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Veterinary Immunology and Immunopathology 99 (2004) 237–243 Modulation of the cytokine responses in equine Joris J. Wijnker, Sarah BullPaul van DijkJanine N. Veenman Victor P. Rutten, Wim R. KleinJohanna Fink-Gremmels aDepartment of Equine Sciences, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 12, 3584 CM Utrecht, The Netherlands bDepartment of Veterinary Pharmacology, Pharmacy and Toxicology, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 12, 3584 CM Utrecht, The Netherlands cDepartment of Infectious Diseases, Division of Immunology, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 12, 3584 CM Utrecht, The Netherlands Received 23 April 2003; received in revised form 20 November 2003; accepted 26 January 2004 The detrimental effects of the pro-inflammatory cytokine TNF-a during equine acute abdominal disease are well known. Its pivotal role in many human diseases has led to various in-depth studies regarding its release mechanism, in particular by TNF-aconverting enzyme (TACE). In this study we investigated the inhibitory effect of a TACE-inhibitor on cytokine release (TNF-a,IL-1a and IL-6) in three different cell models, including U937 cells, a recently established equine macrophage cell line, knownas eCAS, and primary equine PBMC. The aim was to show the similarity of TNF-a release through the TACE mechanism inhuman and equine cells after stimulation with LPS. Results indicate that, by using a TACE-inhibitor, TNF-a, IL-1a and IL-6release can be reduced in both equine cell models and achieved comparable results in the human U937 cells. These resultssuggest that equine TNF-a, like its human homologue, is released from its membrane-bound position by TACE.
# 2004 Elsevier B.V. All rights reserved.
Keywords: Equine acute abdominal disease; TACE; Cytokines; eCAS cells The involvement of the pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-a) during EAD Many of the systemic consequences of equine acute instigated many studies that investigated the mechan- abdominal disease (EAD) can be attributed to a dis- isms of its release and its specific effect on subsequent ruption of the intestinal wall barrier caused by inflam- pathology. Data obtained from these studies confirmed matory or ischemic lesions, allowing the translocation TNF-a as being a pivotal factor in equine endotoxemia of enteric bacteria and endotoxins into the circulation regulation of TNF-a release led to the discovery of theenzyme responsible for the release of TNF-a, namely Corresponding author. Tel.: þ31-30-2533562; fax: þ31-30-2534125.
E-mail address: (S. Bull).
described the unique binding between TACE and 0165-2427/$ – see front matter # 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.vetimm.2004.01.005 J.J. Wijnker et al. / Veterinary Immunology and Immunopathology 99 (2004) 237–243 from Sigma Chemicals (St. Louis, MO, USA). Non-essential amino acids, glutamine, fetal calf serum(FCS), horse serum (HS), penicillin/streptomycin,sodium pyruvate, RPMI 1640 medium, and Dulbec-co’s modified eagle medium (DMEM) were purchased Fig. 1. Chemical structure of compound 6a (Fur-Leu-PTrp(OH)2, from Invitrogen (Breda, The Netherlands). Ficoll- paqueTM plus was obtained from Pharmacia Biotech,Amersham Biosciences (Uppsala, Sweden). Gentami- pro-TNF-a (membrane-bound TNF-a). Based on cin was purchased from Eurovet (Bladel, The Nether- these and comparable findings from other groups lands), and disposable 24-well plates were bought from Corning Inc. Costar (Badhoevedorp, The Nether- determined that TACE belongs to a larger family of lands). Alamar Blue was acquired from BioSource cell surface proteins (a desintegrin and metallopro- International (Etten-Leur, The Netherlands). Com- tease, or ADAM family), belonging to a superfamily pound 6a, the phosphonate analogue N-(Furan-2- of Zn-dependent metalloproteases known as metzin- yl)carbonyl-Leu-PTrp(OH)2, was a kind gift from cins, which also includes the matrix metalloprotei- Dr. Gallina, Uni. G. d’Annunzio, Chieti It. and Poli- Several zinc protease inhibitors have been devel- oped which inhibit MMP release. A newly designed protease inhibitor (designated compound 6a, showed an evident inhibitory effect against adamaly- The human monoblastoid tumor cell line or U937 sin II and several MMPs, as evaluated by the residual enzyme activity by continuous fluorimetric assays 85011440, UK) were cultured in RPMI 1640 medium, supplemented with 10% FCS, 100 U/ml penicillin and pound 6a may exert inhibitory effects on adamalysin 100 mg/ml streptomycin, according to the protocol tions. The U937 cells were plated in 24-well plates release of membrane-bound TNF-a. Furthermore, (2 Â 105 cells/ml) and incubated at 37 8C, 5% CO2 for MMPs have been shown to activate the precursor of 24 h. Subsequently, cells were induced to differentiate IL-1b but to inactivate the mature cytokine, thus may along the monocyte/macrophage lineage by the addi- be involved in the regulation of IL-1b activities at sites tion of PMA (50 ng per well) for 24 h at 37 8C, 5% Therefore the aim of this study was to investigate the inhibitory effects of a TACE inhibitor (compound 6a) on cytokine (TNF-a, IL-1a and IL-6) releasefollowing LPS stimulation in two equine and one The equine bone-marrow-derived macrophage cell human in vitro cell model as a prelude to further were subcultured every 3–4 days. Cells were seeded in24-well plates (2 Â 105 cells/ml) and incubated inRPMI 1640 medium, supplemented with 10% HS, 100 U/ml penicillin, 100 mg/ml streptomycin, 1%non-essential amino acids and 1 mM sodium pyruvate, for 24 h at 37 8C, 5% CO2 prior to testing.
Phosphate buffer saline (PBS), lipopolysaccharide (Escherichia coli, O111: B4) (LPS), dimethyl sulf-oxide (DMSO), phorbol myristate acetate (PMA) and Blood was collected in heparinized tubes from three methyl-thiazolyl tertrazolium (MTT) were obtained healthy horses. Blood was layered on pre-warmed J.J. Wijnker et al. / Veterinary Immunology and Immunopathology 99 (2004) 237–243 Ficoll-paqueTM plus (3:4 ratio) and centrifuged at and at an emission at 590 Æ 10 nm. Results are 400 Â g for 30 min at 22 8C. The buffy coat was transferred to a 15 ml centrifuge tube and the cells washed twice with PBS at 650 Â g for 10 min at 22 8C.
The cell pellet was re-suspended in 1–2 ml DMEM supplemented with 10% FCS, 0.1 mg/ml gentamicin,2 mM glutamine and seeded into 24-well plates (1 Â 106 cells/ml per well). Cells were incubated at37 8C and 5% CO2, and the medium replaced after 2 h TNF-a concentrations in cell culture supernatants were measured in a cytotoxicity assay using a porcinekidney cell line (PK-15) according to the method of Validation of the TNF-a bioassayfor its use in horses was described by In order to establish the appropriate concentration . Interleukin-1a and IL-6 concentrations were of compound 6a and DMSO for further experiments, a measured using a murine T helper cell line (D10), dose–response curve was constructed in PBMC, trea- ted with different concentrations of both compound 6a murine B cell hybridoma cell line (7-TD1), described and DMSO. From preliminary data, 500 mM com- pound 6a and 0.063% DMSO were selected. At this induced cytotoxicity in PK-15 cells and the IL-1a concentration, DMSO had no inhibitory effect on and IL-6-induced proliferation in D10 and 7-TD1 LPS-induced TNF-a release, and neither DMSO nor cells, were determined by using the MTT assay. A compound 6a caused significant cytotoxicity in standard curve using rat (for TNF-a) and mouse (for IL-1a and IL-6) cytokines was generated that acted asreference with which to calculate the cytokine con- centrations in the samples. The range of these standardcurves was 0.256–20.000, 0.113–20.000 and 0.056– Following the initial incubation period, the med- 10.000 U for TNF-a, IL-1a and IL-6, respectively.
ium was removed and replaced by fresh medium,supplemented with LPS (1 mg/ml). Cells were simul- taneously incubated with either DMSO (0.063%,positive control) or compound 6a (500 mM) for 4 A Student’s t-test was used to assess if LPS induced or 24 h, after which time supernatants were collected a significant cytokine response in all three models.
for TNF-a or IL-1a and IL-6 analysis, respectively.
Data are expressed as mean Æ standard deviation Prior to analysis, all samples were stored at (S.D.) of three independent experiments carried out in duplicate. Data analyzed for statistical significanceby one-way ANOVA, followed by a Dunnett’s multi- ple comparison test (Graph Pad Prism1 used asstatistical software). LPS with DMSO was regarded Cell viability following treatment with compound as positive control and set at 100%, and PBS with 6a was measured by using the Alamar Blue reduction DMSO as the negative control. Data showing a prob- ability value of P < 0:05 was considered statistically brief, following treatment of eCAS cells, U937 cells or PBMC to LPS for 24 h, the medium was removed andthe cells were washed twice with warmed PBS. Mediacontaining Alamar Blue (diluted 1:10) was incubated with the cells at 37 8C for 3 h. Resorufin, the fluor-escent reduced Alamar Blue product, was measured U937 cells, eCAS cells and PBMC were stimulated fluorometrically with an excitation at 530 Æ 15 nm with LPS following which the cytokine responses J.J. Wijnker et al. / Veterinary Immunology and Immunopathology 99 (2004) 237–243 Following compound 6a administration, the cyto- kine modulation was measured in the three cell mod-els. In the U937 cells, compound 6a caused asignificant reduction in the release of TNF-a to 20 Æ27% and IL-1a to 67 Æ 4% whereas no significantreduction of IL-6 release was observed. A significantreduction in the release of TNF-a to 22 Æ 10%, andIL-1a to 48 Æ 26% in eCAS cells was observed,following compound 6a treatment. Compound 6aexerted no significant reduction on IL-6 release. InPBMC, compound 6a significantly decreased therelease of TNF-a to 46 Æ 12% and IL-6 to 67 Æ10% The established importance of TNF-a during equine endotoxaemia, and the recent identificationof a specific enzyme responsible for TNF-a release(TACE) in humans, led to our investigation towardsthe effects of a synthetic TACE-inhibitor, compound6a (), in LPS-stimulated equineand human cells. Considering the possible differ-ences in catalytic activity of TACE in humans andhorses, the TACE inhibitor was tested in humanU937 cells, equine PBMC and a recently developedequine macrophage cell line, eCAS cells. The U937human macrophage cell line was selected for com-parison with the eCAS cells, on account of themechanism of TACE-inhibitors being previouslydescribed in human cells. usedMono Mac-6 cells and human PBMC to test theTACE-inhibitor GW3333. Additionally, U937 cellsand human PBMC were used to test the influence of Fig. 2. Effect of compound 6a on LPS-induced cytokine release in histamine on TNF-a and TACE expression and three model systems: (A) U937 cells, (B) eCAS cells and (C) PBMC. Cells were treated with LPS (1 mg/ml) and compound 6a The choice for the PBMC was based on the ease of (500 mM), and cytokine release measured. Positive controls are setat 100%. Data are presented as mean Æ S:D: and are calculated availability of equine blood samples and due to PBMC from three independent experiments carried out in duplicate. Data being extensively used in human studies. A major are considered significantly different when *P < 0:05.
drawback of the equine primary cells is the highvariability in magnitude of responses to LPS stimula- were measured. U937 cells showed a significant tion between the different test subjects and the variable increase in TNF-a, IL-1a and IL-6 response (336, response within one subject over time (Bull, unpub- 24410, 5613 U/ml, respectively) following LPS sti- lished data). This variability in response was observed mulation (). Similarly, TNF-a, IL-1a and IL-6 within the group of horses used. In addition, un- also increased (23 330, 2816, 36 240 000 U/ml, stimulated, negative control cells produced relatively respectively) in eCAS cells. In PBMC only TNF-a high background levels of cytokines, due to the con- (1532 U/ml) and IL-6 (7196 U/ml) were significantly tinuous challenge by external stimulants under field increased. There was no significant loss of cell via- conditions. Both of these factors may account for the bility in any of the cells tested following LPS admin- lack of significant increase of IL-1a following LPS J.J. Wijnker et al. / Veterinary Immunology and Immunopathology 99 (2004) 237–243 The equine macrophage cell line (eCAS cells, the transcription of other inflammatory mediators, ity-orientated study, showed less variability in decreased to a lesser extent as compared to TNF-a, response than the model based on primary equine possibly due to it being produced, not only by TNF-a- stimulated mechanisms, but also under direct influ- standardized tests were carried out to confirm these ence of LPS. Matrix metalloproteinases have been eCAS cells as mononuclear phagocytes. This eCAS cell line provided the opportunity to study the species- It is therefore conceivable that the TACE specific effects of the TACE-inhibitor compound 6a on inhibitor elicits some MMP inhibitory effects that the reduction of TNF-a, IL-1a and IL-6 release.
may decrease the amount of IL-1 produced. However, Due to the limited availability of compound 6a, detailed enzyme kinetic studies could not be carried showed IC50 (mM) values for adamalysin II, out. Preliminary dose–response curves showed a max- MMP-2, MMP-9 and MMP-3 to be 0.4, 60, >100 and imal inhibitory effect on TNF-a release at 500 mM >100, respectively, indicating the specificity of com- (data not shown), without compromising cytotoxicity, pound 6a for adamalysin II rather than MMPs. A hence this concentration was selected for further use.
reduction in IL-6 release was not evident, possibly Compound 6a, according to manufacturer’s instruc- due to the synergistic effects of the remaining TNF-a tions, is sparingly soluble in water, making it neces- and IL-1a, even after TACE inhibition, on its tran- sary to use a co-solvent. DMSO was chosen despite its known inhibitory effect on TNF-a production ( In the PBMC model, LPS-induced TNF-a and IL-6 its negligible inhibitory effects on cytokine production release were significantly reduced following treatment at the used concentration. Furthermore, mass spectro- with compound 6a. LPS failed to stimulate a signifi- cant increase in IL-1a release in PBMC accounting for (0.063%) and compound 6a (500 mM; Wijnker and the lack in reduction after TACE inhibition. Following a stringent isolation procedure, the final cell popula- In our experiments, we demonstrated the inhibitory tion still represents a mixed cell population consisting effect of compound 6a on TNF-a release in the eCAS of monocytes, lymphocytes and other contaminants, and U937 cells. This response to a TACE-inhibitor as differential counts indicated that a culture of mono- strongly suggests the presence of a TACE-releasing cytes of 90–95% purity was obtained. Following the mechanism for TNF-a in eCAS cells, similar to that of LPS induction, early activation of Nf-kB may result in U937 cells, and possibly also in equine PBMC. To our the production of TNF-a and IL-1a, due to increased knowledge, compound 6a has not been previously gene expression and protein synthesis, which occurs shown to block TNF-a release and TACE activity in During the 24 h incubation period in this study, the plete inhibition in TNF-a release due to a TACE- production of IL-1a may have been restored, resulting inhibitor will, however, be difficult to achieve due to in higher IL-1a release. TNF-a levels remain inhib- the existence of alternative release-pathways of TNF- ited, as de novo synthesis of TACE is required to restore ‘normal’ levels. In previous studies, IL-6 was ), or using a different secretase than TACE such produced during the late phase of Nf-kB activation, as collagenase, stromelysin, gelatinase and matrilysin due to this late response, decreased levels are still Inhibition of LPS-induced IL-1a release was also evident at 24 h. Delayed IL-6 production was also observed in the eCAS and U937 cells following treatment with compound 6a. A reduction in TNF-a nificant production of IL-6 only after 24 h.
release following TACE inhibition may lead to the In conclusion, we showed that TNF-a release in diminished activation of signal transduction pathways, an equine cell model can be reduced by using a such as the Nf-kB pathway, which in turn diminishes TNF-a converting-enzyme-inhibitor, and achieved J.J. Wijnker et al. / Veterinary Immunology and Immunopathology 99 (2004) 237–243 comparable results to those seen in a human cell model.
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