key: cord-0010627-0yabira3
authors: ARTURSSON, K.; LINDERSSON, M.; VARELA, N.; SCHEYNIUS, A.; ALM, G. V.
title: Interferon‐α Production and Tissue Localization of Interferon‐α/β Producing Cells after Intradermal Administration of Aujeszky's Disease Virus–Infected Cells in Pigs
date: 2006-06-29
journal: Scand J Immunol
DOI: 10.1111/j.1365-3083.1995.tb03543.x
sha: cb2959b0789ee3a4c6fa91e8c24b5701955e0779
doc_id: 10627
cord_uid: 0yabira3

Intradermal administration of glutaraldehyde–fixed Aujeszky's disease virus (ADV) infected autologous or allogeneic cells resulted in the induction of an interferon(IFN)–α/β response in pigs. Using a sensitive dissociation–enhanced lanthanide fluoroimmunoassay (DELFIA), IFN–α/β was detected in blood at 8 and 24 h after injection of ADV–infected cells. In parallel, by means of in situ hybridization, IFN–α/β mRNA containing cells were demonstrated in regional lymph nodes. Occasional IFN–α/β mRNA positive cells were also seen in injected dermal areas, but not in contralateral lymph nodes, spleen, bone marrow, blood or liver. The ability of leucocytes in whole blood cultures to produce IFN–α/β upon stimulation by ADV was markedly diminished 3–7 days after intradermal injection of ADV–infected cells. In contrast, cultures of purified peripheral blood mononuclear cells (PBMC) had intact IFN–α/β responses. Further, serum from ADV–injected pigs inhibited the in vitro ADV–induced IFN‐α/β responses in PBMC from control pigs, most likely due to the demonstrated presence of anti–ADV antibodies. We suggest that the IFN‐α/β producing cells in lymph nodes may participate in the development of antiviral immunity and could be equivalent to Natural IFN–α/β producing (NIP) cells.

Viral infections usually cause prompt production ofthe type-I interferons (IFN), IFN-o-and -13, which can directly limit the viral replication in cells by activating several intracellular mechanisms [1] . The type I IFN also have many immunomodulatory effects [1] , and recently IFN-Q has been implicated in promoting the development of Thl lymphocytes and IgG2a antibody production that are important in antiviral immunity [2, 3] .

Many different cell types can produce IFN [1] . However, in human mononuclear leucocytes stimulated by RNA viruses, monocytes are the main producers of IFN-Q//? [4] [5] [6] . In contrast, certain DNA viruses, such as the herpes viruses, free or glutaraldehyde-fixed and cell-associated, stimulate IFN-a//? responses in infrequent, but highly productive, mononuclear blood leucocytes. In humans, these cells lack markers typical of monocytes as well as T and B lymphocytes, but express for instance MHC class II antigens, CD4 and CD36 [4, [7] [8] [9] [10] [11] [12] [13] . They have provisionally been designated natural IFN-a//3 producing cells (NIP cells) [11] . Although the NIP cells have some resemblance to antigen presenting dendritic cells, they have, in one study at least, been separated from such cells [14] . The corresponding cells in pigs, which can be triggered by free or cell-associated transmissible gastroenteritis virus (TGEV), a coronavirus. have also been well characterized [15. 16] . The same cell population appears to be stimulated by cells infected by Aujeszky's disease virus (ADV), also termed Pseudorabies virus [17] .

With regard to the IFN-a/^ response in vivo, little or no information is available regarding its cellular basis or the distribution of IFN-a//? producing cells at sites of viral infections and in lymphoid tissues. In patients with hepatitis, IFN-Q producing ceils were demonstrated by immunohistochemical staining in liver sections [18] . In mice injected with polyinosinic-polycytidylic acid (poIy-IC), IFN-/? producing cells were demonstrated in the spleens within a few hours [19] . To further study the lFN-a/0 response in vivo.

we developed an experimental model, where specific pathogen free (SPF) pigs were injected intrademially with glutaraldehyde-fixed, ADV-infected (and thus non-infectious) autologous or allogeneic cells. Because significant levels of IFN-a were found in serum samples 8 and 24 h after the injections, we examined injected dermal tissue, regional and contralateral lymph nodes, the spleen, bone marrow and the liver for the presence of IFN-a and lYU-Li mRNA expressing cells, respectively, at the corresponding time points. We also studied the ability of peripheral blood mononuclear cells (PBMC) from injected animals to produce IFN-a in vitro.

A total of 16 pigs were studied. They were crossbreed gilts (Swedish Yorkshire x Swedish Landrace) of SPF origin, 12 weeks old at the start of the study. Two to three pigs were kept per room at the animal department of the National Veterinary Institute, Uppsala and all pigs were allowed I week's acclimatization prior to the study.

Preparation of PBMC. Blood samples were collected by jugular vein puncture in vacutainer tubes (Becton Dickinson. Grenoble, France) with or without the addition of Sodium Heparin (150USP/10ml blood). The blood was diluted I: I in phosphate buffered saline (PBS) and centrifuged for 30 min, at 550 x g, on Ficoll-Paque (Kabi-Pharmacia, Uppsala, Sweden). The PBMC were collected, washed in PBS and resuspended in complete medium, that is RPMI 1640 (Flow Laboratories, Irvine. UK) supplemented with penicillin (60/ig per ml), streptomycin (100/ig/ ml). L-glutamine (2mM) and 5% Myoclone^"^ fetal calf serum (FCS; Gibco, Paisley, UK).

Virus. Aujeszky's disease virus of the Phylaxia strain (kindly provided by Dr Sandor Belak, National Veterinary Institute, Uppsala. Sweden) was grown in porcine kidney (PK15) cells (Flow Laboratories) in Dulbecco's modification of Eagle's medium (DMEM: Flow Laboratories) supplemented as described above. The virus-containing supernatants were harvested and centrifuged for 5 min at 400 x g lo remove cell debris. The virus titre in PKI5 cells was determined to be 10*' TCID^u per ml.

To study the inhibitory effects of immune serum on the in vitro ADV-induced IFN-Q response, we used purified ADV astheinducer oflFN. Cell culture supernatants. containing the virus, were clarified by centrifugation at 2000 x g for 30 min at 4X. The virus was concentrated by ultraflltration using a 300 kDa cut off tangential How filter (Filtron Corp., Clinton, MA, USA) . The virus was pelleted by centrifugation at 85000 x g for 2h at 4''C. Pellets were resuspended in PBS and stored in aliquots at -80°C. The ADV was used at a concentration of lO' TCID;,,, per ml and was prior to use inactivated by UV irradiation, approximately 0.5 J/cm'. The absence of infectious viral particles was confirmed in PK15 cell cultures.

Preparation of ADV-infected ,stimulatory cells. In experiment 1, PBMC were prepared from the experimental pigs 1 week before start of the experiment. A total of 50 x 10^ autologous PBMC were incubated with ADV, 4 x lo'' TCID50. in 14 ml complete RPMI medium in 25cm^ culture bottles (Nunc, Roskilde, Denmark) for 2 h at 37 'C and 7% CO2 air concentration. The cells were harvested and then washed in PBS by centrifugation, fixed in 0.05% glutaraldehyde in PBS for 15 min at room temperature, washed again in 0.15 M NaCl and stored at 4^ in 0.15 M NaCl with 10% swine serum, penicillin (60^g/ml) and streptomycin (lOO/ig/ml) until use. The swine serum did not contain antibodies to ADV. These cells are designated ADV-PBMC. Control cells not incubated with ADV were concurrently prepared. Before use, all stimulatory cells were washed and diluted to a concentration of 50 x 10^ cells per ml in 0,15 M NaCl.

In experiments 2 and 3. confluent monolayers of PK15 cells in 225 cm" culture bottles (Nunc) were trypsinized, washed with PBS and resuspended at 0.4 x JO'' cells per ml in DMEM complete medium containing HEPES (lOmM). The cells were incubated in slowly shaking culture bottles for 2h at 37 "C and then for another 2h at 37 C with the addition of 8 x 10* TCIDso ADV per ml. The cells were then washed in PBS, glutaraldehyde-fixed as in experiment 1. treated with 3% glycine in PBS for 15 min at room temperature, washed again in PBS and then treated with 0.5 J/cm^ UV-irradiation. These cells are designated ADV-PK15 cells. Control cells were prepared in parallel, omitting the ADV. Al! cells were stored as described above for ADV-PBMC and the cell concentrations were adjusted to 75 x 10^ cells per ml in 0.15 M NaCl prior to injection.

Before injection into the pigs, al! stimulatory cell preparations were verified to be free of ADV infectious to PK15 cells and free of significant levels of endotoxin by employing a limulus assay using a chromogenic substrate (Kabi-Pharmacia, Stockholm. Sweden).

Injection of cells and .mmptings. In experiment 1, four pigs were injected intradermally in the flanking region of the abdomen with approximately 60 x 10" fixed ADV-PBMC and 60 x 10^ control PBMC, each distributed within six marked adjacent sites. Serum samples were collected immediately prior to injections and at 8, 24. 48 and 72 h as well as 1 week after injection. Concurrently, skin biopsies were taken at cell-injection sites and immediately frozen in isopentane chilled by dry ice in acetone.

In experiment 2, six pigs were injected intradermally in the flanking region with approximately 60 x 10*" fixed ADV-PKI5 cells distributed within four adjacent sites in the right flanking region and 7 days later in the same manner with control PK15 cells, but in the left flank. Serum samples were collected immediately before injection and at 8, 24. 48 and 72 h after each set of injections.

In experiment 3, four pigs were injected intradermally in the flanking region with 60 x lO*" fixed ADV-PK 15 cells as described in experiment 2. Two pigs were used as non-injected controls. Blood samples were taken before cell injection and then immediately before anaesthesia (see below). At 6 and 24 h after injection, two pigs. injected with ADV-PK 15 cells, and one non-injected pig were anaesthetized by the use of the combination azaperone (2 mg/kg bodyweight i.m.) and methomidate (10 mg/kg body weight i.p). Skin biopsies (circular, 3 mm diameter) were taken both at the sites of injection and on the contralateral flank of the pigs. The regional subiliac lymph nodes, draining the area of injection [20] , and the contralateral lymph nodes were procured, as well as small parts of the spleens and livers, before the pigs were killed. Bone marrow specimens were removed after death and immediately mixed with an equal volume ofa solution containing 90% FCS and 10% DMSO. All samples were immediately frozen, as described above, and stored in liquid nitrogen.

Preparation of specimen. Cryostat sections, 6/im thick, were prepared from collected tissues. Sections from lymph nodes, spleens and liver measured about I cm". The skin biopsies were sectioned longitudinally. Sections for in situ hybridization were fixed on slides pretreated with 2% gamma-aminopropyltriethoxy-silane in acetone, for I min in 4% paraformaldehyde in PBS. The slides were then stored in 70% ethanol in diethylpyrocarbonate (DEPC)-treated distilled water at 4X until hybridized.

A volume of 4 ml DEPC-treated distilled water was added to 0.9 ml of heparinized blood to lyse erythrocytes. After a 1 min incubation, 5 ml of PBS was added. The leucocytes were pelleted and treated for 5 min with 5 ml of 1% paraformaldehyde in PBS. After washing in PBS, the cells were resuspended in 0.3 ml of PBS and 25/tl were added per well of ethanol-pretreated eight-well slides with hydrophobic coating (8 mm well diameter), dried and stored in 70% ethanol at 4^.

In situ hybridization. The cRNA probes labelled by a-^^S-UTP were prepared by in vitro transcription of DNA as described previously fl7]. For detection of IFN-a mRNA, a 829-bp Eco Rl-Hpa I fragment of the porcine (po) IFN-aj gene [211 *^s transcribed. For detection of 1FN-/3 mRNA a 7I2-bp Eco Rl-Pst 1 fragment of the poIFN-tf gene [17] was transcribed.

The RN A-RN A in situ hybridizations were performed as previously described [17] . that is, at high stringency for 3-4 hat SO'C with probes in 50% formamide and 2xSSC. followed by RNase treatment. The slides were covered with NTB-2 nuclear track emulsion (Eastman-Kodak, Rochester, NY, USA), and developed after 10 days exposure. The hybridized sections were stained by haematoxyiin-eosin according to standard procedures, in total, two to eight sections of each specimen were microscopically examined and the mean number of labelled cells per section was estimated.

In vitro induction oflFN-a ri'.spon,ses. At each sampling occasion in experiment 2. heparinized blood from each pig was diluted 5 x in RPMI complete medium and concurrently PBMC were prepared and diluted to a concentration of 4 x lO" cells per ml in RPMI complete medium. Quadruplicate cultures were established by adding 0.1 ml per well, of either diluted blood or purified PBMC, in flat bottom 96-well microtitre plates (Nunc). To each type of culture, O.I ml of ADV-PKI5 cells or control PK15 cells, 0.8 x 10*" per ml in RPMI eomplete medium, were added. The cultures were incubated for 20 h at 37''C in 7% COi air concentration, and the supernatants were then harvested for IFN-« determinations.

To study the effect of immune sera on the IFN-a response, PBMC cultures from a control pig, not previously used in the experiments and free from antibodies to ADV, were established as described above, except that the FCS was excluded from the medium. The medium was supplemented with 5% of individual sera collected from the pigs in experiment 2, as indicated. The cultures were stimulated by adding 0.1 ml volumes per well of purified, UV-treated ADV diluted 200 x in complete RPMi 1640 medium without FCS. Supernatants were collected for lFN-cv assays after 20 h of culture.

Mca.surement of antibodies to AD V in sera. Antibodies to ADV in sera were detennined by an ELISA, based on the ability of serum anti-ADV antibodies to bind to ADV. In brief, 96-well microtitre plates (Immuno Plate Maxisorp; Nunc) were coated overnight at room temperature with 100/tl per well of purified ADV. protein concentration 6/ig per m! in 50 mM Tris HCl pH9.5. Unspecific binding sites were blocked by post-coating for 1 h at 37°C with ELISA dilution buffer, that is PBS with 0.5% (w/v) BSA, 0.25% (w/v) merthiolate and 0.05% (v/v) Tween 20. After washes, serum samples diluted 500 x in ELISA dilution buffer, were added in volumes of 100^1 per well, incubated for 1 h at 37''C and washed. Bound serum antibodies were detected by incubation for ! h at 37 C with 100/xl per well of HRP-conjugated rabbit anti-swine Ig antibodies (DAKO Corp., Glostrup, Denmark), diluted 10000 x in ELISA dilution buffer. After washing, 200/il per well of substrate consisting of tetramethylbenzidine (I mM) in O.I M pH4.25 potassium citrate buffer containing 2mM H2O2 was added. After 10 min at room temperature, reactions were stopped by adding I M H2SO4. 100/tl per well, and the absorbance was read at 450 nm in a plate spectrophotometer.

Immunoassay of IFN-a. An immunoassay for porcine lFN-a was established, based on the principle of dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA) [22] . It is based on two murine MoAbs, designated K9 and F17 [23, 241 . directed to independent epitopes on polFN-o and used previously in an ELISA for polFN-o [25] . The MoAbs were precipitated from mouse ascitic fluid by ammonium sulphate, and further purified by ion exchange ehromatography (Mono S; Kabi-Pharmacia Biotechnology. Uppsala, Sweden). The K9 MoAb was labelled with Europeum lanthanide chelate according to the manufacturer's recommendations (Wallac Oy, Turku, Finland), and purified by gel filtration on a Superose 12 column (Kabi-Pharmacia Biotechnology). The Europeum-labelled MoAb was used in the assay at a concentration of 0.1 /ig per ml in dilution buffer.

The F17 MoAb was treated with 50mM HCl and diluted to 3/ig per ml in 0.2 M NaHjPOj (pH4.5) with 0.005% NaNj, and used to coat flat bottomed microtitre plates (Immuno Plate Maxisorp; Nunc). Volumes of O.I ml per well were used, and plates were incubated overnight at room temperature. They were washed in PBS with 0.05% Tween 20, and postcoated for I h at 37^ with 0.3 ml per well ofa buffer (pH4.5) containing 6% sorbitol, 0.15M NaCl. 0.1% BSA, 50 mM NaH2pO4. 0.1 mM CaCl; x 2H2O, 4/iM EDTA and 0.005% NaN3, The plates were washed once in 50 mM Tris buffer (pH7.8) with 0.15 M NaCl, 0.05% Tween 20 and 0.005% NaN3. The samples were diluted in dilution buffer, that is, 50mM Tris buffer (pH 7.8) with 0.15 M NaCl. 0.005% NaN,. 0.5mM CaCl: x 2H2O, 20//M EDTA and 0.5% BSA, and 0.2ml volumes were added per well. When serum samples were assayed, 0.5% normal mouse serum was included in the dilution buffer. The samples were incubated for 2h while undergoing slow shaking at room temperature, and the plates were then washed three limes.

Europeum-iabelled K9 MoAb was then added, 0.2 ml volumes per well, plates were incubated for I h and washed six times. Enhancement solution (Wallac Oy), 0.2 ml per well, was added. After 20 min the fluorescence (counts per second) was measured in a DELFIA Fluorometer (Wallac Oy). The IFN-« concentrations in antiviral units [U] per ml was calculated using standard curves constructed by the use of a porcine IFN-a preparation obtained from Sendai virusstimulated leucocytes [26] .

Statistics. The paired Student's /-test was used to determine the significance of differences, using the STATVIEW 512+ program (Brainpower, Inc., Calabasas, CA. USA) on a Macintosh computer.

The DELFIA developed for measurement of IFN-Q in this study proved to be highly sensitive and IFN-a levels as low as 0.1 U per ml were routinely detected. In serially acquired samples from individual animals, even lower IFN-Q levels produced specific signals (see below). With appropriate fitting of Standard curves in log/log plots, linear ranges up to at least 800 U per ml were obtained. Coefficients of variation for the measurements of samples throughout this range were usually below 10%. Two separate experiments were performed to study the IFN-Q levels in serum of pigs injected with ADV-infected cells. In the first (Fig. 1) , four pigs were injected intradermally with 60 X 10^ glutaraldehyde-fixed autologous ADV-PBMC and an identical number of control cells. Serum from all four pigs, obtained at 8 and 24 h post injection, displayed increased IFN-a concentrations measured as counts per second (cps). The two pigs with the highest IFN-a responses had maximal levels corresponding to 0.9 and 3.4 U per ml, respectively.

Iti the second experiment (Table 1) , a group of six pigs were first injected intradermally with 60 x 10^ glutaraldehydefixed ADV-PK 15 cells and I week later with control PK15 cells, using the same method. Serum samples from all animals, obtained 8 and 24 h after injection with ADV-PK 15, displayed increased cps-values in the DELFIA for IFN-fi compared to the values for sera before injection (Oh) and at 48 h or later. The values at 8 and 24 h were significantly increased {P -0.03 and P -0.005, respectively), compared to Oh values. The highest IFN-a concentration, caused by ADV-PK. 15, was in this experiment 1.9 U per ml serum. No evidence of IFN-a response after injection of control PK15 cells was seen.

In experiment 1, skin biopsies obtained from the sites of injection ofthe fixed autologous ADV-PBMC were analysed for the presence of cells containing IFN-a mRNA by means of a "S-labelled cRNA probe applied to cryostat sections. Positive cells were found at a low frequency, only one or two per section at 24, 48 and 72 h after injection. These cells were localized adjacent to injected ADV-PBMC, which could readily be discerned in the sections. Sections of biopsies at other time points were all negative, as were sections from control injection sites receiving control PBMC. In .situ hybridizations with a cRNA probe for human /3-actin mRNA were positive, indicating little degradation of RNA. Examination of skin sections revealed little or no infiltration of inflammatory cells.

It was unlikely that the low number of IFN-a producing cells detected in the skin accounted for the significant systemic IFN-a response in the pigs. Therefore other tissues were examined in addition to skin for presence of IFN-a or IFN-(3 producing cells. In this experiment four pigs were injected with 60 X 10* ADV-PK15 cells and two non-injected pigs were used as controls. Two injected and one control pig were killed at each 6 and 24 h. The results of the evaluation of the collected material from skin and lymph nodes are summarized in Table 2 .

Infrequent cells containing mRNA for IFN-a or IFN-/? were detected in sections of skin taken 6 and 24 h after injection of fixed ADV-PK 15 celis (Fig. 2a) . The labelled cells appeared in the vicinity of the injected ADV-PK15 cells, easily identified at both time points. No significant e mean number of IFN-a/^ mRNA expressing cells per section is indicated, measured in the DELFIA assay, described in Materials and Methods. biopsies were taken at the site of injection (regional), which was in the flanking region, and control biopsies were taken from non-injected skin (control) in the opposite flank.

"^ Lymph node biopsies were taken from the subiliac lymph node (regional) draining the site of injection and from the corresponding contralateral lymph node (control). ND is not done.

infiltrates of infiammatory cells were observed in the skin sections.

Larger number of cells positive for IFN-a or IFN-m RNA were found in the subiliac lymph nodes which drain the sites of injections of ADV-PK 15 cells. The IFN-a and IFN-/? mRNA positive cells were found both at 6 and 24h after injection (Fig. 2 b-d) . Clusters of injected ADV-PK 15 cells were seen along the lymphonodular trabeculae in limited parts of the nodes. The IFN mRNA containing cells essentially lined such ADV-PK 15 cell aggregates on the sides facing the cortex, but were also seen deeper in the cortex. In the lymph node of pig number 14, in which very few IFN mRNA positive cells were detected, no ADV-PK 15 cells were found. A mean of 109 (range 62-178) IFN-Q mRNA positive cells were detected per whole lymph node section from pigs 11, 12 and 13 ( Table 2 ). The IFN-/J mRNA positive cells were less heavily labelled, fewer in number and had a mean of 28 (range 7-43) positive cells per section. Examination of sections from the contralateral subiliac lymph nodes, sections of spleen and liver or smears of bone marrow or peripheral blood did not reveal any clearly positive IFN-a or IFN-/? mRNA containing cells. Also, similar preparations from the two non-injected control pigs were negative.

The six pigs injected with ADV-PK 15 cells in experiment 2 were also examined for the ability of leucocytes in whole blood cultures, or as purified PBMC, to produce IFN-o when exposed in vitro to glutaraldehyde-fixed ADV-PK 15 cells. The results are summarized in Fig. 3 .

The mean concentration of IFN-a produced in cultures of leucocytes derived from pigs before the injection of ADV-PK 15 cells were in PBMC cultures 497 ± 97U/mt (Fig. 3a) and in whole blood cultures 43 ± 31 U/ml (Fig. 3b) . One week later, immediately before the injection of the control PK15 cells, the corresponding IFN-a concentrations were 455 ± 162U/mi and 2.2 ± 5.3 U/ml respectively.

The IFN-a production in purified PBMC at 24 h after injection of the ADV-PK 15 cells was significantly higher than that at Oh (/* = 0.02). Further, the IFN-a production at 48 and 72h was significantly lower than that at Oh {P < 0.05). During the second week, when the pigs were injected with control PK15 cells, the ability of PBMC to produce IFN-a was relatively constant. No IFN-a was produced in vitro when the PBMC were cocultured with control PK 15 cells. Wheti the induction in whole blood cultures was studied, the IFN-a responses were getierally lower, about one tenth of those of purified PBMC, as would be expected from their lower number of tnononuclear leucocytes. A tendeticy towards decreased IFTV-n production (P = 0,09) was seen already at 8 h after the administration of infected cells. The production at 24 and 48 h was significantly lower (_P < 0.05) than that seen at Oh. Furthermore, immediately before injection of the control PK15 cells and at all times thereafter throughout the second week, whole blood cultures of all animals essentially failed to produce IFN-a when stimulated with ADV-PK 15 ceUs.

As shown in Fig, 4 . antibodies to ADV were first detected in serum samples 7 days after the injection of ADV-PK15 cells and their levels further increased during the following 3 days.

Anti-ADV antibodies or other serum factors could possibly cause the differences in the IFN-a producing ability between leucocytes in whole blood cultures and as purified PBMC. Therefore, purified PBMC from a control pig, not previously used in the experiments, were cultured in the presence of 5% of the sera of each of the six pigs used in experiment 2. As shown in Table 3 , sera collected 7 and 10 days after injection ofthe ADV-PK 15 cells markedly inhibited IFNa responses, compared to sera collected before and 3 days after injection. Thus, the suppressive effect appeared concomitantly with the first detection of anti-ADV antibodies.

The results of our study demonstrate that an intradermal injection in pigs of allogeneic or autologous cells, infected by Aujeszky's disease virus and glutaraldehyde-fixed, induces the production of IFN-o of such quantities that low but significant levels can be detected in blood within 8 h. Instrumental in the detection of such low levels of IFN-Q in serum was the development of the highly sensitive immunoassay, using the DELFIA principle, which was able to detect concentrations at 0.1 U per ml.

Significant levels of IFN-a in blood were seen both after the administration of autologous ADV-PBMC and after the administration of allogeneic ADV-PK15 cells, but not with the non-infected control cells. Since no infectious virus was present, our results confirm in vivo that glutaraldehyde-fixed cell-associated virus can induce IFN-a/^, as has been demonstrated previously in vitro [17. 26-29] .

After a single intradermal administration of ADV-infected inducer cells, maximal IFN-a levels in blood were present Attempts were made to trace cells that were responsible for the production of IFN-a or -f3 in vivo in response to the intradermally injected ADV-infected cells. However, in Table 3 . The effect of sera' from pigs injected with ADV-infected glutaraldchyde-lixcd PK-15 cells on ADV-induced production of Sera were obtained prior to injection with ADV-infected, glutaraldehyde-fixed PK-15 cells (day 0) and at indicated days post injection (p.i.).

'The in vitro induction of IFN-Q production was carried out as described in Materials and Methods, but the FCS was substituted with 5% serum, collected from experimental pigs number 5-10 at indicated days p.i. The amount of IFN-a produced was measured by the DELFIA described in Materials and Methods and is expressed as units per ml culture medium, " Cells obtained from a control pig, verified to be free of serum antibodies to PRV.

•* Due to the experimental design the pigs were injected with noninfected control cells on day 7. injected skin, no more than one to two cells expressing IFN-a or -fi mRNA were identified per section. One possible explanation for the low dermal IFN-a//? responses is a poor recruitment of IFN-a//3 producers from the circulation, which is supported by the observed weak inflammatory response. By extrapolating the low frequencies of IFN-tt//j positive cells observed in skin sections, less than 1000 IFN-Q/ /? producing cells should be present per injection site. Assuming that these cells correspond to NIP cells and each cell produces as much as 10 U of IFN [17, 30] before production is down-regulated at 24 h after stimulation [31] . the total amount of IFN-tf produced can have significant local effects. However, the produced IFN-a can only account for a minor part ofthe IFN-a detected in serum since the half-life of IFN-fi here is a matter of minutes [32] .

In contrast to the dermal injection sites, relatively high numbers of IFN-a and -/? mRNA containing cells were demonstrated in the regional lymph nodes 6 and 24 h after administration of ADV-PK 15 cells. Based on their frequency in sections, and size ofthe nodes, approximately 10* IFN-a and 3 x 10"* IFN-/? mRNA positive cells were present at one time. These cells should be able to produce in the order of 1-10 X 10" U IFN-a. which could well result in the serum levels of IFN-a detected by the immunoassays. Furthermore, no IFN-a or IFN-/3 producing cells were observed in other tissues, including contralateral lymph nodes, spleen, bone marrow, blood and liver. It is therefore likely that most of the IFN-Q//? production in the pigs actually occurred in the regional lymph nodes.

The IFN-a//3 mRNA positive cells were localized near aggregates of ADV-PK 15 cells in the lymph node tissue and tended to be positioned preferentially in lymphocyte-dense cortical areas and not in the medulla. The fact that each porcine lymph node consists of accumulations of several independent lymphonoduli. each with separate afilerent lymph vessels [33] , may explain why IFN mRNA positive cells and ADV-infected cells were only seen in parts of each subiliac lymph node. Also, the eflerent lymph of porcine lymph nodes Is free of lymphocytes, which instead must exit via efferent blood. This may not be possible for ADV-PK 15 cells which instead may be trapped in the nodes, offering one explanation for the lack of IFN-a//3 mRNA positive cells in other tissues and organs.

A reasonable hypothesis is that the IFN-a//3 mRNA containing cells identified in vivo in the present study correspond to the NIP cells previously characterized in pigs and in humans [4. 7-13, 15, 30] , but this awaits confirmation. In vitro studies indicate that NIP cells are the only leucocytes capable of IFN-a//^ production after exposure to inducers of the type represented by glutaraldehyde-fixed ADV-PK 15 cells [10, 11, 15, 17, 22, 34] .

The production and very high concentration of IFN-o//5 in the regional lymph nodes could well have a significant impact on early antiviral immune responses. Thus, IFN-a//? has been implicated in the regulation of immune reactions, for instance, promoting development of T^l cells and IgG2a production [2, 3] . Further evidence that locally produced IFN has a significant efifect on a lymphoid organ are the findings that poly-IC injected intraperitoneally in mice, via induction of cells producing IFN-/3. caused dramatic changes in tissue architecture of the spleen and redistribution of lymphoid cells [19] . Furthermore, IFN causes enlargement of lymph nodes by increasing their content of lymphocytes [35] . In addition, the high local IFN-a//^ concentrations should efiScientiy block viral replication in lymph nodes and prevent negative viral impacts on early immune responses.

When purified PBMC derived from pigs injected with ADV-PK 15 was studied, their IFN-Q responses, induced by ADV-PK 15 cells in culture, were increased 24 h after injection ofthe ADV-infected cells. The alterations could refiect IFNinduced effects on PBMC, like distributional changes of leucocytes [19, 35] or priming effects [I]. In contrast, when the leucocytes were tested in whole blood cultures, all animals showed decreased IFN-a responses 7 days p.i. Since it was shown that serum collected from the animals at this time suppressed ADV-induced IFN-a responses of normal PBMC, one or several suppressive serum factors may be involved. Sera from the experimental pigs in the present study had high levels of antibodies to ADV about 1 week after injection of ADV-PK 15 cells. Such antibodies may well be responsible for the suppression, because both polyclonal and certain MoAbs to ADV have been shown to efficiently inhibit the ADV-induced IFN-a response by porcine PBMC in vitro (Artursson et al.., unpublished observations) . Also, antibodies to other viruses can inhibit their interferogenic activity [28. 29. 36 ].

Since antiviral antibodies during a viral infection initially are produced and are present in high concentrations in those n;gional lymph nodes that obviously also are main sites for the IFN-a/^;r esponse, we suggest that the anti-interferogenic activity of antibodies is a physiologically relevant negative feedback mechanism, which limits IFN-a//i responses and might in this way influence the development of antiviral immunity.

Inierferons and other regulatory cytokines

Regulation by inter-Interferon-aff Producing Cells In Vivo 129

of immunoglobulin isotype selection and lymphokine production in mice

IL-4 and IFN (n and -y) exert opposite regulatory effects on the development of cytolytic potential by Thl or T[,2 human T cell clones

Human lymphocytes involved in cv-interferon production can be identified by monoclonal antibodies against cell surface antigens

Virusinduced interferon production by human macrophages

Monocyte is the main producer of human alpha interferons following Sendai virus induction

Human peripheral null lymphocytes 11. Producers of type-1 interferon upon stimulation with tumor cells, Herpes simplex virus and Corynebacterium parvum

Human natural interferon-a producing cells

Cooperation between CD 16 {Leu-llb)+ NK cells and HLA-DR+ cells in natural killing of Herpesvirus-infected fibroblasts

Characterization of Herpes simplex virus stimulated inierferon-a producing blood mononuclear Ieucocyte.s, using combined immunohistochemical staining and in .titu RNA-RNA hybridization

A distinct population of nonphagocytic and CD4^ null lymphocytes produce interferon-n after stimulation by Herpes simplex virus infected cells

Flow cytometric analysis of natural interferon-a producing cells

A leukocyte subset bearing HLA-DR antigens is responsible for in vitro alpha interferon production in response to viruses

Dendritic cells and interleron-alpha-producing cells are two functionally distinct non-B, non-monocytic HLA-DR + cell subsets in human peripheral blood

Characterization of blood mononuclear cells producing IFN-ct following induction by Coronavirusinfected cells (Transmissible porcine gastroenteritis virus)

Age-related increase ot porcine interferon alpha producing cell frequency and of interferon yield per cell

Molecular cloning of a gene encoding porcine IFN-/?

Cellular localization of a-interferon in hepatitis B virus-infected liver tissue

IFN induction and associated changes in splenic leukocyte distribution

Molecular cloning and sequencing of a gene encoding biologically active porcine o-interferon

Europeum as a label in time-resolved immunofluorometric assays

Production, purification and biological properties of an Escherichia coli-derived recombinant porcine alpha interferon

Production of a hybridoma library to recombinant porcine alpha I interferon: a very sensitive assay (ISBBA) allows the detection ofa large number of clones

A sensitive immunoassay for porcine interferon-ft

Appearance of interferon-o in serum and signs of reduced immune function in pigs after transport and installation in a fattening farm

Induction of alpha interferon by membrane interaction between viral surface and peripheral blood mononuclear cells

Inhibition of Herpes simplex virus type 1-induced interferon synthesis by monoclonal antibodies against viral glycoprotein D and by lysosomotropic drugs

Induction of alpha interferon by transmissible gastroenteritis corona virus: Role of transmembrane glycoprotein EI

Enrichment ot corona virus-induced interferon-producing blood leucocytes increases the interferon yield per cell: a study with pig leucocytes

Different patterns of mRNA for IFN-a and -/? in human mononuclear leucocytes after in vitro stimulation with Herpes Simplex virus-infected fibroblasts and Sendai virus

Metabolism of protein anticancer agents

Veterinary Immunology. An introduction, Philadelphia

Infrequent but highly efficient interferona producing human mononuciear leucocytes induced by herpes simplex virus in vitro studied by immuno-ptaque and limiting dilution assays

Interferon induces peripheral lymphadenopathy in mice

Cherbonnel M-Interferon-induction in mouse spleen cells by the Newcastle disease virus (NDV) HN protein

This study was supported by grants from the Swedish Council for Forestry and Agricultural Research, the Swedish Medical Research Council, the Swedish Agency for Research Cooperation with Developing Countries. Ihc Swedish Work Environment Fund and the Hjarre fund. We thank Catharina Johansson and Marianne Carlsson for excellent technical assistance. Special thanks to Per Wallgren for help with the sample collection and Caroline Fossum for supporting the work in many ways, including invaluable discussions and review of the manuscript.