Increased intestinal TNF-α, IL-1β and IL-6 expression in ovine paratuberculosis

Veterinary

ELSEVIER

Immunology and Immunopathology 49 (1996) 331-345

Veterinary immunology and immunopathology

Increased intestinal TNF-a, IL-l /? and IL-6 expression in ovine paratuberculosis H.M. Alzuherri, C.J. Woodall, C.J. Clarke Department

*

of Veterinary Pathology, UniuersiQ of Edinburgh, Veterinary Field Station, Easter Bush, Roslin EH2.5 9RG. CJK Accepted

3 May 1995

Abstract Mycobacterium avium subspecies paratuberculosis is an intracellular parasite of intestinal macrophages and causes a chronic granulomatous enteritis in sheep and other ruminants (paratuberculosis or Johne’s disease). Macrophages can produce a variety of immunoregulatory cytokines that may influence mycobacterial killing and produce disordered inflammation within the gut. In this study, messenger RNA (mRNA1 was extracted from intestinal tissue from control and multibacillary diseased sheep and profiles for the cytokines tumour necrosis factor-a (TNF-a 1, interleukin-1 p (IL-1 /3 1, IL-6, transforming growth factor-p 1 (TGF-fi 1) and granulocyte-macrophage colony stimulating factor (GM-CSF) were semi-quantified using reverse transcriptase polymerase chain reactions (RT-PCR). Infected intestinal tissues had significantly increased mRNA for TNF-cu, IL-lp and IL-6 but TGF-Pl and GM-CSF mRNA levels were not significantly different from controls. Supernatants from in vitro intestinal cultures were assayed for TNF-(r activity using the PK(lSl-1512 cytotoxicity bioassay and levels were significantly raised in diseased samples. TNF-cx was not detected in any serum samples. Further analysis on intestinal tissues from sheep with the different, paucibacillary, form of disease showed significant elevation of TNF-a mRNA but not other cytokines tested. Increased pro-inflammatory cytokine expression in the intestine coincident with a failed or misdirected immune response may contribute to the pathogenesis of paratuberculosis and the persistence of a chronic inflammatory state. Keywords: Ovine; Cytokine; Intestine; Paratuberculosis; Mycobacteria

* Corresponding author: Tel. 0131-650 8802; Fax. 0131445 5770; E-mail [email protected]. 0165-2427/96/$15.00 0 1996 Elsevier Science B.V. All rights reserved SSDI 0165-2427(95)05477-4

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1. Abbreviations cDNA, ComplementaryDNA; GM-CSF, Granulocyte-macrophage colony stimulating factor; IL, Interleukin; mRNA, Messenger RNA; PCR, Polymerase chain reaction; TGF-P 1, Transforming growth factor-pl; TNF-(u, Tumour necrosis factor-a.

2. Introduction Paratuberculosis or Johne’s disease is a chronic granulomatous enteritis of sheep and other ruminants caused by Mycobacterium avium subspecies paratuberculosis. The disease causes subclinical production losses, chronic weight loss and death and is of worldwide economic importance. Following oral infection, mycobacteria invade intestinal macrophages and are capable of resisting host defences and multiplying to reach very high numbers intracellularly (multibacillary form). Such events may take years to develop and are usually accompanied by marked inflammatory changes in the intestine including a profound monocyte/ macrophage infiltration. Many of these recruited cells subsequently become infected (Chiodini et al., 1984). The macrophage has a central role in mycobacterial killing, antigen processing and presentation, and directing inflammatory and immune processes. These mechanisms are influenced strongly by macrophage cytokines such as tumour necrosis factor-a (TNF-(~1, interleukin-l/3 (IL-l /3>, IL-6, granulocyte-macrophage colony stimulating factor (GM-CSF) and transforming growth factor p (TGF-P 1. These have pleiotropic effects but TNF-(Y, IL-lp, IL-6, and GM-CSF are generally pro-inflammatory and upregulate macrophage killing mechanisms. Inflammatory cell recruitment, acute phase protein induction, protein catabolism and lymphocyte activation and differentiation are also stimulated. TNF-(Y is produced mainly by activated macrophages and has previously been shown to be associated with cachexia, granulomatous inflammation and enteropathy (Beutler and Cerami, 1989; Amiri et al., 1992; Garside and Mowat, 1993). IL-1 is also a major macrophage product with a fundamental role in immunoregulation and inflammation (Dinarello, 1991). Monocytes, macrophages and stromal cells can produce IL-6 and lead particularly to acute phase protein production (Gross et al., 1992) whilst GM-CSF enhances granulocyte and macrophage activities (McInnes, 1993). Mycobacteria may stimulate macrophage cytokine production but they are also refractory to many intracellular killing mechanisms (Neil1 and Klebanoff, 1988; Sibley et al., 1988, Bradbury and Moreno, 19931. Their persistence in high numbers may thus encourage the continued production of pro-inflammatory cytokines leading to a state of chronic enteritis and clinical disease. In other mycobacterial diseases such as tuberculosis and leprosy, raised cytokine expression by macrophages has been implicated in the aetiopathogenesis of the chronic inflammatory lesions (Arnoldi et al., 1990; Barnes et al., 1990; Parida and Grau, 1993). Investigations into inflammatory bowel diseases of man have also suggested a pathogenic role for macrophage-derived cytokines. In particular, elevated TNF-ty, IL-lp and IL-6 have been found in intestinal tissues and blood from Crohn’s disease and ulcerative colitis patients (Mazlam and Hodgson, 1992; Cappello et al., 1992; Gross et al., 1992; Isaacs et al., 1992; Reinecker et al., 1993). Cytokine studies in ruminant paratuberculo-

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sis are limited but increased IL-1 activity of blood monocytes from chronically infected cattle and TNF-a, IL-l/3 and IL-6 activity in in vitro infected bovine monocytes has been recorded (Kreeger et al., 1991; Adams and Czuprynski, 1994). Lesions in paratuberculosis are usually confined to the intestine and associated lymphoid tissues but the cytokine profile at the site of disease has not been previously reported. In this study we have investigated intestinal cytokine expression in eight sheep with the multibacillary form of paratuberculosis and eight healthy control sheep by mRNA extraction and reverse transcription-polymerase chain reaction (RT-PCR). In addition TNF-(Y bioactivity from intestinal culture supernatants and serum has been measured in vitro. A further six sheep with the different, paucibacillary form of disease have also been investigated by these methods.

3. Materials and methods 3.1. Animals and sampling procedures Diseased adult Scottish Blackface ewes from southern Scotland were confirmed as infected on the basis of clinical signs (e.g. progressive weight loss), serological testing (agar gel immunodiffusion test; AGID), gross pathology, histopathology and PCR analysis of intestinal tissue for the IS-900 specific DNA sequence of M.a.paratuberculosis. Both multibacillary (n = 8) and paucibacillary (n = 6) forms of pathology were recognised on histopathological examination of intestine. Age, breed and sex-matched normal healthy sheep were selected as controls (n = 8). Serum samples were collected immediately prior to death. Sheep were killed by intravenous barbiturates and small samples of fresh distal ileum were immediately snap frozen in isopentane/dry ice slush and stored at - 70°C until used for mRNA extraction. Other ileal samples were washed in ice-cold Hank’s balanced salt solution (HBSS; Gibco BRL, Paisley, UK) and divided into 100 mg pieces for the gut explant culture. Each sheep underwent a full necropsy with associated histopathology of tissues. 3.2. RNA preparation Total cellular mRNA was isolated from frozen tissues using the acid guanidium thiocyanate-phenol-chloroform extraction procedure (Chomczynski and Sacchi, 1987). RNA was reverse-transcribed into complementary DNA (cDNA) using murine leukemia virus (MLV) reverse transcriptase (Gibco BRL) for 45 min at 37°C and stored at - 20°C until use. 3.3. Polymerase chain reaction Ovine oligonucleotide primers specific for ATPase (Woodall et al., 1994a), TNF-a, IL-l p (Woodall et al., unpublished data), IL-6 (Ebrahimi et al., 1995), TGF-1 (Woodall et al., 1994b), GM-CSF (McInnes and Haig, 1991) and the IS-900 sequence of M.a.paratubercu1o.G (Vary et al., 1990) were prepared.

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For each PCR assay, 3 ~1 of cDNA were added to a 47 ,ul reaction premix consisting of 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2.5 mM MgCl? and 20 mM of a mixture of dATP, dGTP, dCTP, dTTP, and Wl detergent (Gibco BRL) that was overlaid with mineral oil. Samples were heated to 95°C for 5 min then reduced to 80°C and 0.3 ~1 (1.5 unit) of Taq polymerase added. Samples were amplified by denaturation at 95°C for 1 min; annealing at 55°C (ATPase, GM-CSF and IS-900>, 61°C (TNF-a and TGF-Pl) or 58°C (IL-lp); primers extension at 72°C for 2 min. The final cycle extension was at 72°C for 5 min. Samples for ATPase, IL-l& GM-CSF and IS-900 were run for 33 cycles and TNF-a and TGF-Pl for 30 cycles. Amplification procedures for IL-6 differed as follows: denaturation at 95°C for 1 min, annealing at 55°C for 1 min and extension at 72°C for 3 min, for two cycles, then for 33 cycles as follows: denaturation at 95°C for 1 min, annealing at 65°C for 1 min and extension at 72°C for 2 min, and a final cycle extension of 5 min. For each assay, a negative control sample containing only sterile water (Sigma, Poole, UK) and an appropriate positive control for each cytokine was run in parallel. These PCR parameters were designed to produce subsaturation levels of PCR product. 3.4. Analysis

of PCR products

Aliquots of 7 ~1 PCR product were loaded onto a 2% agarose gel in Tris-acetateethylenediaminetetraacetic acid buffer (pH 8.0). A 1 kbp ladder (Gibco BRL) was used to assess sample size. Gels were stained with ethidium bromide and photographed under ultraviolet light. ATPase was assayed on all samples to verify efficient cDNA synthesis from the extracted RNA. 3.5. Southern

blotting and hybridisation

Gels were Southern blotted onto Gene Screen plus nylon membrane (NEN) by alkaline transfer according to the manufacturer’s instructions. After capillary transfer, membranes were UV-cross linked and hybridised at 65°C overnight with specific radiolabeiled internal ovine probes for ATPase and each cytokine. Membranes were autoradiographed with Kodak X-OMAT film for 4 h. Autoradiographs were analysed by measuring the integrated intensity of each band using a Bio Image analyzer (Millipore, Ann Arbor, USA). Integrated intensities for each cytokine were normalised according to the ATPase results to give a semi-quantitative assessment of each cytokine. 3.6. In vitro intestinal culture Procedures were based on similar techniques employed in man and mouse (Crabtree et al., 1991; Clarke and Stokes, 1992). Fresh, HBSS-washed ileal tissue was cut into 100 mg pieces and each was immediately placed into 2 ml complete culture media (RPM1 1640 medium containing 10% foetal calf serum (FCS), 2 mM L-glutamine, 50 pg ml-’ amphotericin B and 100 pg ml-’ penicillin/ streptomycin) in 24-well culture plates (all from Gibco BRL). Negative control culture media without tissue were also used. Cultures were incubated in quadruplet at 37°C in a 5% CO, humidified atmosphere for

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24 h. Supernatants were harvested, clarified by centrifugation, Millipore filter and stored at -70°C until assayed. 3.7. PK(15)-1512

cytotoxicity

filtered

through

335

a

assay for TNF-a

Cytotoxicity assays were performed as described previously (Green et al., 1993). Briefly, 2.5 X lo4 PK(15) cells were cultured in wells of 96-well flat-bottomed cell culture plates in 100 ~1 Eagle’s modified essential medium (EMEM) with 7% FCS (Gibco BRL) in a humidified 5% CO, atmosphere at 37°C. After 24 h, the medium was removed and replaced with 50 ~1 basal Iscove’s medium with 7.5% FCS (Gibco BRL) and 3 pg ml-’ Actinomycin D (Sigma). After 2 h, 50 ~1 of serially diluted samples, recombinant ovine TNF-(Y (rovTNF-cu) standards and media controls were added in duplicate. After a further 18 h incubation wells were pulsed with 50 ~1 of a 3 mg ml I solution of 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MIT) (S’g 1 ma ) m . d’stll I I e d water for 3 h. The medium was aspirated and the well contents were solubilised with 36 mM aqueous HCI in isopropanol. Optical density (OD) readings for each well were determined at 570 nm using a spectrophotometer. Using a plot of log concentration of rovTNF-a against OD values and allowing for subtraction of negative control values, a TNF-ar activity value for the samples was calculated. 3.8. Statistical

analysis

Data for each cytokine appeared to be distributed non-parametrically so sample comparisons between diseased and control animal groups were made using the Kruskal-Wallis analysis of variance rank test in the first instance for comparisons between three groups, and the Mann-Whitney rank test for comparing two groups, with the significance level taken as 5% probability.

4. Results 4.1. Clinical signs, serology and pathology All diseased sheep had a history of progressive weight loss and were in poor condition. Serological testing for paratuberculosis (AGID) gave a positive result for all sheep with multibacillary lesions. Only l/6 sheep with paucibacillary lesions was positive by AGID whilst all controls were negative. Distinctive gross lesions of paratuberculosis were recognised in all diseased sheep and included intestinal thickening and mucosal ridging, luminal dilation, intestinal lymphangitis and lymphadenitis. Most multibacillary forms had pigmented yellow intestinal mucosae. Histopathological examination of intestine from multibacillary cases showed a very marked mucosal and submucosal infiltration by sheets of predominantly epithelioid-type cells with abundant cytoplasm and fewer other inflammatory cell types such as lymphocytes, neutrophil polymorphs and Giant cells (Fig. l(a)). Villous atrophy and disruption of the normal mucosal architecture was marked. Ziehl-Neelsen (ZN) staining of sections showed the

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Fig. 1. Typical histological lesions in the ileum of a sheep with the multibacillary form of paratuberculosis. (a) Marked infiltration of macrophages with abundant cytoplasm (M) into the lamina propria with separation of crypt epithelium (El. HE stain, high power. (b) Large numbers of mycobacteria (arrows) within the macrophage cytoplasm. ZN stain, high power.

epithelioid cell cytoplasm to be packed with large numbers of acid-fast organisms (Fig. l(b)). Paucibacillary forms of disease showed a less pronounced epithelioid cell infiltration with a multifocal distribution of small granulomas. Lymphocytic infiltration was more marked and Giant cells were more common (Fig. 2(a)). Acid-fast organisms were very few or absent in this form of disease (Fig. 2(b)). Control sheep were found to be free of significant disease by gross and histological examination.

4.2. PCR analysis of intestinal cytokines and Mycobacterium

paratuberculosis

PCR amplification of segments of cDNA from frozen gut showed positive signals for ATPase that were of similar autoradiograph intensity. Internal negative and positive

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controls indicated no contamination. In the multibacillary group, TNF-o, IL-lp, IL-6 and TGF-/31 mRNA were detected in all eight samples whilst the number of controls expressing mRNAs were 7/S, 6/8, 5/8 and 6/8 respectively (Figs. 3(a)-3(d)). To improve the comparability of data the integrated intensity readings of autoradiographs for each cytokine were quantified after normalising them on the basis of an empirical value of 1000 units for ATPase expression (Fig. 4). Mann-Whitney rank testing of the data showed that infected animals had significantly increased levels of expression of TNF-a, IL-l/3 and IL-6 compared with controls (P < 0.005, P < 0.01 and P < 0.05 respectively). TGF-j31 expression was not significantly different between groups although the diseased group median value was higher. GM-CSF was expressed in only

Fig. 2. Typical histological lesions in the ileum of a sheep with the paucibacilhuy form of paratuberculosis. (a) Few macrophages forming a small focal granuloma (~3) and a marked lymphocyte (L) infiltration into the lamina propria. HE stain, medium power. (b) Very small number of intracellular mycobacteria (arrow). ZN stain. high power.

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2/S diseased samples and was not detected in any controls (Fig. 3(e)) and no significant difference between groups was detected (Table 1). All the multibacillary samples showed strong expression of PCR-amplified IS-900 cDNA indicating the specific presence of M.a.paratubercu1o.G whilst control samples all gave a negative result (Fig. 5). Tissues from the paucibacillary group (n = 6) were assayed with the same controls and values normalised with multibacillary samples on this basis. Kruskal-Wallis analysis showed significant differences between the three groups for TNF-a and IL-l p mRNA (P < 0.05 for both). The paucibacillary group had a higher median expression of TNF-a and IL-lp mRNA compared with controls, but only TNF-a was significantly

(8)

(b)

N Fig. 3. Intestinal cytokine mRNA expression in diseased and control sheep. Autoradiographs of cDNA PCR products analysed on a 2% agarose gel, blotted onto nitrocellulose membrane and hybridised with specific radiolabelled internal primers. (a) TNF-a; (b) IL-lo; (c) IL-6; (d) TGF-PI; (e) GM-CSF. For each cytokine: top rows show diseased (multibacillary) samples, lanes 1-8; bottom rows show control sheep samples, lanes 1-8; P, positive control PCR products; N, negative controls; lanes labelled with arrowheads indicate sheep not used in the analysis because of significant intercurrent disease.

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elevated (P < 0.05) (Table 1). There was no significant difference between TNF-(Y and IL-lp mRNA levels in multibacillary and paucibacillary groups (P > 0.05). None of the paucibacillary group gave a positive result with the M.a.paratuberculosis PCR test, indicating the absence of mycobacteria in some sites or a presence below the sensitivity level of the assay.

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Fig. 4. Scattergram of mRNA levels for various cytokines in the intestines of individual sheep. Values are based on normalised densitometry readings of autoradiographs. Filled circles, control sheep (n = 8); open circles, multibaciilary sheep (n = 8); open triangles, paucibacillary sheep (n = 6).

4.3. TNF-CY cytotoxicity

assay

Cytotoxic TNF-a activity in supernatant from in vitro intestinal cultures was present in 4/7 samples from multibacillary sheep (median value 0.30 ng ml-‘, range O-124 ng ml-‘) but in only l/9 samples from control sheep (0.05 ng ml-’ ). The samples from

Table 1 Intestinal cytokine group a

mRNA expression

in diseased and control sheep. Values given as median (range) for each

Cytokine

Control (n = 8) Multibacillary (n = 8) Paucibacillary (n=6)

TNF-(U b

IL-ID h

IL-6

TGF-P 1

GM-CSF

552 (O-855) 1112 * * * (827-1587) 1515 * (936-2030)

561 (O-1 109) 1292 * * (748-1996) 1031 (47-2336)

87 (O-6081 738 * (92-1587) ND

477 10-1830~ 1160 (591-2414) ND

0 (0) 80 (O-417) 0 (O-189)

a Values are calculated on the basis of normalising the ATPase expression for each individual (integrated intensity on autoradiograph) to 1000 units. h Significant differences found between groups by the Kruskal-Wallis test (I’ < 0.05). Significant differences were found between diseased and control groups using the Mann-Whitney test: * * * P < 0.005: * * P < 0.01; * P < 0.05. ND, not done.

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Fig. 5. Mycobacrerium auium subspecies paratuberculosis IS-900 cDNA PCR products from sheep intestine. Ethidium bromide-stained agarose gel. Top row, control samples, lanes 1-8; bottom row. diseased (multibacillary) samples, lanes l-8; p, positive control; n. negative controls; I, molecular weight ladder. Side arrows indicate the 1200 bp PCR product size.

the diseased group had significantly higher TNF-a activity than controls (P < 0.05). Assays on the paucibacillary group showed activity in only l/5 samples (0.01 ng ml-‘). Activity was not detected in any serum sample.

5. Discussion This study demonstrates the presence of significantly elevated TNF-(w, IL-I /? and IL-6 mRNA and elevated TNF-(r like activity in the intestines of sheep with the multibacillary form of Mycobacterium auium subspecies paratuberculosis infection. Cells of the monocyte-macrophage series have been shown to be major producers of these pro-inflammatory cytokines and the results do correlate with the lesions of granulomatous enteritis associated with a considerable infiltration of macrophages (Beutler and Cerami, 1989; Beuscher et al., 1990; Ohmann et al., 1990). These findings are also in agreement with other workers who detected elevated levels of TNF-(Y, IL-1 p and IL-6 in bovine peripheral blood mononuclear cells incubated in vitro with M.a.paratuberculosis antigens (Adams and Czuprynski, 1994). Moreover, monocytes from cattle naturally infected with M.u. paratuberculosis spontaneously released high levels of IL-1 (Kreeger et al., 1991). TNF-cu and IL-1 are implicated in the pathogenesis of granulomatous inflammation and cachexia, both major features of paratuberculosis. In addition, these cytokines cause inflammatory cell recruitment, including monocytes. and the induction of acute phase proteins that are also recognised in the disease (Beutler and

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Cerami, 1989). IL-6 is associated with B cell and plasmacyte development and its elevation in the multibacillary type of the disease correlates well with the strong (but ineffective) antibody response in this form of disease (Van Snick, 1990). IL-6 production has also been shown to be stimulated by TNF-a and IL-l/3 (Elias and Lentz, 1990: Chaplin and Hogquist, 1992). Mycobacterial antigens such as lipoarabinomannan (LAM), peptidoglycans and heat shock proteins (hsp) are potent inducers of macrophage cytokine production, with different bacterial strains sometimes inducing quite different levels of cytokine (Sullivan et al., 1991; Moreno and Rees, 1993). Such events often upregulate macrophage intracellular killing activity, for example by influencing the respiratory burst and nitric oxide pathways (Nathan, 1987; Denis, 1991). Mycobacterium auium paratuberculosis can resist these effector mechanisms and persist and multiply in ruminant macrophages (Bendixen et al., 1981; Zurbrick and Czuprynski, 1987). The high bacillary load in paratuberculosis may therefore provide a prolonged and effective stimulus for cytokine production by the parasitised host macrophages. In concert with effective resistance strategies of mycobacteria to the host defences, such excessive cytokine production may contribute significantly to the formation of chronic inflammatory lesions in the disease (Neil1 and Klebanoff, 1988; Celada and Nathan, 1994). Cytokine-associated immunopathology has been reported in other mycobacterial conditions such as tuberculosis and leprosy. The histopathology of borderline lepromatous leprosy is similar to the multibacillary lesions of paratuberculosis and lesions have also been associated with elevated TNF-a, IL-l /3 and IL-6 (Pisa et al., 1990; Parida and Grau, 1993). True granulomas are not usually seen in paratuberculosis but such lesions in tuberculoid leprosy and in tuberculosis are coincident with high levels of TNF-a and IL-l mRNA and product in situ (Arnoldi et al., 1990; Yamamura et al., 1992). Inflammatory bowel diseases (IBD) of man such as ulcerative colitis and Crohn’s disease have also been associated with high levels of TNF-o, IL-I and IL-6 (Cappello et al., 1992; Isaacs et al., 1992; Reinecker et al., 1993). Crohn’s disease has clinical and histological similarities with paratuberculosis and M.a.paratuberculosis has been implicated in its aetiology by some workers (Chiodini, 1989; Sanderson et al., 1992). In our study, TNF-a activity was not found in the serum of affected animals. This could be a consequence of plasma dilution of a product distant from its production site or a reflection of different cytokine profiles recognised between mucosal and systemic sites (Barnes et al., 1993). A number of control animals were shown to express cytokine mRNA in the intestine. This has also been reported in healthy human controls, albeit at a lesser level than diseased intestine (Isaacs et al., 1992; Reinecker et al., 1993). It is perhaps not surprising that the intestine of a grazing animal, exposed to a vast array of antigenic material should display some cytokine activity. GM-CSF mRNA levels were weak or undetectable in most samples in this study, in agreement with other studies on human intestine (Pullman et al., 1992). This may be a reflection of GM-CSF being a predominantly T-cell, rather than a macrophage product. Levels of TGF-/?l mRNA were raised, but not significantly, in the multibacillary lesions. TGF-/3 is considered to have down-regulatory functions for the immune and inflammatory systems so its presence in the intestine could be essential. Furthermore TGF-j3 could also play a role in

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suppressing macrophage defence activities in paratuberculosis (Adams and Hamilton, 1992). Assays on the paucibacillary form of intestinal lesions in paratuberculosis demonstrated a significant increase in TNF-(U mRNA but not in IL-lp. The histologic granulomatous lesions in this form of paratuberculosis tend to be more scattered compared with the multibacillary form (Carrigan and Seaman, 1990). Also the mycobacterial burden is often very low or even undetectable. Macrophage cytokines may therefore be less apparent in the paucibacillary form although the significant lymphocyte infiltration may be a source of TNF-a. We have shown that TNF-a, IL-lfl and IL-6 mRNA and TNF-a activity is elevated at the site of disease in the multibacillary form of paratuberculosis. These essentially macrophage cytokines and other lymphocyte-derived cytokines produced locally are likely to exert a major influence on the type of inflammatory and immune response developed during infection. Our results lend support to the hypothesis that elevated production of these cytokines, coincident with a failure to clear a heavy bacterial burden, may be one factor encouraging the persistent chronic inflammatory state seen in paratuberculosis. Further studies to localise cytokine production in situ are currently underway and should allow a greater understanding of the role of cytokines in the pathology of this disease.

Acknowledgements The authors would like to thank Dr D.R. Sargan (currently at University of Cambridge Veterinary School) and B. Ebrahimi (Department of Veterinary Pathology, University of Edinburgh) for the gift of IL-6 primers and rovTNF-a, Dr R. Bujdoso (currently at University of Cambridge Veterinary School) for the gift of IS-900 primers and Dr R. Boid (CTVM, University of Edinburgh) for the use of the Bio Image analyser. This study was funded by the Biotechnology and Biological Sciences Research Council of the UK.

References Adams, J.L. and Czuprynski, C.J., 1994. Mycobacterial cell wall components induce the production of TNF-o, IL-l and IL-6 by bovine monocytes and the murine macrophage cell line RAW 264.7. Microb. Pathogen. 16: 401-411. Adams, D.O. and Hamilton, T.A., 1992. Molecular mechanisms regulating macrophage activation In: C.E. Lewis and J.O.D. McGee (Editors), The Natural Immune System: The Macrophage. IRL Press, Oxford, UK, pp. 77-114. Amiri, P.. Locksley, R.M., Parslow, T.G., Sadick, M., Rector, E., Ritter, D. and McKerrow, J.H., 1992. Tumour necrosis factor restores granulomas and induces parasite egg-laying in schistosome-infected SCID mice. Nature, 356: 604-607. Amoldi, J., Gerdes, .I. and Flad, H-D., 1990. Immunologic assessment of cytokine production of infiltrating cells in various forms of leprosy. Am. J. Pathol., 137(4): 749-753. Barnes, P.F., Fong, S-J., Brennan, P.J., Twomey, P.E., Mazumder, A. and Modlin. R., 1990. Local production of tumor necrosis factor and IFN-y in tuberculous pleuritis. J. Immunol.. 145: 149-154.

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