key: cord-0007800-5c6a831z
authors: Myers, T.J.; Schat, K.A.
title: Natural killer cell activity of chicken intraepithelial leukocytes against rotavirus-infected target cells
date: 2002-11-13
journal: Vet Immunol Immunopathol
DOI: 10.1016/0165-2427(90)90064-y
sha: 05b34d83dc67745e49e17b3e31899bfadd160a88
doc_id: 7800
cord_uid: 5c6a831z

Intraepithelial leukocytes (IEL) and splenocytes collected from uninfected and rotavirus-infected chickens were evaluated for cytotoxic activity against a natural killer (NK) cell-susceptible lymphoblastoid cell line (LSCC-RP9) and against rotavirus-infected chick kidney cells in 4-h chromium-release assays. Both splenocytes and IELs from uninfected and rotavirus-infected chickens were cytotoxic for LSCC-RP9, and the levels of this NK cell activity were not altered by infection of the host with rotavirus. IELs but not splenocytes from uninfected and rotavirus-infected chickens were cytotoxic for rotavirus-infected but not for uninfected chick kidney cell targets. Because this cytotoxic activity was not induced nor altered by rotavirus infection of the host, and was not major histocompatibility complex-restricted, it was considered to be due to NK cell activity. The cytotoxicity of IELs against rotavirus-infected target cells was dose-dependent; however, there was some suppression of cytotoxic activity at high effector to target cell ratios. There were no differences in the cytotoxic activities of IELs collected from the duodenum versus the jejunum. The in vitro cytotoxic activity of IELs against rotavirus-infected target cells suggested that NK cell activity may be an important immune response to rotavirus infections in vivo. The absence of cytotoxic activity by splenocytes against rotavirus-infected target cells indicated that there may be different subpopulations of NK cells in the spleen and intestinal epithelium of chickens.

The immune response to rotavirus infections has been the subject of much study since the recognition of the etiological significance of rotaviruses in human and domestic animal diarrheas (Kurstak et al., 1981) . Most of these studies have examined antibody-mediated responses. In mammals, rotavirusspecific intestinal antibodies clearly mediate passive immunity (Offit and Clark, 1985; Archambault et al., 1988) , and are thought to be important in the active immune response to primary (Sheridan et al., 1983; Van Zaane et al., 1986 ) and secondary (Bishop et al., 1986 ) rotavirus infections. However, there is evidence to suggest that mechanisms besides rotavirus-specific intestinal antibodies may participate in recovery and resistance. Clinical studies in humans show that the presence of rotavirus-specific fecal IgA does not always correlate with resistance to diarrhea following challenge (Bernstein et al., 1986) . Also, studies in experimentally infected calves have shown that calves lacking coproantibodies to a heterologous secondary challenge virus can still be protected against developing diarrhea (Bridger and Oldham, 1987) . Furthermore, neonatal nude (athymic) mice are able to recover normally from rotavirus infection despite the inability to mount a T cell or an intestinal antibody response (Eiden et al., 1986) . Conversely, neonatal mice with severe combined immunodeficiency, which are also incapable of normal T and B cell responses, exhibit persistent rotavirus infections following inoculation (Riepenhoff-Talty et al., 1987 ) .

A limited number of studies have examined non-antibody responses to rotavirus infections. Offit and Dudzik (1988) have demonstrated MHC-restricted, rotavirus-specific cytotoxic T cell activity in vitro using splenocytes from rotavirus-infected mice at 7 DPI. This cytotoxic activity was directed against target cells infected with rotaviruses either homotypic or heterotopic to the virus inoculum. Kohl et al. (1983) demonstrated that human interferon can induce in vitro NK cell activity of human peripheral blood leukocytes (collected from randomly selected individuals) against rotavirus-infected target cells. In calves, La Bonnardiere et al. (1981) showed that intestinal interferon is produced during rotavirus infection and Van den Broecke et al. (1984) found a correlation between high levels of intestinal interferon and reduced clinical disease. Thus, there is evidence to indicate that cytotoxic T cells, NK cells, and interferon may participate with intestinal antibodies in mediating rotavirus immunity.

Recent studies in our laboratory demonstrated that embryonally bursectomized (B cell-deficient) chickens showed a slightly delayed but complete recovery from, and resistance to, avian rotavirus infection (Myers and Schat, 1990) , providing further evidence for non-antibody mechanisms of rotavirus immunity. This report characterizes the cytotoxic activity of intestinal IELs from rotavirus-infected chickens using a 4-h CRA.

The Ch2 strain (Yason and Schat, 1985 ) of avian rotavirus was propagated and semipurified as described .

P2a and N2a strain SPF chickens homozygous for the MHC alleles B 19 and B 21, respectively (Weinstock and Schat, 1987) , were obtained from departmental flocks. Effector cell donor birds were P2a chickens inoculated either with 105 TCIDso Ch2 rotavirus in 1 ml PBS, pH 7.2, by esophageal cannulation or with a sham inoculation of PBS alone. Infected and uninfected birds were housed in separate isolation units. At specified times following inoculation, serum samples and duodenal, splenic, and thymic tissues were collected. Exposure to infectious rotavirus was confirmed by evaluating serum samples for the presence of rotavirus-specific IgM in an enzyme-linked immunosorbent assay .

Duodenal and jejunal IELs were isolated by the procedure of Chai and Lillehoj (1988) . Briefly, the duodenum or jejunum from each bird was removed, cut into 1 cm pieces, and incubated first with 5 mMdithiothreitol (to remove mucus) and then with 10 -4 M EDTA (to remove the epithelial cells from the mucosal basement membrane). IELs were separated from this population of cells by passing the cell suspensions twice through a nylon wool column, followed by purification of the IELs on a discontinuous Percoll gradient. Thymocytes and splenocytes were isolated as described by Schat and Calnek (1980) . All effector cells were suspended in LM-Hahn basal medium (Calnek et al., 1981 ) supplemented with 20% heat-inactivated FBS (LM20) prior to their addition to target cell cultures.

Lymphoblastoid cell line targets: LSCC-RP9 is an avian leukosis virustransformed lymphoblastoid cell line (MHC alleles B2B 15) sensitive to in vitro NK cell lysis (Sharma and Okazaki, 1981 ) . RECC-CU60 is a reticuloendotheliosis virus-transformed lymphoblastoid cell line (MHC alleles B 19B 19 ) refractory to in vitro NK cell lysis (Weinstock et al., 1989) . These two cell lines were grown in LM-Hahn basal medium supplemented with 10% heatinactivated FBS (LM10) at 41 °C in a humidified atmosphere of 5% CO2. Prior to their use in a CRA, these cells were centrifuged over Ficoll-Hypaque (Pharmacia, Piscataway, NJ) and labeled with 300/zCi NaS1CrO4 per 107 cells as described (Schat and Calnek, 1980) . Labeled cells were resuspended in LM20 and dispensed to round-bottomed 96-well tissue culture plates (Corning Glass Works, Corning, NY) at 5 × 10 4 cells per well just prior to the addition of effector cells.

CKC targets. The development of CKC targets was modeled after a procedure described by Offit and Dudzik (1988) . Primary CKC cultures were established from 2-to 3-week-old SPF P2a and N2a chicks as described by Calnek and Madin (1969) . The growth medium (M25) used for CKC cultures has been described . These cultures were trypsinized at 48 h in culture and frozen in liquid nitrogen for use as secondary CKCs. Twenty-four hours prior to their use in a CRA, an ampule of CKCs was thawed, and cells were counted, resuspended at 0.5 × 106 cells per ml in M25 medium, dispensed into a 96-well fiat-bottomed tissue culture plate (Coming) at 200 Ftl per well, and allowed to grow at 38°C in a humidified atmosphere of 5% CO2. The next day, cultures in a row of 12 wells were trypsinized and these cells were collected and counted to determine the number of target cells per well (counts averaged about 5 X 104 cells per well). The remaining wells were then washed twice in PBS, and inoculated with 7.5 × 105 TCIDso Ch2 in 25 ~tl M20 (M25 without FCS) medium (or with 25/~1 M20 medium alone) resulting in an average multiplicity of infection of 15 : 1. Prior to inoculation of cultures, the purified Ch2 virus had been diluted 1:3 in M20 supplemented with 5/~g/ml trypsin and incubated in a 37 °C waterbath for 1 h. The cultures were incubated at 38 °C for 1 h, the inoculum was aspirated, and, after washing once with PBS, the wells received 200/~1 of M20. Each well then received 2.5/tCi Na 51CrO4 in 10 #1 M20 and the cultures were incubated at 38°C for 3 h (Schat and Heller, 1985) . Wells were washed three times in M20 with 30 min between washes. Each well then received 100/zl LM20 just prior to the addition of effector cells. CKC cultures grown on glass coverslips in 24-well tissue culture plates (Coming) in parallel with the 96-well cultures and evaluated with a direct fluorescent antibody assay (Yason and Schat, 1985) demonstrated from 75 to 90% rotavirus-positive cells 5 h post inoculation when the CKC cultures were ready to receive the effector cell suspensions.

Effector cells were added to target cells at an effector to target cell (E:T) ratio of 50: 1 unless otherwise noted. Effector cells added to CKC targets were centrifuged onto the CKC monolayers at a low speed for 1 rain while effector cells added to suspensions of lymphoblastoid cell targets were not. Cultures were set up in duplicate or in triplicate and were incubated at 38 °C for 4 h. Culture supernatant fluids and pellets were harvested separately and counted in a gamma counter as described (Weinstock et al., 1989) . Percent release was calculated for each well as follows: 100X (cpm of supernatant fluid)/ (cpm of supernatant fluid+ cpm of pellet). Mean percent spontaneous release was determined to be the mean percent release of 12 wells containing target cells without the addition of effector cells. The average spontaneous release values observed were for LSCC-RP9: 26.81%; uninfected P2a cells: 14.78%; rotavirus-infected P2a cells: 20.88%; uninfected N2a cells: 14.53%; rotavirus-infected N2a cells: 21.23%. Percent specific release was determined for duplicate or triplicate experimental wells as follows: mean percent release experimental -mean percent spontaneous release. Mean percent specific release values were compared using Student's t test (Bishop, 1966) .

Cytotoxic activity against lymphoblastoid cells. Duodenal IELs (experiment 1 ) and splenocytes (experiment 2) from four or five uninfected and four or five rotavirus-infected chickens were evaluated approximately every other day for 10 DPI in a CRA using LSCC-RP9 target cells. Thymocytes from uninfected birds, which have been shown to exhibit little or no NK cell activity against LSCC-RP9 (Sharma and Coulson, 1979 ) were used as negative controls in both experiments. Two additional control trials, each consisting of IELs from four uninfected and four rotavirus-infected (7 DPI) birds incubated either with LSCC-RP9 or RECC-CU60 target cells, were also performed.

In experiment 3, duodenal IELs from four uninfected and four rotavirusinfected birds were assessed approximately every other day for 8 DPI in a CRA against uninfected and rotavirus-infected P2a and N2a CKC targets. In experiment 4, duodenal IELs and splenocytes from five uninfected and five rotavirus-infected birds were evaluated at 5, 7, and 9 DPI in a CRA against uninfected and rotavirus-infected P2a CKC targets. Rotavirus-infected birds were re-inoculated with the virus at 14 DPI, and IELs and splenocytes from five uninfected and five rotavirus-infected birds were then evaluated at 16 and 19 DPI. In experiment 5, duodenal IELs collected from uninfected birds (two birds in trial 1 and four birds in trial 2 ) were tested at various E: T ratios against uninfected and rotavirus-infected P2a target cells. Experiment 6 compared the cytotoxic activities of IELs collected from the duodenum versus the jejunum of uninfected birds (four birds in each trial) in a CRA against uninfected and rotavirus-infected P2a CKC targets.

The ability of IELs and splenocytes from rotavirus-infected chickens to lyse a known avian NK cell target (LSCC-RP9) was assessed in experiments 1 and 2 (see Fig. 1 ). The cytotoxic activity oflELs and splenocytes from both uninfected and rotavirus-infected birds was significantly (P< 0.05) greater than that seen with thymocytes for all assay days. With the exception of IELs Fig. 1 . Cytotoxic activity of thymocytes, IELs, and splenocytes against LSCC-RP9 target cells at an E:T ratio of 50: 1. Each bar represents the mean percent specific release ( + standard deviation) for five birds in experiment 1 and four birds in experiment 2. IELs and splenocytes were collected from uninfected control (UC) and rotavirus-infected (Inf) chickens in separate experiments, while thymocytes were collected from UC chickens in both experiments. *Significantly different at P< 0.05. evaluated at 10 DPI, there was no significant difference between the mean % specific release values from uninfected versus infected birds at any DPI for IELs in experiment 1 or splenocytes in experiment 2. Cytotoxic activity was not detectable for IELs from uninfected or rotavirus-infected birds incubated with RECC-CU60 target cells (data not shown). Serum samples collected from birds at 10 and 8 DPI in experiments 1 and 2, respectively, were tested for rotavirus-specific IgM. These assays demonstrated rotavirus-specific IgM re-o 10 ¢D 0 -5 

The cytotoxic activity of IELs and splenocytes for rotavirus-infected CKC targets was evaluated in experiments 3 through 6. Experiment 3 (Fig. 2 ) 

that the rotavirus-inoculated birds were positive for rotavirus-specific IgM while the uninoculated control birds were not. Experiment 4 (Fig. 3 ) showed that while IELs from uninfected and rotavirus-infected P2a chickens were cytotoxic for rotavirus-infected P2a CKCs, splenocytes from uninfected and rotavirus-infected chickens were not. The mean percent specific release val- ues for rotavirus-infected target cells was significantly greater (P< 0.05 ) than for uninfected target cells in assays using IELs but not those using splenocytes. The levels ofcytotoxic activity induced by IELs from uninfected versus rotavirus-infected birds were not significantly different at any DPI for rotavirus-infected target cells, despite reinoculation of the birds at 14 DPI. Serum samples collected from rotavirus-inoculated birds at 19 DPI were positive for rotavirus-specific IgM while sera from the uninoculated control birds were negative. Experiment 5 (Fig 4) demonstrated that the cytotoxic activity of IELs from uninfected P2a chickens for rotavirus-infected P2a cells was dose dependent. Cytotoxic activity peaked at E: T ratios of 25: 1 and 50: 1, and had decreased at 100: 1. The mean percent specific release values for rotavirusnegative versus rotavirus-infected target cells were significantly different at all E:T ratios in both trials except at 12.5:1 (P< 0.05). Experiment 6 ( Fig.  5 ) compared the cytotoxic activity of IELs collected from the duodenum and jejunum. While the mean percent specific release values for uninfected versus rotavirus-infected target cells were significantly different (P< 0.01 ) in both trials, the mean percent specific release values for duodenum versus jejunum were not.

This study demonstrated that NK cell-like activity was expressed by chicken IELs against rotavirus-infected target cells. Furthermore, we confirmed the NK cell activity of chicken IELs against the lymphoblastoid target cell LSCC-RP9 demonstrated by Chai and Lillehoj ( 1988 ) .

The results of experiments 1 and 2 showed that both IELs and splenocytes expressed cytotoxic activity against the accepted chicken NK target cell, LSCC-RP9. The absence of significant chromium release from RECC-CU60 target cells incubated with IELs demonstrated that the IEL preparations did not exhibit non-specific toxicity for lymphoblastoid cells. These experiments confirmed the reports by Chai and LiUehoj ( 1988 ) and LiUehoj and Chai ( 1988 ) which first demonstrated NK cell activity for chicken IELs against LSCC-RP9 and other lymphoblastoid cell lines. We also demonstrated that the NK activity of splenocytes and IELs measured against LSCC-RP9 cells was not affected by primary rotavirus infection of the host. This was in contrast to work performed by Lillehoj (personal communication) who showed that splenic and IEL NK activity was transiently depressed during the early phase of Eimeria primary infections. The subclinical nature of Ch2 rotavirus infections in chickens (Yason and Schat, 1986 ) is one possible explanation for the difference seen between these two systems.

Experiments 3 through 6 demonstrated that IELs exhibited cytotoxic activity against rotavirus-infected target ceils. Because this activity was not induced by rotavirus infection of the host and was not MHC-restricted, it was considered by definition to be NK cell rather than cytotoxic T cell activity. Furthermore, it is unlikely that the cytotoxic activity observed in these experiments was due to macrophages or antibody-dependent cellular cytotoxicity. IELs isolated by this procedure exhibit the characteristic morphology of lymphocytes but not macrophages (Chai and Lillehoj, 1988 ) . Also, cytotoxic activity in our study was demonstrated for IEL preparations from uninoculated control chickens which were negative for antibodies to rotaviruses. While Kohl et al. (1983) reported NK activity for human peripheral blood leukocytes against rotavirus-infected target cells, our demonstration of IEL cytotoxicity for rotavirus-infected cells extend these findings to suggest that NK cell activity may indeed be an important intestinal response to rotavirus infection. In similar studies, in vitro spontaneous cytotoxic activity has been demonstrated for murine (Carman et al., 1986) and porcine (Cepica and Derbyshire, 1983 ) IELs against emetic coronavirus-infected target cells. Our results were in contrast, however, to the findings of Offit and Dudzik (1988 ) , who demonstrated both primary and secondary (after in vitro stimulation) cytotoxic T cell activity for splenocytes against rotavirus-infected target cells. We were unable to demonstrate rotavirus-specific cytotoxic T cell activity for IELs or splenocytes following primary or secondary infection of the host. This does not necessarily indicate that a cytotoxic T cell response was absent from infected birds. First, the response could be masked by a higher level of NK activity in the splenocyte or IEL preparations. Furthermore, MHC-restricted cytotoxic T cell activity has only been documented for chicken splenocytes against reticuloendotheliosis virus-infected cells (MacCubbin and Schierman, 1986; Weinstock et al., 1989) and has yet to be demonstrated for chicken IELs.

While splenocytes were able to lyse LSCC-RP9 cells in an NK fashion, they were unable to lyse rotavirus-infected target cells in this study. This would suggest that there are different NK subpopulations in the spleen and intestine of chickens. This finding is supported by experiments in mice demonstrating that while both IEL and spleen cells exhibit NK activity against YAC-1 tumor cells, there are distinct differences in surface markers between these two populations (Tagliabue et al., 1982; Klein, 1986 ) . Studies are currently planned in our laboratory to examine the surface phenotype 0f IELs with NK activity described in this report.

The reduced cytotoxic activity of IELs for rotavirus-infected CKC targets at high E: T ratios suggested there may be a subpopulation of IELs or some factor in the IEL preparations which suppressed NK cytolysis. Lillehoj and Chai ( 1988 ) did not observe reduced NK activity of IELs at high E: T ratios against LSCC-RP9 target cells in 4-h or 16-h assays. However, Mowat et al. (1983) demonstrated that murine IELs could suppress the NK activity of spleen cells, and that this suppression was not mediated by T lymphocytes or macrophages. Further experimentation will be needed to explore and confirm our observation. Chai and Lillehoj (1988) demonstrated that the NK activity of IELs for lymphoblastoid target cells was greater in the jejunum than in the duodenum for the SC strain chickens. We did not observe a difference in NK activity between these two sites using the rotavirus-infected target cell assay. It is unclear if this discrepancy is due to differences between the IELs from the two strains of chickens or to differences between the assay systems. Chai and Lillehoj (1988) commented that the cytotoxic activity of their IEL preparations was critically dependent upon the expeditious handling of intestinal tissues and cell preparations. We observed a similar decrease in cytotoxic activity when tissues or cell suspensions.were not processed quickly. Furthermore, in our assays using rotavirus-infected CKCs, we observed poor cytotoxic activity by IELs if the number of rotavirus-positive CKCs (as demonstrated by the direct fluorescent antibody assay) was less than 50% or if the spontaneous release of chromium was greater than 30% (data not shown). This study did not attempt to determine the importance of NK cell activity to rotavirus immunity in vivo. This question will be difficult to address experimentally in the chicken system. However, the beige mouse, which exhibits a selective impairment of NK function (Roder, 1979 ) , may prove to be a useful model for examining this question.

Immune response of pregnant heifers and cows to bovine rotavirus inoculation and passive protection to rotavirus infection in newborn calves fed colostral antibodies or colostral lymphocytes

Rotavirus fecal IgA antibody response in adults challenged with human rotavirus

Heterologous protection against rotavirus-induced disease in gnotobiotic piglets

Avirulent rotavirns infections protect calves from disease with and without inducing high levels of neutralizing antibody

Characteristics of in vitro infection of chicken kidney cell cultures with a herpesvirus from Marek's disease

Spontaneous and induced herpesvirus genome expression in Marek's disease tumor cell lines

Intraepithelial leukocytes contain a unique subpopulation of NK-like cytotoxic cells active in the defense of gut epithelium to enteric murine coronavirus

Antibody-dependent cell-mediated cytotoxicity and spontaneous cell-mediated cytotoxicity against cells infected with porcine transmissible gastroenteritis virus

Isolation and functional characterization of chicken intestinal intraepithelial lymphocytes showing natural killer cell activity against tumor target cells

T-cell deficient mice desplay normal recovery from experimental rotavirus infection

Ontogeny of the Thy-1", Lyt-2 + murine intestinal intraepithelial lymphocyte: characterization of a unique population of thymus-independent cytotoxic effector cells in the intestinal mucosa

Cytokine-stimulated human natural killer cytotoxicity: response to rotavirus-infected cells

Vertebrate animal and related viruses, part B-RNA viruses

Interferon activity in rotavirus infected newborn calves

Comparative natural killer cell activities of thymic, bursa, splenic, and intestinal intraepithelial lymphocytes of chickens

MHC-restricted cytotoxic response of chicken T cells: expression, augmentation, and clonal characterization

Analysis of natural killer effector and suppressor activity by intraepithelial lymphocytes from mouse small intestine

Propagation of avian rotavirus in primary chick kidney cell and MA104 cell cultures

Intestinal Iga response and immunity to rota virus infection in normal and antibody-deficient chickens

Development of immunoglobulin classspecific enzyme-linked immunosorbent assays for measuring antibodies against avian rotavirus

Protection against rotavirus-induced gastroenteritis in a murine model by passively acquired gastrointestinal but not circulating antibodies

Rotavirus-specific cytotoxic T lymphocytes cross-react with target cells infected with different rotavirus serotypes

Persistent rotavirus infection in mice with severe combined immunodeficiency

The beige mutation in the mouse I. A stem cell predetermined impairment in natural killer cell function

In vitro cytotoxicity of spleen lymphocytes against Marek's disease tumor cells: Induction by SB-1, an apparently non-oncogenic Marek's disease virus

A chromium-release assay for the study of cell-mediated immune responses to Marek's disease antigens

Presence of natural killer cells in specific-pathogen-free chickens

Natural killer cell activity in chickens: target cell analysis and effect of antihymocyte serum on effector cells

Virus-specific immunity in neonatal and adult mouse rotavirus infection

Characterstics of natural killer cells in the murine intestinal epithelium and lamina propria

Interferon response in colostrum-deprived newborn calves infected with bovine rotavirus: its possible role in the control of the pathogenicity

Intestinal antibody response after vaccination and infection with rotavirus of calves fed colostrum with or withour rotavirus antibody

Virus-specific syngeneic killing of reticuloendotheliosis virus transformed cell line target cells by spleen cells

Cytotoxic T lymphocytes in reticuloendotheliosis virus-infected chickens

Isolation and characterization of avian rotaviruses

Experimental infection of specific-pathogen-free chickens with avian rotaviruses

The authors would like to express their sincere thanks to Pricilla O'Connell for her expert technical assistance in these experiments, and to Gwen Troise for her help in the preparation of the manuscript. This work was supported in part by USDA grant 8602162 and by USDA Animal Health Disease Research Section 1433 formula funds.