Proceedings of the Australasian Society for Experimental Pathology Twenty-fourth Annual Meeting 28 September- 1 October 1992 Clunies Ross Lecture Theatre Faculty of Veterinary Science University of Sydney

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1992 Blackwell Scientific Publications

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Abstracts

S5

The following seven papers comprise the first Wilhelm Symposium: "Cells, Mediators and Inflammation" This Symposium has been organised to honour the memory of the Society's founding President, Professor Don Wilhelm. The Society welcomes the family of Professor Wilhelm at this Symposium, and all of his past students, friends and colleagues. The Symposium will be introduced by Professor John V. Hurley.

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ASEP 24th Annual Meeting 1992

THE ACCUMULATION OF LEUKOCYTES IN INFLAMMATORY LESIONS L. P. Bignold. Department of Pathology, University of Adelaide, S. A. 5001. Many aspects of the behaviour of leukocytes in the microcirculation (margination, adhesion, formation of pseudcpod and motility between endothelial cells) which lead to their accumulation in inflammatory lesions have been recognised for over a century (1), However, two major characteristic features of this accumulation have not been explained. First, the type of emigrant leukocyte depends on the nature of the injury. Thus "pyogenic" bacteria evoke neutrophils, parasites and allergic conditions evoke eosinophils, viruses evoke lymphocytes and mycobacteria evoke monocyte/macrophages. Second, when inflammation is established as an abscess or a chronic ulcer, the cells of the inflammatory reaction form zones, comprising a central focus of neutrophils surrounded by granulation tissue with lymphocytes and plasma cells around the outside. Possible roles of endothelium: It has long been suggested that abnormal adhesiveness (stickiness) of endothelium contributes to the emigration of neutrophils (1). However, adhesion to endothelium does not necessarily lead to emigration of neutrophils, since normally 50% of intravascular pool of neutrophils is marginated in the lung and bone marrow without emigration occurring. The abnormality of the endothelium appears to be not simply the interendothelial gaps of increased vascular permeability, since histamine-induced interendothelial gaps are not associated with emigration. Early studies provided no basis for a leukocytespecific role of endothelium, but in the last 10 years, there has been considerable interest in expression of specific leukocyte adhesion molecules ("integrins", 2) by endotheiium under the influence of mediators of inflammation. Such a mechanism could lead to leukocyte-specific trapping by specific endothelial adhesive factors inducible by specific agents, and subsequent random migration of cells into the tissues. Possible roles of chemotaxis Chemotaxis is the active movement of cells according to ambient concentration gradients of chemicals. Chemotaxis could explain emigration of leukocytes since such gradients could form in inflamed tissues if the chemioal was synthesised (and hence achieved highest concentration) in the tissues and was removed by diffusion into the microcirculation (hence lowest concentration) from which the leukocytes emigrate. Nevertheless, leukocyte-type specific chemotactic factors have not been generally identified, even in comparisons of pyogenic and non-pyogenio organisms. Chemotaxis could also explain extravascular tissue zonation of leukocytes in lesions such as abscesses, for which endothelial mechanisms seem difficult to conceive. Miles-Wllhelm criteria for mediators of inflammation and emigration of leukocvtes In the last 10 years, various cytokines (bioactive proteins produced by living cells under the influence of stimulators such as endotoxin) have been reported as having numerous effects on both leukocytes and endothelium. However, some of the reported actions in vitro are not likely to occur in vivo. For example, a substance produced by lymphocytes in the edge of the wall of an abscess is unlikely to be able to afffect the movements of neutrophils in the pus in the centre of the lesion. In the 1950s, a similar type of scientific problem arose from the plethora of endogenous purified compounds which appeared to have pro-inflammatory effects. Miles (3) and Wilhelm (4) (reviewed 5) devised "criteria" for considering the roles of these substances of which Jhe following were most important: a putative substance should be widely distributed among the species and among all individual tissues susceptible to inflammation. Furthermore, it should be potent and present in the tissues at the relevant time of the inflammation. Both inhibitors of the substance and depletion of the substance from the animal should inhibit the relevant aspect of inflammation. A version of last criterion is the study showing that mice genetically deficient in IL-2 have normal lymphocyte-proliferative and other immune responses (6). These criteria seem appropriate to assessing putative chemotactic factors involved in the accumulation of leukocytes in a tissue, with perhaps an additional criterion that they must be present in highest concentration at the histological site of accumulation of cells and form the appropriate concentration gradients to induce the observed direction of migration of the leukocytes. (1) (2) (3) (4) (5) (6)

Florey HW (1962) General Pathology, 3rd edn, Lloyd-Luke, London. Kishimoto TK et al (1989) Adv Immunol 46, 149. Miles AA (1959) Lect Sci Basis Med (Athlone Press, London) 8 198 Wilhelm DL (1971) Ann Rev Med 22, 63. Bignold LP (1989) Pathology 2 1 , 200 Schorle H et al (1991) Nature 352, 621.

Abstracts

S7

BIOLOGICAL ACTIVITIES OF INTERLEUKIN-8 IN VIVO. Ian Colditz. CSIRO Division of Animal Health, Armidale NSW 2350. The neutrophil activating peptide-l/interleukin-8 discovered in 1987 is a member of a family of closely related cytokines with 4 conserved cysteine residues. Other members of the family include platelet basic protein, connective tissue activating peptide III, platelet factor 4, inteferon-7 inducible protein-10, melanocyte growth stimulatory activity and the type II alveolar epithelial cell product ENA-78. IL-8 induces neurophil chemotaxis, enzyme release and the respiratory burst in vitro via stimulation of a selective receptor population distinct from receptors for C5a, f-Met-Leu-Phe, PAF and LTB^. In vivo, IL-8 induces neutrophil accumulation and associated plasma leakage by a mechanism independent of de novo protein synthesis at the site of inflammation. The neutrophil influx induced by IL-8 extends for around 8 hours which is appreciably longer than the neutrophil influx induced by other neutrophil chemotactic agonists. IL-8 induces neutrophil accumulation in skin with similar potency to C5a and LTB^. IL-8 has also been shown to induce chemotaxis of T lymphocytes in vitro. This finding lead us to investigate the capacity of IL-8 to induce lymphocyte accumulation in vivo. Lymphocytes were collected from the efferent prefemoral lymphatic vessel of sheep and labelled in vitro with "^In-oxine before reinjection to donor sheep which had inflammatory lesions in skin. '"in-labelled lymphocytes migrated in large numbers into sites stimulated with TNFa, IFN7 and into delayed type hypersensitivity lesions. In contrast very few lymphocytes migrated into sites stimulated with IL-8 or activated complement despite intense neutrophil migration to these sites. Lesions were also examined by immunohistology which revealed a significant but very small infiltrate of CD5, CD4 and CD8 lymphocytes in IL-8 lesions. These findings suggest that chemotactic agonists such as IL-8, which lack the capacity to induce expression of endothelial adhesion molecules for leucocytes, play a minor role in directing the migration of lymphocytes into inflammatory lesions in skin.

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ASEP 24th Annual Meeting 1992

'INFU\MMATORY TONE': A HEURISTIC CONCEPT FOR MORE RATIONAL THERAPEUTICS? Whilshoiise. M.W.. Department of Pathology, University of Adelaide, S.A. 5001 A local inflammation may elicit svsfennic responses through the mediation of cytokines and other locally produced 'hormones'. Two examples are TNF-S/IL-1 stimulating the release of ACTH and IL-6 inducing additional hepatic synthesis of plasma glycoproteins ('acute phase response'). Some of these distal responses would clearly help to autoregulate the severity of the initial inflammatory reaction e.g. increasing plasma levels of corticosteroids, antoxidant proteins, anti-proteases, etc. Other distal responses may not seem so beneficial at first sight e.g. reduction of plasma zinc and albumen levels. The net effect is to alter, if oniy transiently, the balance of various hormones, trace metals and other natural bioregulants in both the inflammatory locus and its contiguous compartments (circulation, draining lymphoid system). Other, more localised, perturbations of homeostasis can sometimes profoundly alter the properties/performance of endobiotics/xenobiotics within the inflammatory locus, effectively 'conditioning' their action. Some examples are: ( i) Cyanide generation (from thiocyanate or glycine) by activated leukocytes to liberate gold(l), that is either pre-bound to albumen or present in circulating aurothiolate drugs, forming aurocyanide which can stimulate PMN's or suppress proliferating lymphoid cells. ( i i) Non-enzymic oxygenation of salicylate by activated leukocytes forming 2,3-/2,5dihydroxybenzoates that are useful antoxidants and may (unlike salicylate) inhibit prostaglandin production, ( i i i ) Raised levels of cycloxygenase in mononuclear phagocytes, promoting transformation of C20 unsaturated fatty acids to E-prostaglandins which are potent autoregulants of immune responsiveness. Thus, the overall effect of activating inflammatory leukocytes (and other byestanding cells?) is to a) focus potential drug action by local biotransformation of judiciously selected circulating pro-drugs ('Trojan Horse therapy'); and b) aunment local autoregulants after eliciting increased levels of circulating anti-inflammatory factors derived from distal tissues. To comprehend the relative 'worth' of an inflammatory stress for triggering these propharmacological/natural autoregulant phenomena it may be necessary to assess its 'Tone'. Where this is too high, as in fulminant fever, it may over-ride most natural regulants and require drastic downregulation (e.g. high dose i.v. steroids). When it is too low, it may not permit the expression of inflammation-dependent drug activity. It will be important to consider if a given therapy may 'undermine' a beneficial level of Tone e.g. aspirin-like drugs preventing the E-prostaglandins from suppressing the production of inflammatory/immunostimulant cytokines (TNF, IL-1 IFN-g, IL-2). Conversely, it might sometimes help to raise the inflammatory Tone to stimulate corticoid release, allow pro-drug activation etc. Historically, we can trace this latter concept to Cooley's and Shear's anti-cancer 'vaccines' based on pyrogenic materials of microbial origin. The experimental data which triggered the (re-)deveropment of this concept is mainly derived from studying drug action t)Oth In vitro with pro-oxidant/cytokine-forming activated PMN/MNP leukocytes and in vivo using two types of inflamed rats, namely those (a) those developing chronic inflammation, mainly in response to arthritogens, and (b) those developing acute paw oedema.

(i) ( i i) (iii)

(iv)

Some key observations were: Gold drugs were oniy anti-arthritic in DA rats challenged with powerful arthritogens but not in Hooded rats which suffered less arthritis/inflammation; Gold drugs were only anti-arthritic in DA rats given a much less inflammatory arthritogen if coadministered with a synthetic PGEi analogue (Misoprostol) • Many antoxidants only expressed acute/chronic anti-inflammatory activity ii co-administered with Misoprostol and (acute) anti-oedemio activity if animals were pre-inflamed (by tailbase injection) Likewise, low-dose corticosteroids only expressed anti-oedemic activity if co-administered with Misoprostol or the animals were pre-inflamed.

The bottom line is: Hormonal/cytokine/pro-oxidant context(s) of an inflammation, together with other factors contributing to its Tone', may determine its natural/drug-assisted resolution or persistence.

Abstracts

S9

ROLES OF NITRIC OXIDE IN BIOLOGY Clark LA., Rockett, K.A.* and Cowden, W.B.*, Division of Biochemistry and Molecular Biology and *}ohn Curtin School of Medical Research, Australian National University, Canberra ACT 2601. Five years ago the idea that nitric oxide, a free radical gas, with a half-life measured in seconds, would prove to be an essential physiological mediator, and also important in immunology and pathology, was unthinkable. Yet next year will see the third International Conference on this concept. This talk will review the origins of these rapid developments, outlining the present known roles of nitric oxide in biology, and predicting where the field is heading. Nitric oxide has proved to be a cross-roads between fields of biology not previously thought to be connected, and it has intense practical, not just theoretical interest. For instance, it is probably the main physiological control of vascular tone, and bradykinin, acetylchoiine and the nitrovasodilator drugs influence blood pressure through generating nitric oxide. Its cytokineinduced overproduction appears to be responsible for the hypotension of sepsis, and this Information has led to successful clinical trials using analogues of arginine, the amino acid from which nitric oxide is generated, to restore blood pressure in severely hypotensive patients. Nitric oxide can also be toxic, and is now regarded as a major mediator of macrophagemediated control of tumour cells, certain protozoan parasites, and intracellular bacteria. There is also evidence that it can kill liver cells. Pancreatic islet p cells are also vulnerable, and several groups have argued that nitric oxide, released from inflammatory macrophages, is important in the onset of insulin-dependent diabetes. Lower concentrations, in contrast, are thought to be vital for normal control of insulin release from healthy p cells. Perhaps the most intense area of study of nitric oxide is its role in neurophysiology. For thirty years it has been appreciated that the amino acid, L-glutamate, has a central role in excitatory synaptic transmission in the brain. Glutamate's action was known to depend on its capacity to allow calcium entry into post-synaptic cells, and to be associated with an increase in cyclic GMP generation. Evidence is now very strong that the essential intermediate is nitric oxide. Glutamate-induced entry of calcium activates the enzyme necessary for its synthesis, and a reaction between nitric oxide and the iron centre in guanylate cyclase activating this enzyme in turn, leading to cGMP formation. Like oxygen or carbon dioxide, nitric oxide diffuses freely through cell membranes. This has opened up a range of experimental approaches to key questions in neurophysiology, such as the nature of learning and memory, in which nitric oxide is now thought to be a major player. As in the pancreas, excess nitric oxide may be toxic in the brain, in this case induced by the greatly increased release of glutamate from pre-synaptic cells that occurs in cerebral hypoxia and hypoglycaemia. The fields of immunology and immunopathology have also been invaded by nitric oxide. For example, there is now evidence that nitric oxide production by activated macrophages explains why the presence of these cells inhibits nitrogen-induced lymphocyte proliferation. Nitric oxide also inhibits the development of allospecific cytotoxic T lymphocytes, and has been incriminated in Ag/Ab-induced tissue damage, as well as anaphylaxis. We seem destined' to read much more about this mediator in the near future.

ASEP 24th Animal Meeting 1992 EOSINOPHILS Rothwell,—T.L.W. , Department Sydney, N.S.W. 2006.

of Veterinary

Pathology,

University -^

of

Eosinophils are a major and well-known component of the cellular infiltrate in parasitized tissues and some hypersensitivity reactions. Substantial numbers of eosinophils are also present in tissues affected by a diverse group of inflammatory and neoplastic changes as well as apparently normal alimentary and female reproductive tracts. Eosinophils arise in the bone marrow, have a limited life-span in the circulation, then enter the tissues. They have phagocytic and secretory activities and possess membrane receptors for complement components and immunoglobulin, including immunoglobulin E. Among their secreted products are: the granule components, major basic protein, eosinophil cationic protein and eosinophil-derived neurotoxin; toxic oxygen metabolites; various enzymes and membrane-derived lipid mediators such as leukotriene C4. Eosinophil proliferation and differentiation is strongly influenced bv GM-CSF, lL-3 and IL-5. Cells from individuals with circulating eosinophilia may be less dense than cells from non-eosinophilic individuals and because they have some enhanced functions in vitro have been referred to as activated eosinophils. Haemopoietic factor's for eosinophils, as well as some other agents, are capable of eosinophil activation. Eosinophils are thought to be responsible for tissue injury in some disease processes, for example, damage to the respiratory tract epithelium m asthma. However, although convincing in vivo evidence is not available, in vitro studies suggest that eosinophils may have the useful function of acting as effector cells in the immune response against some helminth parasites. In experimental animals, the magnitude of helminth-induced eosinophil responses is genetically-determined and, in some infections, eosinophila IS a trait of parasite resistent animals (or high responders). In these infections there is evidence of a polarization of Th-cell responses to infection, with high responders producing high levels of IL-5 but little IFN-7 and low responders high levels of IFN-7 but little IL-5. In sheep where helminth parasites are of major economic importance, the Identification of high responders would be useful in selective breeding programs for helminth resistant stock. Examination of cytokine production by Th-cells in vitro may be useful in identifying hiqh responders. J ^ •^

Abstracts

Sll

TNF: PROTECTIVE AND PATHOGENIC EFFECTS IN IMMUNITY AND INFLAMMATION Deborah A. Rathjen, Peptide Technology Ltd., 4-10 Inman Road, Dee Why, NSW 2099. Tumour necrosis factor (TNF) is an inflammatory and immunoregulatory cytokine produced mainly by monocytes and macrophages after stimulation with various agents most notably bacterial lipopolysaccharide (LPS). While TNF was originally characterised as an anti-tumour agent and a cytotoxic factor for malignant cells in culture it is now clear that TNF has many regulatory functions in immune reactions and that it plays a pivotal role in the enhancement of inflammation and immunity. TNF has an important role in host defence against viral, bacterial and parasitic infections and in autoimmune responses. Natural induction of TNF is protective, but its overproduction may be detrimental and even lethal to the host. TNF is thought to be the central mediator of pathology observed in Toxic Shock Syndrome, Adult Respiratory Distress Syndrome, cachexia, cerebral malaria, rheumatoid arthritis. Graft vs Host Disease, allograft rejection, hypersensitivity pneumonitis and other pneumopathies such as those resulting from exposure to bleomycin and silica. The central role of TNF in these conditions has been indicated by elevated TNF levels in serum and the abrogation of pathology by neutralising anti-TNF antibodies. Clinical trials of recombinant TNF conducted in cancer patients over the last 7 years have been disappointing. Little tumour regression has been observed in the majority of cases and administration of TNF has been associated with severe dose-limiting side-effects. The side-effects reported include: fever, chills, headaches, nausea, vomiting and diarrhoea with hypotension being the most commonly reported dose-limiting sideeffect. More severe side-effects include disseminated intravascular coagulation, pulmonary haemorrhage and severe hepatic dysfunction. Recent trials in patients with melanoma and ovarian ascites tumours have however, been more encouraging and TNF has been shown to be of significant benefit in the treatment of these tumours. Cellular targets for the expression of TNF toxicity appear to be primarily the neutrophil and the endothelium. The massive accumulation of neutrophils in the lungs of TNF-treated animals reflects the activation of neutrophils by TNF. TNF causes neutrophil degranulation, respiratory burst and enhances the antimicrobiocidal and anti-tumour activity of neutrophils. Endothelial cells treated with TNF display enhanced procoagulant activity and diminished anticoagulant potential. TNF also induces the expression of cell adhesion molecules leading to the accumulation of inflammatory cells on the vascular wall. The molecular basis of the pleiotropic effects of TNF on almost all cells of the body is becoming better understood. We now know that TNF has at least 2 distinct receptors and that three intracellular signalling pathways are involved in signal transduction. It is now possible to modulate TNF activity in vivo through a number of strategies including the development of receptor-specific antagonists.

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ASEP 24th Annual Meeting 1992

NOVEL PROTEIN MEDIATORS OF INFLAMMATION Geczy, C.L.. Heart Research Institute, 145 Missenden Road, Camperdown, N.S.W. 2050. Leukocyte infiltration and fibrin deposition are prominent histological features of inflammation and are evident in numerous pathological conditions in which cellmediated immunity plays a role. Our studies show that monocyte/macrophage (mo/mac) procoagulant activity, principally due to expression of surface tissue factor, is the major initiator of fibrin formation at sites of extravascular coagulation. The dose correlation of mo/mac procoagulant expression and DTH skin test responses to microbial antigens is mediated by a cytokine, macrophage procoagulant inducing factor (MPIF) which is a hydrophobic heparin-binding protein of 17 kD. MPIF directly induces tissue factor, detected 4-6 hr after stimulation of mo/mac. In contrast, a second pathway of procoagulant induction is mediated by interferon y which primes inflammatory macs, but not blood mos, to express procoagulant in response to a second signal (bacterial lipopolysaccharide, some cytotoxic drugs). Highly enriched preparations of murine MPIF induce intense cellular infiltration, with histological and temporal changes typical of a DTH response when injected into normal skin. We separated a novel chemotactic protein (CP-10) from MPIF. CP-10 is an 88 amino acid member of the SlOO protein family. It is an acidic protein of mwt 10.2 kD and contains two Ca2+ -binding EF-hands and lacks putative signal or membrane anchor sequences. CP10 has optimal chemotactic activity for PMN at 10-^2 . -[Q-n M and for mac at lO'i^ - lO-lZ M making it one of the most potent chemotactic factors described. Like related SlOO proteins, CP-10 is highly conserved within the EF-hand domains but differs in the hinge (between the two EF-hands) and C-terminal regions. The chemotactic activity has been located to the hinge region (CP-lOp, 42-55). Native CP-10 induced a sustained (24 hr) influx of PMN and mononuelear cells in vivo whereas CP-lOp elicited a more transient (6-8 hr) response. In accordance with a role in chemotaxis, CP-lOp and CP-10 upregulated Mac 1 (CDllb/CD18) integrin expression on infiltrating neutrophils. Although CP-10 has no direct effect on mo/mac procoagulant induction expression of Mac 1 may contribute to rapid PMN accumulation within fibrin thrombi and to plasma Factor X binding on monocytes. These studies suggest that MPIF and CP-10 play pivotal roles in extravascular fibrin deposition, neutrophil and mo/mac recruitment and activation, all of which are important features of inflammatory responses.

Abstracts

S13

PROFFERED PAPERS THE ANTI-ARTHRITIC ACTIVITY OF CYCLOSPORIN-A IN RATS INVOLVES SUPPRESSING A POPULATION OF CD4 +VE (T-HELPER) LYMPHOCYTES AND 4 CYTOKINES (IL-1, IL-2, TNF AND IFN-y) BUT STIMULATING B LYMPHOCYTES AND IL-6 PRODUCTION. Havnes D.R.. Gadd S.J., Whitehouse M.W., *Mayrho(er G. and Vernon-Roberts B. From the Departments ot Pathology and 'Microbiology and Immunology, University ot Adelaide, GPO Box 498, Adelaide, South Australia, 5001. Cyclosporin-A (CsA) has been used (or many years to prevent graft rejection. Recently it has been used to treat autoimmune and related disorders, including rheumatoid arthritis. Its effects were thought to be mediated by its potent inhibition of lL-2. However, it i.s increasingly obvious that CsA may affect many other aspects of the immune response. CsA treatment at the time of disease initiation completely prevented expression of experimental adjuvant arthritis in rats. Surprisingly, CsA did not prevent lymphoid responses to the arthrogenic adjuvant (MTb/squalane). Immunofluorescence assays with flow cytometry and immunohistology of lymph nodes showed that although CsA treatment significantly reduced CD4 +ve cell numbers, the numbers of B-lymphocytes were significantly increased. Ex vivo and in vitro studies indicated that these changes in lymphocyte populations may be due to suppression ot IL-1, lL-2, TNF, and IFN-7 production (IC50 1-150nM in vitro) whereas lL-6 production was relatively unaffected in vitro (IC50 > lOOOnM) or enhanced ex vivo. These findings indicate that CsA therapy may target those T-helper lymphocytes involved in the cellular immune response. These cells (TH1) produce a characteristic group ot cytokines (IL-1, IL-2, TNF and IFN-7). Other T-helper lymphocytes (TH2) involved in the humoral (B-cell) responses produce a different group of cytokines including lL-6. TH2 cells may be unaffected by CsA or even enhanced as a result of TH1 suppression by CsA. TH1 cells may therefore be strategic targets for txith drug and monoclonal antibody therapy for chronic diseases like this experimental arthritis. A ROLE FOR I L - 4 , IL-6 AND IL-6R IN SJOGREN'S SYNDROME. Aziz, K.E., Markovic, B . , McCluskey, P . J . , Wakefield, D. School of Pathology, U n i v e r s i t y of N.S.W., NSW 2033, AUSTRALIA. Cytokines a r e immunologic mediators and can d e f i n e the functional characteristics of Thi and Th2 lymphocytes. Sjogren's syndrome (SS) is associated with a lymphocytic and monocytic i n f i l t r a t i o n of s a l i v a r y g l a n d s . Minor s a l i v a r y glands (MSG) were examined from 17 p a t i e n t s and 10 c o n t r o l s by In s i t u h y b r i d i z a t i o n . We found t h a t 24.6±3.8% and 3.7^1.1% of p r e s e n t c e l l s produced mRNA of IL-4 and IL-6 r e s p e c t i v e l y i n s a l i v a r y g l a n d s from patients. Only 9.3±5.2% and 1.3±0.26% of p r e s e n t c e l l s were p o s i t i v e for IL-4 and IL-6 probes i n c o n t r o l s . The i n c r e a s e i n t h e number of IL-4 producing cells in patients biopsies reached statistical significance but not in the case of IL-6. The pattern of IL-6R mRNA synthesis corelated with IL-6. The percentage of infiltrating mononuclear c e l l s producing IL-4 and IL-6 m-RNA was 31 .9±5.45% 2.4^0.55%. The r e s u l t s indicate a major role for IL-4 and a possible role for IL-6 in the immunopathogenesis of SS. IGSSPOT: AN IMPROVED METHOD FOR DETECTION OF INDIVIDUAL CYTOKINE-SECRETING CELLS. Kumar. R.K., Velan, G.M., L i , W. and O'Grady, R., School of Pathology, U n i v e r s i t y of New South Wales, P.O. Box 1, Kensington, NSW 2033. The ELISPOT and reverse haemolytic plaque assays have recently been described f o r the detection of cytokine secretion by single c e l l s i n c u l t u r e . We present a modification of the former technique, i n which c o l l o i d a l gold-labelled antibodies and s i l v e r enhancement are used as the reporter system. Compared t o the ELISPOT assay, development of "spots" surrounding i n d i v i d u a l cytokine-secreting c e l l s is rendered t e c h n i c a l l y straightforward and the high contrast o f the reaction product makes enumeration simple. To evaluate the method, the proportion of splenic lymphocytes secreting interferon-gamma was assessed by IGSSPOT. In p a r a l l e l , the concentration of t h i s cytokine i n c u l t u r e supernatants of splenic lymphocytes was measured by enzyme immunoassay. Both values increased i n p a r a l l e l following s t i m u l a t i o n i n v i t r o with concanavalin A. The technical advantages of the immunogoid-silver technique suggest that i t may be superior t o enzymatic reporter methods f o r the detection o f i n d i v i d u a l cytokine-secreting c e l l s .

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ASEP 24th Annnal Meeting 1992

INFLAMMATION IN FOETAL SHEEP

Crowe. P.M. Hurley, J.V., Kumta, S., Ritz, M. and Romeo R., Microsurgery Research Centre, St Vincent's Hospital, Fitzroy, Vic, 3065. Open skin wounds in foetal sheep excite much less inflammatory response than similar wounds in adult animals but otherwise heal in a very similar manner. However almost nothing is known of the response of foetal sheep to more severe inflammatory stimuli. To investigate this, 0.25 ml of Oil of Turpentine, either neat or diluted with paraffin oil, was injected un'der the skin of foetal sheep of 75, 100 and 120 days gestation (gestation period 150 days) and of adult sheep. The injection site was examined histologically 3-28 days after injection. At all stages examined turpentine, both neat and diluted, excites a severe initial cellular reaction which is followed by granulation tissue formation and fibrosis. In the 75 day foetus, where the blood contains very few neutrophils, the early lesions are composed of large and small mononuclear cells and only occasional neutrophils. As the foetus matures the blood neutrophil level rises and the number of neutrophDs in the early lesions increases until in the 120 day foetus the reaction resembles closely that of adult sheep. PLASMINOGEN ACTIVATOR lNHIBITOR-2 SYNTHESIS BY CYTOKINE STIMULATED ENDOTHELIAL CELLS. Zoellner H.. *Wojta, J,, *Gallicchlo M., *McGrath K. and Hamilton J.A., Departments of Medicine, University of Melbourne, and *Diagnostic Haematology, Royal Melbourne Hospital Parkville VIC 3050. Fibrinolysis depends on conversion of plasminogen to the highly active proteinase plasmin by plasminogen activators (PA). The activity of PA is in turn regulated by PA inhibitors 1 and 2 (PAI-1 and - 2 ) . Vascular endothelial cells (EC) secrete large amounts of PAI-1, thought to contribute to the stability of thrombi. IL-1 and TNFa increase PAI-1 synthesis by EC, of proposed importance in the development of thrombi. We describe the synthesis of PAI-2 by EC in response to IL-1 and T N F Q , in comparison with the synthesis of PAI-1. Cultured human EC were stimulated with I L - 1 Q or T N F Q , and the conditioned media and cell lysates assayed for PAI-1 and PAI-2 by ELISA. Most PAI-1 was secreted, while FAI-2 was cell associated. IL-lo and TNFa increased the synthesis of PAI-2 and PAI-1 by EC in a dose-dependent manner. IL-la was a stronger stimulus for PAI-2 synthesis than TNFa, while both cytokines were equally effective in induction of PAI-1 synthesis. Northern blot analysis revealed similar changes in mRNA levels to those in antigen levels. PAI-1 is currently thought to be the major EC product affecting thrombi, while our observations raise the possibility of a significant contribution by PAI-2. PAI-2 has recently been found to protect cells from T N F Q cytotoxicity. PAI-2 in EC may initially protect cells from the cytotoxic effects of T N F Q , and then contribute to the stability of early thrombi following EC death in sites of vascular injury.

T H E R O L E O F P O L Y M O R P H O N U C L E A R L E U C O C Y T E S IN GASTRIC ISCHAEMIA/REPERFUSION INJURY. Andrews. F.J.. Malcontenti-Wilson, C. and O'Brien, P.E., Departmenl of Surgery, Monash University, Alfred Hospital, Prahran, VIC, 3181.

Gastric mucosal injury has been associated with periods of ischaemia in several different clinical settings including trauma, ethanol exposure and aspirin administration. Studies of the small bowel, heart and skeletal muscle demonstrate that reperfusion of ischaemic tissue induces infiltration of polymorhonuclear leucocytes (PMN) that contribute significantly to tissue injury. The aim of this study was to determine the role of PMN infiltration in gastric ischaemia/reperfusion injury. Rats were subjected to 30 minutes gastric ischemia followed by reperfusion. Injury was assessed by quantitative histology. Sections of mucosa were also stained for PMN using an immunoperoxjdase technique. In control animals, there were 4 + 2 PMN/mm^ in the superficial and 9 + 4 PMN/mm^ in the deep mucosa. This increased significantly to 67+9 PMN/mm^(p < 0.05) and 160j;53 PMN/mm^ (p < 0.01) respectively at 15 minutes of reperfusion. The percentage of these PMN which were extravasated at this time was 83j;4% in the superficial mucosa and 82 + 4% in the deep mucosa (p