoveries took place. During studies aimed at complete purification of human interferon-γ from crude mitogen-stimulated lymphokine preparations, the use of two different antiviral bioassays for the cytokine yielded disparate results. Analysis revealed the presence of a "contaminant" IFN-like cytokine that was detectable with only one of the two assays. Superficially, the contaminant resembled IFN-ß. However, further analysis showed that it was not an IFN at all but an IFN-inducing cytokine identifiable as interleukin-1.

Identification of a novel IL-1 cytokine family member in teleost fish.

A novel IL-1 family member (nIL-1F) has been discovered in fish, adding a further member to this cytokine family. The unique gene organization of nIL-1F, together with its location in the genome and low homology to known family members, suggests that this molecule is not homologous to known IL-1F. Nevertheless, it contains a predicted C-terminal beta-trefoil structure, an IL-1F signature region within the final exon, a potential IL-1 converting enzyme cut site, and its expression level is clearly increased following infection, or stimulation of macrophages with LPS or IL-1beta. A thrombin cut site is also present and may have functional relevance. The C-terminal recombinant protein antagonized the effects of rainbow trout rIL-1beta on inflammatory gene expression in a trout macrophage cell line, suggesting it is an IL-1beta antagonist. Modeling studies confirmed that nIL-1F has the potential to bind to the trout IL-1RI receptor protein, and may be a novel IL-1 receptor antagonist.

beta(2)-Microglobulin-selective direct hemoperfusion column for the treatment of dialysis-related amyloidosis.

Lixelle is a direct hemoperfusion-type adsorption column that was developed to selectively eliminate beta2-microglobulin (beta2-m) from the circulating blood of patients with dialysis-related amyloidosis (DRA). The adsorbent in Lixelle comprises porous cellulose beads to which hydrophobic hexadecyl alkyl chain is covalently bound. One milliliter of wet Lixelle beads eliminates more than 1 mg of beta2-m in vitro. In hemodialysis patients who were treated with Lixelle, Lixelle improved joint pain, nocturnal awakening, pinch strength, motor terminal latency, and their activity of daily living. The adsorbent adsorbs beta2-m selectively but not specifically, as well as inflammatory cytokines such as interleukin-1beta and IL-6 which are considered to be involved in the development of DRA. Lixelle treatments reduce the circulating levels of beta2-m and inflammatory cytokines, thereby improving the symptoms of patients with DRA.

Synthesis and biological activities in vitro and in vivo of glycosylated human interleukin-1alpha, neoglyco IL-1alpha, coupled with N -acetylneuraminyl-galactose.

In order to study the effect of glycosylation on its biological activities, and to develop IL-1alpha with less deleterious effects, recombinant human IL-1alpha was chemically coupled with N -acetylneuraminic acid (alpha1-6) galactose (Neu5Ac-Gal). Glycosylated IL-1alpha (Neu5Ac-Gal-IL-1alpha) was purified by anion-exchange chromatography and average number of carbohydrate molecules introduced per molecule of IL-1alpha was 2.5. Neu5Ac-Gal-IL-1alpha exhibited reduced activities about 1/15-fold compared to IL-1alpha in all the activities performed in vitro. Binding affinities of Neu5Ac-Gal-IL-1alpha to Type I and Type II IL-1 receptors were decreased to 1/15 and 1/10, respectively. Neu5Ac-Gal-IL-1alpha exhibited reduction in activities in vivo, including induction of serum amyloid A and NOx, and down-regulation of serum glucose. However, Neu5Ac-Gal-IL-1alpha exhibited comparable activity to IL-1alpha in improvement of the recovery of peripheral white blood cells from myelosuppression in 5-fluorouracil-treated mice. In addition, tissue level of Neu5Ac-Gal-IL-1alpha was relatively high compared to IL-1alpha. These results indicate that coupling with Neu5Ac-Gal enabled us to develop neoIL-1alpha with selective activities in vivo.

Super high flux hemodialysis at high dialysate flows: an ex vivo assessment.

BACKGROUND AND OBJECTIVES: The removal of cytokines by standard hemofiltration is limited. Super high flux membranes may significantly improve removal even when used in dialysis mode. We sought to measure cytokine clearance using a large surface super high-flux membrane and a standard hemodialysis setting. SETTING: ICU laboratory of a tertiary institution. SUBJECTS: Six healthy volunteers. METHODS: Blood form healthy volunteers was incubated for 4 hours with E. coli endotoxin to stimulate cytokine production. Cytokine containing blood was then circulated through a dialysis circuit at 3 different dialysate flow rates. Blood and dialysate were sampled for cytokine and albumin measurements and calculation of clearances. RESULTS: Super high-flux dialysis achieved high median cytokine clearances (IL-1 clearance of 106 ml/min, IL-6 clearance of 66.8 ml/min, IL-8 clearance of 61.7 ml/min and TNF clearance of 36.1 ml/min). Increasing dialysate flow rate from 300 to 500 ml/min did not significantly increase cytokine clearances. Albumin clearances however were between 2.7 and 5.4 ml/min. CONCLUSIONS: Cytokine dialysis is feasible at high dialysate flow rates yielding high cytokine clearances. Albumin loss, however, is appreciable and may require separate supplementation in the clinical setting.

Preparation of novel mesoporous carbons for the adsorption of an inflammatory cytokine (IL-1 beta).

Mesoporous carbons derived from two types of sulphonated styrene divinylbenzene copolymers (Macronet MN500HS and CT275, Purolite International Ltd) were produced and their adsorptive capacity for the proinflammatory cytokine IL-1 beta (MW 14.4 kDa) determined. The carbons produced had surface areas from 400 to 1200 m(2)g(-1) and pore volume between 0.2 and 1.4 cm(3)g(-1). The mechanical strength of the carbon beads with surface area values up to 800 m(2)g(-1) were robust. The highest adsorption value of IL-1 beta was 150 ng g(-1) for a mesoporous carbon with surface area around 900 m(2)g(-1) and pore volume around 1.3 cm(3)g(-1). However, there was a trade-off between adsorptive capacity and mechanical strength. When used in conjunction with existing treatment modalities, the materials produced have the potential to enhance the removal of uraemic toxins.

Expression in Escherchia coli and purification of sea bass (Dicentrarchus labrax) interleukin 1beta, a possible immunoadjuvant in aquaculture.

Interleukin 1beta (IL-1beta) is a pleiotropic cytokine that plays a pivotal role in regulating immune responses. Our group has recently cloned IL-1beta from sea bass (Dicentrarchus labrax), one of the main Mediterranean aquacultured fish species. The cDNA is 1292 bp and codes for a deduced peptide of 29.4 kDa with a pI of 5.1. As for trout and carp IL-1beta precursor sequence, no candidate cut site for ICE (IL-1beta converting enzyme) enzyme was apparent in the alignments of sea bass IL-1beta with other mammalian IL-1betas. Nevertheless, a possible mature peptide could start at Ala86, giving a protein of 176 amino acids. The nucleotide sequence coding for this polypeptide was cloned into a pQE-30 expression vector. The plasmid was then transformed in Escherichia coli, and the recombinant protein was purified. Finally, we demonstrated that this purified recombinant IL-1beta was able to induce IL-1beta gene expression in a dose-dependent manner on cells purified from sea bass head kidney and could have immunoadjuvant effects in sea bass vaccination experiments.

Cytokine dialysis: an ex vivo study.

To test the hypothesis that dialysis using a new large pore membrane would achieve effective cytokine removal, blood from six volunteers was incubated with endotoxin (1 mg) and then circulated through a closed circuit with a polyamide membrane (nominal cut-off: 100 kDa). Hemodialysis was conducted at 1 or 9 L/hr of dialysate flow at the start of circulation and after 2 and 4 hours. The peak dialysate/plasma concentration ratios were 0.92 for interleukin (IL)-1beta, 0.67 for IL-6, 0.94 for IL-8, 0.33 for tumor necrosis factor (TNF)-a, and 0.11 for albumin. The dialysate/plasma ratios for all cytokines and albumin were decreased with increased dialysate flow from 1 to 9 L/hr (p < 0.05). Clearances for IL-1beta, IL-6, and IL-8, however, were significantly improved with increased dialysate flow (p < 0.01). There was no increase in TNF-a clearance (not significant) and a decrease in albumin clearance (p < 0.01). The peak clearance at 9 L/hr was 33 ml/min for IL-1beta, 19 for IL-6, 51 for IL-8, 11 for TNF-alpha, and 1.2 for albumin. No adsorption of cytokines was observed. We conclude that cytokine dialysis is achievable through a membrane with a high cut-off point with negligible albumin loss. These findings support the technical feasibility of this new approach to blood purification in sepsis.

Production and secretion of interleukin-1alpha proteins by rat testis.

The present study characterizes constitutively expressed rat testicular interleukin-1alpha (IL-1alpha) proteins. IL-1 bioactivity of crude testis protein was completely neutralized by IL-1alpha antiserum, IL-1 receptor antagonist, and soluble type I IL-1 receptor. Upon non-denaturating gel permeation chromatography, bioactive IL-1 eluted at molecular sizes of 45, 31, and 17kDa and at charges of pH 5.7 and 6.0 after chromatofocusing. SDS-PAGE/Western blot analysis of proteins extracted from whole testis, seminiferous tubules, interstitial, and seminiferous tubule fluids all demonstrated IL-1alpha immunoreactivity at 45, 24, and 19kDa. Activated macrophages and tissue proteins from endotoxin treated rats showed immunoreactive 31 and 19kDa IL-1alpha. The results indicate that the testis produces three isoforms of IL-1alpha proteins that are secreted into the interstitial compartment and tubular lumen where they may exert paracrine functions. The testicular IL-1alpha isoforms may represent posttranslationally modified precursor, mature IL-1alpha, and a 24-kDa alternate splice form.

Effects of equine recombinant interleukin-1alpha and interleukin-1beta on proteoglycan metabolism and prostaglandin E2 synthesis in equine articular cartilage explants.

OBJECTIVES: To evaluate the effects of equine recombinant interleukin-1alpha (rEqIL-1alpha) and recombinant interleukin-1beta (rEqIL-1beta) on proteoglycan metabolism and prostaglandin E2 (PGE2) synthesis by equine articular chondrocytes in explant culture. SAMPLE POPULATION: Near full-thickness articular cartilage explants (approx 50 mg) harvested from stifle joints of a 3-year-old and a 5-year-old horse. PROCEDURE: Expression constructs containing cDNA sequences encoding EqIL-1alpha and EqIL-1beta were generated, prokaryotically expressed, and the recombinant protein purified. Near full-thickness articular cartilage explants (approx 50 mg) harvested from stifle joints of a 3-year-old and a 5-year-old horse were separately randomized to receive rEqIL-1alpha or rEqIL-1beta treatments 10 to 500 ng/ml). Proteoglycan release was evaluated by 1,9-dimethylmethylene blue spectrophotometric analysis of explant media glycosaminoglycan (GAG) concentration and release of 35S-sulfate-labeled GAG to explant media. Proteoglycan synthesis was assessed by quantification of 35S-sulfate incorporation into proteoglycan. Explant media PGE2 concentrations were evaluated using a PGE2-specific enzyme-linked immunoassay. Data were collected at 48-hour intervals and normalized by DNA content. RESULTS: Proteoglycan release was induced by rEqIL-1alpha and rEqIL-1beta at concentrations > or =0.1 ng/ml, with 38 to 76% and 88 to 98% of total GAG released by 4 and 6 days, respectively. Inhibition of proteoglycan synthesis (42 to 64%) was observed at IL-1 concentrations > or = 0.1 ng/ml at 2 and 4 days. Increased PGE2 concentrations were observed at IL-1 concentrations > or = 0.1 ng/ml at 2 and 4 days. CONCLUSIONS AND CLINICAL RELEVANCE: The rEqIL-1 induced potent concentration-dependent derangement of equine chondrocyte metabolism in vitro. These findings suggest this model may be suitable for the in vitro study of the pathogenesis and treatment of joint disease in horses.

The first cytokine sequence within cartilaginous fish: IL-1 beta in the small spotted catshark (Scyliorhinus canicula).

Cartilaginous fish are considered the most primitive living jawed vertebrates with a complex immune system typical of all jawed vertebrates. Cytokine homologs are found within jawless and bony fish, although no cytokine or cytokine receptor genes have been sequenced in cartilaginous fish. In this study the complete coding sequence of the small spotted catshark (Scyliorhinus canicula) IL-1beta gene is presented that contains a short 5' untranslated region (54 bp), a 903-bp open reading frame, a 379-bp 3' untranslated region, a polyadenylation signal, and eight mRNA instability motifs. The predicted translation (301 amino acids) has highest identity to trout IL-1beta (31.7%), with greatest homology within the putative 12 beta-sheets. The IL-1 family signature is also present, but there is no apparent signal peptide. As with other nonmammalian IL-1beta sequences, the IL-1-converting enzyme cut site is absent. Expression of the IL-1beta transcript is detectable by RT-PCR in the spleen and testes, induced in vivo with LPS. Furthermore, a 7-fold increase of transcript levels in splenocytes incubated for 5 h with LPS was seen. The genomic organization comprises six exons and five introns with highest homology seen in exons encoding the largest amount of secondary structure per amino acid. Southern blot analysis suggests at least two copies of the IL-1beta gene or genes related to the 3' end of the IL-1beta sequence are present in the catshark. The cloning of IL-1beta in S. canicula, the first cytokine sequenced within cartilaginous fish, verifies previous bioactivity evidence for the presence of inflammatory cytokines.

Fas/FasL mediated apoptosis of thyrocytes in Graves' disease.

We examined in the present study the possible involvement of Fas and its ligand (FasL) in the process of Graves' disease. Immunohistochemical analysis showed that few normal thyrocytes expressed Fas but many thyrocytes in Graves' disease expressed this molecule. The percentage of FasL-positive thyrocytes in Graves' thyroids was, however, less than in normal thyroids. Several apoptotic thyrocytes and infiltrating mononuclear cells (MNCs) were detected scattered throughout Graves' thyroid tissues and abundant proliferating cell nuclear antigen (PCNA)-positive thyrocytes were present. Apoptotic cells, as well as PCNA-positive cells, were scarcely detectable in normal thyroid glands, however. In vitro treatment of thyrocytes by IL-1beta a cytokine found to be expressed in Graves' thyroid glands, increased Fas but reduced FasL expression. IL-1beta-stimulated thyrocytes became sensitive to apoptosis by anti-Fas IgM monoclonal antibody (mAb). Activated T cells, which strongly expressed FasL, showed cytotoxic activity toward IL-1beta-stimulated thyrocytes but not toward unstimulated thyrocytes. This cytotoxic activity involved the Fas/FasL pathway. Importantly, unstimulated thyrocytes could kill activated, but not resting, T cells. IL-1beta-stimulated thyrocytes, with down-regulated FasL expression, could not efficiently kill activated T cells. The cytotoxic activity of unstimulated thyrocytes toward activated T cells was inhibited by anti-FasL mAb. Interestingly, unstimulated thyrocytes induced apoptosis in IL-1beta-stimulated thyrocytes but not in unstimulated thyrocytes. These interactions were also blocked by anti-FasL mAb. Our results suggest that the apoptotic cell death of both thyrocytes and infiltrating MNCs found in Graves' thyroid glands is regulated by IL-1beta through Fas/FasL interactions.

Immunohistochemical staining of IL-1 alpha and IL-1 receptor antagonist but not IL-1 beta in cultures of sertoli cells.

The interleukin-1 (IL-1) system has been suggested to be involved in the cell cell cross talk within the testis. To identify a testicular cell source of IL-1 alpha, IL-1 beta and IL-1 receptor antagonist (IL-1ra), immature mouse Sertoli cells were isolated, purified, cultured and examined for the cellular compartment localization of these cytokines by immunohistochemical staining. Our results show that both Germ cells and Sertoli cells in unpurified Sertoli cell cultures (before hypotonic shock) and purified culture of Sertoli cells (after hypotonic shock) were stained for IL-1 alpha. The levels of this cytokine were increased in Sertoli cells when the purified cultures were stimulated with lipopolysaccharide (LPS) (5 microg/mL). However, we could not identify a positive staining for IL-1 beta when Sertoli cell cultures were stained for this cytokine, even after stimulation with various concentrations of LPS (0.1-10 microg/mL). On the other hand, immunohistochemical staining of isolated Sertoli cells without treatment with hypotonic shock (cultures containing Sertoli cells and Germ cells) for IL-1ra showed constitutive positive staining of both cell types (Sertoli cells and Germ cells). Our results, using immunohistochemical staining, may indicate the different expression of IL-1 alpha, IL-1 beta and IL-1ra in Sertoli cells. These results may suggest the involvement of IL-1 system in the autocrine and paracrine regulation of testicular cell functions.

Detection of monokines in paraffin-embedded tissues of pigs using polyclonal antibodies.

Monokines are glycoproteins, synthesised by macrophages, which exert various effects on the organism. The most important monokines are interleukin (IL)-1alpha, IL-1beta, tumor necrosis factor (TNF)-alpha and IL-6. This paper reports on immunohistochemical techniques developed for the detection of IL-alpha, IL-1beta, IL-6 and TNF-alpha in fixed and paraffin-embedded pig tissues (spleen, lymph nodes, thymus, liver and kidney). Different fixatives (buffered formalin, acetic formalin, paraformadehyde-lysine-periodate and Bouin solution), and antigen unmasking techniques (permeabilisation with Tween 20, pronase enzymatic digestion and microwave-citrate buffer) were used. We describe different protocols for detection of monokines using polyclonal antibodies against the studied monokines. No signal was obtained with monoclonal antibodies against pig-TNF-alpha and human IL-1alpha. Bouin solution was shown to be the best fixative for immunohistochemical detection of IL-1alpha, TNF-alpha, and IL-6, using permeabilisation with Tween 20 as an unmasking antigen method. Acetic formalin was shown to be the best fixative for IL-1beta detection, not needing antigen retrieval techniques. Macrophages were identified as the main cytokine-producing cells, although other types of cells also stained positively to some cytokines. These techniques represent valuable tools for studies of the pathogenesis of viral and bacterial diseases, and of the immune system of the pigs.

Can cytokines be removed by hemofiltration or hemoadsorption?

To study the removability of pro-inflammatory cytokines by hemofiltration (HF), we performed experimental HF with various high-flux membranes (HFM) using a closed circuit system filled with monocyte-free human plasma, which contained TNFalpha, IL-1beta, and IL-6. Plasma and filtrate samples were taken before and 1, 2, 3, and 4 hours after the initiation of HF, and each cytokine was determined by enzyme-linked immunosorbent assay. IL-1beta was well removed through filtration during experimental HF using HFM (PAN>CTA>PMMA>PS). TNFalpha and IL-6 were only minimally filtered out by HF using HFM. TNFalpha was removed to some extent by using PS, and IL-6 was partially removed by using PMMA during experimental HF through other mechanisms, such as adsorption, than the filtration. IL-1beta and IL-6 were effectively removed by HA using charcoal adsorbent column, especially during the first 2 hours, while TNFalpha was only partly removed.