key: cord-1051967-5j2hf7wb authors: Charley, B.; Petit, E.; Laude, H.; La Bonnardière, C. title: Myxovirus- and coronavirus-induced in vitro stimulation of spontaneous cell-mediated cytotoxicity by porcine blood leukocytes date: 1983-03-31 journal: Annales de l'Institut Pasteur / Virologie DOI: 10.1016/s0769-2617(83)80047-1 sha: e6d86dd9fa56d9d01412e3dbd4924881fe58894f doc_id: 1051967 cord_uid: 5j2hf7wb Summary Spontaneous cell-mediated cytotoxicity (SCMC) by porcine blood leukocytes toward human myeloid tumour target cells (K562) was shown by Koren and coworkers in 1978, using a 4-h 51Cr-release assay. In the present paper, SCMC was found to be enhanced following a 24-h incubation of leukocytes with swine influenza virus (SIV) of the H3N2 type, and with the transmissible gastroenteritis virus (TGEV), a porcine coronavirus. In most of our experiments, both SIV and TGEV induced interferon (IFN) production by leukocytes (titres ranging from 30-7,500 units/ml). When porcine leukocytes were incubated with TGEV in the presence of anti-IFN antiserum, no SCMC stimulation occurred, suggesting that the virus-induced SCMC stimulation was mainly due to endogenously produced IFN. On the other hand, when either human αIFN or porcine virus-induced IFN were added to porcine leukocytes, a rapid and marked SCMC increase was observed. Both the SIV-induced SCMC increase and IFN production were suppressed when SIV was UV-inactivated. On the contrary, UV-inactivated TGEV was still able to enhance SCMC and to induce IFN production by porcine leukocytes. On the whole, these results suggest that myxovirus and coronavirus are able to activate porcine leukocyte SCMC via endogenously produced IFN. In the last few years, increasing interest has been devoted to viruses causing neonatal diarrhea in man and animals, and to the defenee mechanisms of the newborn against these infections. The pig appears to be a convenient animal model for studies on the pathogenesis of viral enteritis, as well as on the nature of early non-specific defence mechanisms against neonatal diarrhea; indeed, the transmissible gastroenteritis virus (TGEV), a porcine coronavirus, produces an acute and fatal disease and induces high amounts of intestinal interferon (IFN) [7] . In addition to IFN, the presence of spontaneous cell-mediated cytotoxicity (SCMC) was investigated as a potential non-specific defence against enteropathogenic viruses. SCMC, or natural killing (NK), has been described in various mammalian and avian species [5, 4, 9] , and there is increasing interest in its possible role in host defence against disease [5] . The treatment of rodent or human lymphocytes with IFN, or the infection of rodents with different viruses greatly increases SCMC [2, 3, 4, 13] but to our knowledge no similar data are available for other animal species. In the present report, we provide results concerning the in vitro effects of TGEV on the spontaneous cytotoxicity of porcine leukocytes, using a short-term 51chromium-release assay against human myeloid tumour target cells (Ks,~) as described by Koren el aI. [6] . We compare these data to the in vitro effects of a myxovirus which was previously shown to induce NK in mice [10] . Furthermore, we present results on the influence of porcine as well as human IFN on SCMC via porcine leukocytes. Conventionally reared pigs, 2-8 months old, were used. As a source of virus-induced porcine IFN, a serum obtained from a TGEVinfected neonate (IFN titre = 7,500 U/ml) was used. Human ~IFN, with a titre of 4 x 106 U/ml, was kindly provided by Institut Pasteur Production (Garches, France). IFN was assayed on bovine MDBK cells using vesicular stomatitis virus as a challenge [7] . IFN titres were expressed with reference to an internal bovine standard IFN, yielding a mean titre of 2,500 Ug/ml by the 50 % plaque-reduction method. Anti-human aIFN antiserum was kindly provided by Dr Chany (Paris); after absorption on porcine leukoeytes, it was used at a final dilution of 1 [200, which was able to neutralize _~ 750 units of porcine IFN. Mononuclear cells were separated from heparinized blood by a Ficoll density centrifugation method: 20 ml of diluted blood (1 ]4 in 9 %0 NaC1) were layered onto 7.5 ml of a mixture containing 14 % Ficoll (Pharmaeia, Uppsala, Sweden) and 32.8 % Telebrix (Guerbet, Aulnay-sous-Bois, France) {121. After eentrifugation for 15 rain at 2,000 g at room temperature, the leukoeytes were pipetted out, washed twice and resuspended in RPMI medium supplemented with 10 % foetal calf serum (FCS), 2 mM L-glutamine and antibiotics (penicillin and streptomycin). A strain of influenza virus type A (H3N~) isolated from pig lung (Swine influenza virus: SIV), provided by Dr Hannoun (Paris), was passaged in embryonated eggs. Infectivity was expressed as the 50 % egg infective doses (EIDs0) per ml. As a source of the TGEV cell-adapted Purdue 115 strain was used. Methods for propagation and titration (plaque-forming units: PFU) of this virus have previously been described [8] . Human myeloid leukaemia cells (K5,2, kindly provided by Dr Gutner, Villejuif, France) were labelled with Na~lCrO~ (Amersham, UK. 40 ~Ci/2 • 10 ~ cells in 250 ~1 RPMI) for 1 h. The cells were then washed three times with RPMI-10 % FCS and adjusted to a final concentration of 105/ml in RPMI-10 % FCS. Porcine leukocytes were incubated in round-bottomed microtitre plates (Nunc, Roskild, Denmark) for 24 h at 37 ~ C in a humidified atmosphere containing 7 % CO2. Triplicate cultures were prepared with 108 leukocytes per well in RPMI-10 % FCS. Then, 104 labelled target cells were added in a total volume of 0.2 ml (leukocyte: target ratio of 100), and plates were centrifuged for 5 min at 70 g as described by Korea et al. [6] . After 4 h of incubation, the assay was terminated by spinning the plates for 10 rain at 300 g, and samples (0.1 ml) of the supernatants were collected for radioactivity measurement. Th'e % cytotoxicity was calculated as follows: where SR (spontaneous release) is defined as the radioactivity released from target cells incubated in medium alone (RPMI-10 % FCS) and MR (maximal release) as epm in the supernatants of targets lysed with Triton • 100. It was checked that SIV (2.5 • 106 EIDs0/ml) was unable to increase 51Chromium SR by labelled target cells within 4 h. When porcine blood leukocytes were incubated with SIV 24 h before the addition of labelled K562 cells, SCMC was found to increase: this stimulation was significant (p < 0.001, paired t test) in results from 17 different leukocyte preparations. An IFN production was detectable in the supernatants of infected leukoeytes, with IFN titres ranging from 275 to 7,500 Ug/ ml (table I: exp. 1) . Furthermore, when an aliquot fraction of the virus preparation was completely inactivated by UV irradiation, it no longer enhanced SCMC, and IFN production was markedly reduced (table I: Controls showed that TGEV (1.2 • 107 PFU/ml) and absorbed anti-IFN antiserum did not increase SR by labelled K562 within 4 h. SCMC increased significantly following 24 h incubation of porcine leukoeytes with TGEV (p < 0.001, paired t test, with data from 12 different cell suspensions). IFN was detected, with titres ranging from 30 to 2,500 ~.g/ ml. Since virus-induced porcine IFN was shown to be neutralized by antihuman ~IFN antiserum (La Bonnardi~re and Laude, manuscript in preparation), we were able to show that anti-~IFN antiserum, when added to porcine leukoeytes with TGEV, suppressed virus-induced SCMC enhanceinent and IFN production (table II) . Control experiments showed that ultraeentrifuged virus suspensions were no longer able to stimulate SCMC, indicating that the enhancing effect was related to the virus particle; however, contrary to findings with SIV, when TGEV was UV-inactivated, it was still able to induce SCMC enhancement and IFN production (table II1) . Since the above results suggested that SCMC enhancement by SIV and TGEV was related to IFN production, we tried to determine whether the addition of IFN to porcine leukocytes would affect their cytotoxieity: table IV shows that l0 s units of porcine IFN per ml increased the SCMC of two cell suspensions but was inactive on a third preparation. Human alFN (2 • 10 ~ Ug/ml) signitieantly increased SCMC (p < 0.01 in paired t test with data froln nine different cell preparations). This enhancement was dose-dependent and had already been observed with 2 • 10 ~ Ug/ml ( fig. 1) . Furthermore, the IFN, boosting ,, effect was also observed against two other hulnan tumour cell lines (CEM and PI)e-B1 [9] ) and was inhibited by anti ~IFN antiserum (data not shown). Controls showed that human and porcine IFN did not increase"SR by labelled target cells. Each curve is related 1o one leukocyte preparation. This demonstrates that SIV and TGEV increase the SCMC of porcine leukoeytes. Both viruses induced IFN production by leukocytes, and moreover, anti-human MFN antisernm, which has been shown to neutralize porcine IFN (La Bonnardi~re and Laude, in preparation) suppressed the TGEV-indueed SCMC stimulation. Both porcine and human IFN were also shown to stimulate SCMC in vitro. The present data are in agreement with previous studies showing that different viruses (including lymphocytic ehoriomeningitis, mumps, measles and herpes simplex viruses) induced IFN production by leukoeytes, and that NK cells in these cell preparations became activated (reviewed by Welsh [131) . Influenza virus has also been shown to induce IFN production by infected human leukocytes [11] and to stimulate NK cells in vitro [13] and in vivo [10] . However, to our knowledge, this the first demonstration that a coronavirus is able to induce IFN production by blood leukocytes and to increase SCMC. UV-inactivation experiments produced distinct results with the two viruses, since inactivated SIV was unable to stimulate SCMC or to induce IFN, whereas inactivated TGEV retained its inducing effect: this data further indicated that virus-induced SCMC enhancement is correlated with IFN production. The discrepancy in the results of UV-inactivation experiments using the two viruses is probably due to differences in the doses of UV treatment: in order to obtain almost total SIV inactivation, it was necessary to irradiate the virus suspension up to 4 h, whereas the TGEV was fully inactivated following 2 min of UV treatment. It therefore appears that prolonged UV irradiation abolishes the ability of the virus to induce IFN and SCMC enhancement. Indeed, such a result was observed in preliminary experiments with TGEV. However, it is worth noting that some exceptions to this rule do exist, since Casali et al. [1] described increased SCMC by a measles virus glyeoprotein, with no detectable IFN production. Finally, porcine leukocytes were found to express activated SCMC when incubated with IFN, as was shown for rodents and man [9., 3, 4] . According to our results, porcine IFN seemed less active than human IFN. This seems to be consistent with the finding that porcine IFN has a more pronounced antiviral effect uFon heterologous (bovine) cells than upon homologous cells [7] . The role of SCMC-and IFN-(< boosted )) SCMC in vivo in TGEV-induced enteritis is currently under investigation in our laboratory. Ces deux virus ont induit une production d'interf~ron par les leucocytes. Quand les leucocytes ont ~t~ incub6s avec le virus de la GET en pr5sence de s~rum anti-interferon, aucune stimulation de la cytotoxicit~ spontan6e n'est apparue, ce qui sugg~re que cette stimulation ~tait principalement due ~ de l'interf6ron produit par les leucocytes. Par ailleurs, quand de l'interf~ron ~ humain ou de l'interf~ron viro-induit porcin est ajout6 aux leucocytes de pore, on observe une stimulation rapide et importante de la cytotoxieit~ spontande. La stimulation de cytotoxieit~ spontan~e et la production d'interf~ron sont supprimfies quand le virus grippal porcin est inaetiv~ par les rayons ultraviolets, mais non quand le virus de la GET est inactive. L'ensemble de ces rdsultats sugg~re que le myxovirus oa le coronavirus peuvent stimuler l'activit~ cytotoxique spontan~e des leucocytes de pore par production d'interf~ron endog~ne. MOTS-CL~S : Cyt0t0xicitfi, Myxovirus, Coronavirus, Interfgron; K562, Leucocytes, Pore. We are grateful to R. Scherrer for his help in preparing this manuscript. 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