Immunology 197937231
Immune mechanisms against canine distemper I.
IDENTIFICATION OF K CELL AGAINST CANINE DISTEMPER VIRUS INFECTED TARGET CELLS IN VITRO
CHI KUAN HO I LORNE A. BABIUK Department of Veterinary Microbiology, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada
Received 2 October 1978; acceptedfor publication 26 October 1978
in the recovery from, as well as resistance to, viral infections (Allison, 1974; Notkins, 1974; Rouse, Wardley & Babuik 1976a,b). Because of its potentially important role in defence, considerable interest has been generated to determine the particular cell type(s) involved in this activity. These studies have utilized cell separation techniques, surface marker studies and functional analysis in attempts to define the properties of the effector cells against various target cells. In all the studies conducted to date the effector cell in ADCC has always been found to possess Fc receptors for IgG regardless of other surface markers present. Since the Fc receptor is of crucial importance it is not surprising that neutrophils (Gale & Zighelboim 1975; Rouse et al., 1976b; Rouse, Grewal, Babiuk & Fujimiya, 1977a; Rouse, Wardley, Babiuk & Mukkur, 1977b), mononuclear phagocytic cells (Dennert & Lennox, 1972), B lymphocytes (Perlmann, Perlmann & Wizzell, 1972; Zighelboim, Bonavida & Fahey, 1973) as well as the recently described null (Greenberg, Hudson, Shen & Roitt, 1973, Melewicz, Shore, Ades & Phillips, 1977) and presumably natural killer (NK) cells (Kay, Bonnard, West & Herberman, 1977; Oehler, Lindsay, Nunn, Holden & Herberman, 1978) have been shown to be cytotoxic to sensitized target cells in at least some systems. Although it appears that the most effective cells in mediating ADCC differ among animal species, the effectiveness of different cell types may also vary with the target cells. Thus, in cattle the neutrophil is very effective against both virus
Summary. Canine peripheral blood lymphocytes, polymorphonuclear leucocytes (PMN) and monocytes (macrophages) were obtained by various cell separation techniques and were tested for their cytotoxic capacity against antibody-sensitized canine distemper virus (CDV) infected Vero cells by an in vitro chromium release assay. Canine lymphocytes were found to destroy CDV infected target cells effectively, while neither PMN nor monocytes (macrophages) could do so. The active lymphocyte was characterized by various rosetting techniques to be a non-T and a non-B lymphocyte. These cells bear no surface immunoglobulin (Slg-) but possessed both Fc receptors (Fc+) and complement receptors (EAC+) suggesting that these cells are neither classical T nor B cells. The possible roles of this K cell in the resistance against canine distemper are discussed. INTRODUCTION Antibody-dependent cell-mediated cytotoxicity (ADCC) represents an in vitro model of antiviral activity which may be an important immune mechanism Correspondence: Dr Lorne A. Babiuk, Department of Veterinary Microbiology, W.C.V.M., University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N OWO. 00 1 9-2805/79/0500-0231$02.00 ) 1979 Blackwell Scientific Publications
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Chi Kuan Ho & Lorne A. Babiuk
infected cells and heterologous erythrocytes whereas the peripheral blood lymphocyte can kill erythrocytes but not virus infected cells (Rouse et al., 1976a). Despite the fact that dogs are often used as experimental transplantation models very little is known about the cytotoxic potential of canine leucocytes. We have recently initiated studies in this regard to demonstrate the ability of canine T lymphocytes to mediate direct cytotoxic activity against vaccinia virus infected cells in vitro (Ho, Babiuk & Rouse, 1978). The present communication reports the extension of these studies of canine effector cell function to ADCC and the characterization of the specific cell type(s) involved in this activity. MATERIALS AND METHODS
Animals Six healthy male and female mongrel dogs (2-6 months old) were immunized with a single subcutaneous injection of live canine distemper virus (Nordon Laboratories, Lincoln, Neb.) to guard against natural infection with virulent virus. These dogs were then used as blood donors 4 months post-vaccination. Virus Vero cell adapted canine distemper virus (Green strain) kindly provided by Dr D. J. Shen (Washington State University, Pullman, Washington) was used
throughout this study. Subconfluent monolayers of Vero cells (African Green Monkey kidney cells) were infected with canine distemper virus (5 x 105 TCID50/ml) at a multiplicity of infection (MOI) of 0-1 by incubation at 350 for 4 h with occasional shaking. The virus was removed and replaced by Eagle's minimum essential medium (MEM) supplemented with 0-1 mM glutamine, 1% non-essential amino acids, 5% foetal calf serum (FCS) and 50 ig/ml of gentamycin. The infected cells were incubated at 350 in a humidified 5% CO2 incubator. After 48 h of incubation, when extensive cell degeneration occurred, the culture supernatant was harvested, cell debris was removed by centrifugation at 200 g for 10 min and the virus suspension was dispensed into small vials which were kept at - 700 until used. Due to the labile nature of the virus, a fresh batch of virus was prepared every 2-3 months.
Preparation of F(ab')2 fragment of rabbit anti-dog immunoglobulin (IgG) Rabbit IgG F(ab')2 fragment was prepared by a method similar to that described by Campbell, Gar-
vey, Cremer & Sussdorf (1977). Rabbit IgG from pooled antisera was precipitated by ammonium sulphate (50% (NH4)2SO4, pH 7-8) followed by passage through a DEAE-cellulose column equilibrated with 0-01 M phosphate buffer, pH 7-5+005 M sodium chloride (NaCl). The eluted protein (IgG fraction) was concentrated by pervaporation to a concentration of 10 mg/ml and dialysed against 0-07 M acetate buffer+ 0-05 M sodium chloride, pH 4 0. Crystallized pepsin (Sigma Chem. Company, Lot No. 83C-8080, St Louis, Mo.) was added to the dialysate at a concentration of 0 3 mg/ml and the mixture was incubated at 370 for 18 h. The F(ab')2 fragments in the digest were separated from the intact IgG and Fc molecules by Sephadex G-100 column filtration. Antibody activity of the purified F(ab')2 was tested by immunodiffusion. Preparation of lymphocyte effector cells Dog peripheral blood was collected from the cephalic vein into syringes containing preservative free heparin (15 i.u./ml of blood). The blood was diluted 1: I with Hanks's balanced salt solution (HBSS), layered onto Ficoll-Hypaque (specific gravity 1-077 at 250) and centrifuged at 400 g for 30 min as described elsewhere (Ho et al., 1978). Interface peripheral blood leucocytes (PBL) were harvested and contaminating monocytes were removed by incubating the PBL in plastic culture dishes for 30 min at 37°. To remove possible cytophilic antibody, the nonadherent PBL were washed four times in HBSS, prewarmed at 370, and resuspended in MEM + 5% FCS. Viability of this cell preparation was > 99% as assessed by trypan blue exclusion. These cells contained about 83% lymphocytes and were used as effector cells (PBL) or employed for the following cell separations.
Removal of surface immunoglobulin (SIg+) bearing cells. Sheep red blood cells (SRBC), coated with rabbit anti-dog Ig F(ab')2 were prepared by the method of Parish & Hayward (1974) with slight modification. Briefly, a suspension of 5% SRBC in 0 9 NaCl containing 0-2 mg/ml of F(ab')2 and 0-00 I% chromium chloride was incubated at room temperature for 5 min. The Ig-SRBC were then washed once in 0-1 M phosphate buffered saline, pH 7-2 and twice in HBSS prior to being resuspended to a 2% solution in HBSS+2% FCS. To remove SIg-SRBC rosetting cells, equal volumes of PBL (3 x 106 cells/ml) and Ig-SRBC were gently mixed, incubated for 30 min at 40 and centrifuged at 200g for 10 min. The pelleted cells were gently resuspended to half the original volume in MEM + 2%
Canine K-cell activity
FCS, layered onto Ficoll-Hypaque (specific gravity 1-082 g/ml at 250) and centrifuged for 20 min at 400 g. The interface cells were harvested, washed once, and resuspended at a concentration of 3 x 106 cells/ml in MEM + 5% FCS. These cells represented the surface immunoglobulin depleted (SIg-) population. Depletion of lymphocytes bearing Fc receptors (Fc depleted populations). The preparation of antibody coated SRBC (EA) and the depletion of Fc receptor bearing lymphocytes has been described in detail previously (Ho & Babiuk, 1978b). Briefly, washed 5% SRBC were coated with a sub-haemagglutinating level of canine anti-SRBC hyperimmune serum by incubation at 370 for 15 min. The EA was washed and incubated with canine leucocytes at 370 for 20 min, the EA and leucocyte suspension was then layered onto Ficoll-Hypaque and centrifuged for 20 min at 400 g as described above. The cells present at the interface were referred to as the Fc depleted (Fc-) cells. Depletion of lymphocytes bearing complement receptors (EAC+-depletedpopulations). The preparation of antibody-complement coated SRBC (EAC) was similar to that described elsewhere (Ho & Babiuk, 1978b). Briefly, SRBC were sensitized with a sub-haemagglutinating level of rabbit anti-SRBC IgM (Cordis No. 768-990, Miami, Fl.) followed by reaction with fresh AKR mouse serum which served as a source of complement. Following sensitization of the SRBC, equal volumes of lymphocytes (3 x 106 cells/ml) and 2% EAC were mixed and incubated for 20 min at 37°. Non-rosetting lymphocytes were removed from the rosetting cells by Ficoll-Hypaque flotation (specific gravity 1-082 g/ml) as described above. The interface non-rosetting cells were harvested and were referred to as complement depleted (EAC-) cells.
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viously (Ho & Babiuk, 1978a). Erythrocytes were sedimented from canine peripheral blood by mixing with a 1/5 volume of 6% dextran in Tris-EDTA (0 01 M Tris, 0.5% EDTA) at room temperature for 30 min. The few contaminating erythrocytes present in the cell rich plasma were removed by washing the cells with 5 volumes of cold 0.87% ammonium chloride. The contaminating B cells were removed by sedimentation of SIg rosetting cells as described above. The nonrosetting cells were washed four times in HBSS, and resuspended in MEM + 5% FCS. These cells contained 70-80% neutrophils and constituted the PMN population.
Preparation of monocytes Peripheral blood leucocytes were incubated in plastic petri dishes at a concentration of 5-8 x 106 cells/ml for 30 min at 370. The non-adherent cells were removed at the end of the incubation period. The adherent cells were washed three times with 0.025% trypsin in 0-01% EDTA+0 85 M NaCl (prewarmed at 370) and then removed from the petri dishes by gentle scraping with a rubber policeman. The dislodged cells were washed four times in HBSS prewarmed at 370, resuspended in MEM + 15% FCS and were kept at 40 until used. In some cases, 0-2 ml of PBL were incubated (10 x 106 cells/ml) in wells of microtitre plates and adherent cells were used directly as effector cells. The number of effector cells present was estimated from the percentage of mononuclear phagocytic cells (latex ingesting) in the PBL population and ratio of effector to target cells were adjusted by varying the number of target cells. Viability of the dislodged cells was about 80% as assessed by trypan blue exclusion and > 80% of the adherent cells were identified as monocytes.
T-lymphocyte enriched populations. Peripheral blood leucocytes (PBL) were passed through a nylon wool column which had been equilibrated at 370 with HBSS+10% autologous plasma as described previously (Ho & Babiuk, 1978a). The effluent cells were further depleted of complement receptor (EAC) bearing cells by rosetting and removal of rosetted cells as described above. The non-rosetting cells were referred to as enriched T lymphocytes.
Preparation of macrophages Pure canine macrophage cultures were established from peripheral blood leucocytes as described in detail elsewhere (Ho & Babiuk, 1978a, b). Canine peripheral blood leucocytes were incubated in plastic tissue culture dishes in a humidified 5% CO2 incubator. Culture fluid was MEM + 20% horse serum supplemented with 1% autologous red cell lysate. Following 10 days of incubation, when purity of the macrophages reached > 99% as judged by various criteria, these cells were mechanically dislodged as above and were used as effector cells.
Preparation ofpolymorphonuclear leucocytes Polymorphonuclear leucocytes (PMN) were obtained by slight modification of the method described pre-
Immunofluorescent staining of surface immunoglobulin (SIg) bearing cells Surface Ig bearing cells were detected by a modifi-
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Chi Kuan Ho & Lorne A. Babiuk
cation of an indirect immunofluorescence technique described elsewhere (Ho & Babiuk, 1978b). Washed lymphocytes (3 x 106 cells/ml) were incubated at 40 for 30 min with 200 yig/ml of rabbit anti-dog F(ab')2 fragments. The cells were washed and further incubated with FITC-conjugated goat anti-rabbit IgG (heavy and light chains specific, Cappel, Downington, Pa.) under the same conditions. After three washings in HBSS the cells were examined with a phase fluorescent microscope. Target cells Subconfluent monolayers of Vero cells grown in Eagle's minimum essential medium (MEM) supplemented with 5% heat inactivated FCS were infected with canine distemper virus (Green strain) at a MOI of 1. After 16-24 h, the infected cells were removed by trypsinization, washed, resuspended in 0-9 ml of medium and 100 pCi of sodium 5"chromate (New England Nuclear, Cat. No. NEZ-030, Boston, Mass.) prior to incubation at 370 for 1 h in a humidified 5% CO2 incubator. Following labelling, the cells were washed three times in HBSS, resuspended in MEM + 5% FCS and were used as target cells. Noninfected Vero cells were labelled with chromium in the same manner to serve as controls. Antibody dependent cell-mediated cytotoxicity assays (ADCC) Cytotoxicity assays were performed in ninety-six-well microtitre plates as described previously (Ho & Babiuk, 1978a), 51chromium labelled CDV virusinfected Vero cells were first incubated with varying amounts of pooled dog anti-CDV antiserum (50% serum neutralizing titre approximately 1250) for 1 h at 4°. The antibody coated target cells were washed twice in HBSS and suspended in MEM + 5% FCS at a concentration of 5 x 104 cells/ml and 0- 1 ml aliquots were dispensed into wells of the microtitre plates. In some cases, antiserum was added directly to the wells. Equal volumes of effector cell preparations were then added to the target cells at different effector to target cells ratios and the plates were incubated at 37° in a humidified 5% CO2 incubator. At various time intervals following incubation, halfthe supernatant from each well was harvested and the isotopic release was measured with the aid of a gamma counter. The amount of cytotoxicity was calculated from the following formula: Percentage specific release= (CPM test - CPM control) v 100 Total releasible assay -CPM control
Controls included effector cells + target cells, effector cells + target cells in the presence of normal dog serum, effector cells + uninfected Vero cells in the presence or absence of antibody. All sera were heat inactivated at 560 for 30 min and absorbed twice with Vero cells (40 for 1 h) before use. Normal serum was obtained from gnotobiotic dogs and had no detectable neutralizing antibody against CDV. In experiments designed to measure the inhibition of ADCC by Staphylococcus aureus protein A, rabbit anti-dog Ig F(ab')2 or heat aggregated (630 for 10 min) normal dog gammaglobulin inhibitor was added at various concentrations to an ADCC assay and harvested as described above.
RESULTS Evidence and conditionsfor demonstrating ADCC in the dog Canine leucocytes were found to destroy antibody sensitized canine distemper virus-infected Vero cells. In the presence of specific anti-CDV serum, the leucocytes were capable of killing infected target cells but not uninfected cells. Normal serum, containing no CDV antibody activity could not sensitize infected cells so that they could be lysed by the effector cells
40[r Ne
301-
aJ
0
0)
20H 1-
a
In
lo0 .1I
io-5
lo
10-3d
10-2
lo-,
Antiserumn dilution
Figure 1. The effect of antiserum concentration on ADCC. Antiserum was added directly to the assay system with an effector (PBL) to target cell ratio of 50:1 and an incubation period of 20 h in the presence of canine distemper antiserum (-) or normal serum (A).
Canine K-cell activity
suggesting that the lytic activity observed was specific antibody dependent especially since the activity was directly proportional to the concentration of antibody (Fig. 1). This mechanism of destruction was extremely sensitive in that only low levels of antibody were required. Furthermore, the number of effector cells required for detecting destruction was very low (Fig. 2), suggesting that this mechanism could possibly be important in preventing CDV spread at an early stage of the disease when antibody levels were low and 50
40
c 30
20 o 20 _
/
0
1
I 0
25
50
100
Effector/target cell ratio
Figure 2. ADCC in the presence of varying effector to CDV infected target cell ratios. Incubation period was 20 h in the presence of a 1/20 dilution of anti-CDV serum (A) and normal serum (-). Uninfected target cells incubated in the presence of effector cells and anti-CDV serum served as controls (o).
40 -
S
30 -
20
ci,10
0
5
10 15 Hours of incubation
20
25
Figure 3. Effect of incubation period on ADCC. PBL effector cells were used at an effector to target cell ratio of 50: 1, in the presence of a 1/20 dilution of anti CDV serum (&), normal serum (o) and with anti-CDV serum in the absence of effector cells (-).
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leucocyte levels were low and leucocyte infiltration was not maximal. For specific lysis to occur the effector and target cells had to be incubated for relatively long periods of time (Fig. 3). Thus, specific 5'Cr release was barely detectable during the first 10 h of incubation but increased rapidly thereafter such that over the next 5-10 h specific release reached maximum levels. If the mechanism of killing was due to ADCC then the addition of heat aggregated dog IgG, Staphylococcal protein A or rabbit anti-dog F(ab')2 might be expected to interfere with the binding of the leucocyte Fc receptor and sensitizing antibody and thereby reduce killing. This was shown to be the case in all three instances (Table 1) suggesting that killing was due to ADCC. Characterization of cells active in ADCC Various populations of leucocytes, obtained from the same animal were assessed for their cytotoxic potential against CDV infected targets. As illustrated in Table 2, lymphocytes were the most effective cells in mediating ADCC with the polymorphonuclear and adherent population being less effective. Further enrichment of macrophages by continued cultivation resulted in death of lymphocytes and a further decrease in cytotoxicity suggesting that macrophages were very inefficient in mediating ADCC as were the polymorphonuclear cells even though both cell types possess Fc receptors (Ho & Babiuk, 1978a). To characterize further the lymphocyte subpopulations capable of mediating ADCC, lymphocytes were separated into various subpopulations by rosetting techniques and nylon wool filtration. Depletion of surface immunoglobulin bearing cells from the PBL population did not alter the degree of cytotoxicity (Table 3) suggesting that a classical B cell was not responsible for the majority of the cytotoxicity observed. The effector cell did, however, possess Fc receptors as one would expect for ADCC to occur, since removal of EA rosetting cells dramatically reduced the degree of cytotoxicity. Further evidence that suggested the K cell was not a B cell was obtained from nylon wool fractionation experiments where the activity was found in the nylon effluent population (Table 3), a population previously shown to be depleted of B cells and enriched in T cells. The active cell was probably also not a classical T cell since the canine K cells expressed C receptors but T cells of most species do not generally express C receptors. Thus, the K cell probably represents an Fc and C receptor, surface immunoglobulin negative nylon
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Chi Kuan Ho & Lorne A. Babiuk
Table 1. Inhibition of antibody dependent cell mediated cytotoxicity by rabbit anti-dog F(ab')2 fragments, staphylococcal protein A and heat aggregated dog immunoglobulin*
Inhibitor None F(ab')2
ProteinA Hagg.
Igt
Concentration (pug) Specific releaset (%) Inhibition (%) 1.0 10 0 100.0 1.0 100 1000 50 500 5000
28 29 5 22 16 271 18 7 28 4 182 126
20 42 -
40 75
35 56
* ADCC assay was performed as described in Materials and Methods with 1/20 dilution of antiserum with effector to target cell ratio of 50:1 and an incubation time of 20 h. Infected Vero cells in the presence ofantiserum and/or inhibitors were used as control spontaneous releases. t Calculated by formula: percentage inhibition= counts per minute in the presence of inhibitors/counts per minute specific release x 100. t Heat aggregated normal dog gammaglobulin (630 for 10 min).
Table 2. Antibody dependent cytotoxic action expressed by different leucocyte
preparations Specific 5 1Cr release + SDt (%)
Cell preparation
Major cell type ± SD* (%)
PBL 85 + 7 lymphocytes Dextran sedimentation 77 + 5 PMN Plastic adherent cells 84 + 6 monocytes > 99 macrophages Macrophage culture
6h
20 h
3.7 + 1 8 4 + 27 3+2 4 2+2
32 + 7 5 11 + 3 4 8.6 + 3 4 + 1.5
* Based on morphological observation by phase contrast microscopy. Cells were stained with 0 5% methylene blue in the absence of serum. SD, standard deviation. t ADCC was performed with PBL as effector cells at an effector to target cell ratio of 25:1; target cells were preabsorbed with 1/20 antiserum and incubation period is 20 h. Spontaneous release (21 %) was subtracted during the calculation of specific release.
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Canine K-cell activity Table 3. Surface markers of lymphocyte effector cells and their activity against CDV infected targets.
Cells with specific surface markers* + SD (%) Cell preparations PBL
SIg depleted PBL Nylon wool effluent EAC depleted Nylon wool effluent EAdepletedPBL EAC depleted PBL
Fc
EAC
SIg
Specific 5ICr release + SD§ (%)
25.1+4 2 215 +4-5 10+3
23 6+3 22-3+5 7 5+2 5
21 +6t 4.3 + 24
26 5+3 255 +4-5 19+2 2
10+3
7 5+2 5
6 5+3.7
1-3+1-5 2 4+ 17
26+2 18 + 27
3+1.5 52 + 34
6 5 + 3-7
19+2-2 43+3 28 + 27
* Based on the percentage of rosetting cells with EA, EAC and SIg respectively. SD, standard deviation. t The same cell preparation had 24 3 + 3.5% SIg+ cells based on immunofluorescence.
tN.D., not done. § ADCC was performed under the same conditions as described in Table 2.
wool non-adherent cell belonging to neither the T- nor B-cell lineage.
DISCUSSION
Antibody-dependent cell-mediated cytotoxicity (ADCC) has been shown to occur in a large number of species by a variety of different cell types all of which bear Fc receptors. Its actual biological role in antiviral defence in vivo, however, remains speculative. In the systems studied to date ADCC appears to be a very effective method of killing virus infected cells in vitro in that cytotoxicity can be detected very rapidly in the presence of low levels of antibody suggesting that it may be an important mechanism in the early stages of disease when antibody levels are low. To date, no reports have been published to suggest that a similar mechanism exists in dogs and could be of any relevance to recovery from canine distemper infections or be involved in post-distemper encephalitis. The present study has demonstrated that such a mechanism may be functional in the dog and that the canine cell type most effective in mediating ADCC against canine distemper virus-infected cells was a lymphocyte while the polymorphonuclear and monocyte, which also express Fc receptors (Ho & Babiuk, 1978a,b) are ineffective. In fact, if it were possible to remove all contaminating lymphocytes from the polymorphonuclear and monocyte populations these cells would probably
be totally incapable of mediating cytotoxicity (Table 1). The reason why these cells are ineffective, even though they possess the required Fc receptor, is not understood but it could be that the number of Fc receptors, the tenacity of binding of immunoglobulin to both target and effector cells as well as the type of antibody and target cell may influence the ability to mediate ADCC. Some evidence for such possibilities has been reported recently (Lustig & Bianco, 1976) which shows that ADCC was influenced by the type of antibody and/or the presence of complement. Some types of antibody and effector cells normally unable to participate in ADCC could do so if low levels of complement were added. These results suggested that possibly the presence of a second bond may increase the tenacity of the binding and also the efficiency of killing. It could be argued that since our effector cells were from immune animals and because the activity was found in T-cell enriched populations we were measuring direct cytotoxicity and not ADCC. This is unlikely for a number of reasons. Firstly, it has been shown that T-cell activity against virus infected cells is transient and short lived in all species studied to date (Doherty, Zinkernagel & Ramshaw, 1974; Ho et al., 1978; Rouse & Babiuk, 1977) including the canine species. Thus, after an initial sensitization, direct T-cell killing is maximal at 1-2 weeks, then declines rapidly to background levels. Since our effector cells were taken 4 months post-distemper immunization one
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Chi Kuan Ho & Lorne A. Babiuk
would expect T-cell cytotoxicity to be minimal. Secondly, cytotoxicity was not detectable unless specific antibody was added. Thirdly, blockage of the Fc receptor by various agents (Table 3) dramatically reduced cytotoxicity. Since most T cells do not normally express Fc receptors (Chiao, Ascensao, Pahwa, Pahwa & Good, 1976; Gy6ngyossy, Arnaiz-Villena, Soteriades-Viachos & Playfair, 1975) we felt that the T cell was not responsible for the activity observed. Unfortunately, it is not yet possible to prove this definitely by depleting T cells with rosetting techniques since other cell types as well as canine T cells form spontaneous E rosettes (Esser, Cosgrove, Scott & Cosimi, 1977; Gyongyossy et al., 1975; Ho & Babiuk, 1978a, b). Furthermore, specific anti-canine T-cell serum is not presently available to eliminate all T cells. The effector cell was also shown not to be a B cell since depletion of surface immunoglobulin bearing lymphocytes has little effect on the level of cytotoxicity. From these observations we are led to conclude that the canine effector cell responsible for cytotoxicity against canine distemper infected cells is a nylon nonadherent or weakly adherent non-T or B lymphocyte which bears both Fc and complement receptors. These cells are probably equivalent to the null cells reported to be the K cells in humans (Perrin, Tishon & Oldstone, 1977) and mice (Ramshaw & Parish, 1976). Although we have shown evidence for the existence of a K cell in the dog it still does not demonstrate that this cell is of any relevance in preventing canine distemper in vivo. Ideally, for any defense mechanism to be effective in this regard the K cell must be present at the site of viral replication and must be able to kill the infected cell prior to viral dissemination. If killing occurs after cell to cell spread of the virus then this mechanism cannot prevent spread even though it may reduce the virus load in the case of enveloped viruses where release occurs over extended periods. The present study demonstrates that cytotoxicity takes a relatively long time (- 10 h) before killing is detectable. Preliminary results with the Green strain of CDV have demonstrated that during a lytic infection, virus maturation and spread occurs at approximately 8 h post-infection (Ho & Babiuk, unpublished-results), therefore, spread would have occurred before the cell was killed. Kinetic experiments with other strains have shown similar results, therefore one would suspect that K cells may not totally inhibit viral spread unless they are cytostatic as well as cytotoxic. It is tempting to speculate that since CDV replicates in canine lymphocytes the K cell does play an im-
portant role in both the early stages of the infection as well as in areas such as the brain where antibody levels are generally low. If the immune response does not eliminate the virus at a critical time in an infection, possibly due to virus replication in lymphocytes and suppression of CMI, the virus may evade the animal's immune responses. Under these conditions, virus persistence may be established and antibody levels increase to such a level that there is antigenic modulation and elimination of the viral antigens on the cell membrane. Once this occurs the K cell can no longer kill the virus infected cell thus permitting continued persistence which may lead to post-distemper encephalitis as has been suggested in measles infected cells (Perrin et al., 1977). If these virus-antigen antibody complexes are shed into the circulation they could block ADCC in a manner similar to blocking by aggregated immunoglobulin (Table 3). This possibility is reinforced by the observation that Ag-Ab complexes are shed from measles virus-infected cells (Perrin & Oldstone, 1977) and that high levels of antibody do block ADCC in SSPE (Steele, Fucillo, Hensen, Vincent & Bellanti, 1976) and CDV infected cell systems (Ho & Babiuk, unpublished results).
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R.A. (1976) An analysis of lymphocytes with both T cell marker and IgG or complement receptors in patients with X-linked gammaglobulin. Clin. Immunol. Immunopathol. 6, 141. DENNERT G. & LENNOX I.S. (1973) Phagocytic cells as effectors in a cell-mediated immunity system. J. Immunol. 111, 1844. DOHERTY P.C., ZINKERNAGEL R.M. & RAMSHAW I.A. (1974) Specificity and development of cytotoxic thymus-derived lymphocytes in lymphocytic choriomeningitis. J. Immunol. 112, 1548. ESSER R.E., COSGROVE M., SCOTT G. & CosIMI A.B. (1977) Rosette formation by canine leucocytes with human erythrocytes. Transplantation, 24, 223. GALE R.F. & ZIGHELBOIM T. (1975) Polymorphonuclear leukocytes in antibody-dependent cellular cytotoxicity. J. Immunol. 114, 1047. GREENBERG A.H., HUDSON L., SHEN L. & ROITT I.M. (1973) Antibody dependent cell-mediated cytotoxicity due to a null lymphoid cell. Nature (New Biol.) 242, 11 1. GYONGYOSSY M.I.C., ARNAIZ-VILLENA A., SOTERIADESVLACHOS C. & PLAYFAIR J.H.L. (1975) Rosette formation
Canine K-cell activity of mouse lymphocytes. IV. Fc and C3 receptors occurring together and separately on T cells and other leucocytes. Clin. exp. Immunol. 19,495. Ho C.K., BABIUK L.A. & ROUSE B.T. (1978) Immune effector cell activity in canines: failure to demonstrate genetic restriction in direct antiviral cytotoxicity. Infect. Immun. 19, 18. Ho C.K. & BABIUK L.A. (1978a) Isolation of various canine leucocytes and their characterization by surface marker analysis. Immunology, 35, 733. Ho C.K. & BABIUK L.A. (1978b) Long term culture of canine peripheral blood monocytes in vitro. Can. J. comp. Med. (In press.) KAY H.D., BONNARD G.D., WEST W.H. & HERBERMAN R.B. (1977) A functional comparison of human Fc receptor bearing lymphocytes active in natural cytotoxicity and antibody dependent cellular cytotoxicity. J. Immunol. 118, 2058. LUSTIG H.J. & BIANCO C.S. (1976) Antibody mediated cell cytotoxicity in a defined system. Regulation by antigen, antibody and complement. J. Immunol. 116,253. MELEWICZ F.M., SHORE S.L., ADES E.M. & PHILLIPS D.J. (1977) The mononuclear cell in human blood which mediated antibody dependent cellular cytotoxicity to virus infected target cells. II. Identification as a K cell. J. Immunol. 118, 567. NOTKINs A.L. (1974) Commentary: immune mechanisms by which the spread of viral infection is stopped. Cell Immunol. 11, 478. OEHLER J.R., LINDSAY L.R., NUNN M.L., HOLDEN H.T. & HERBERMAN R.B. (1978) Natural cell-mediated cytotoxicity in rats. II. In vivo augmentation of NK-cell activity. Int. J. Cancer, 21, 210. PARISH C.R. & HAYWARD J.A. (1974) The lymphocyte 11. Separation of Fc receptor, C receptor and surface immunoglobulin bearing lymphocytes. Proc. R. Soc. Lond. B187, 65. PERLMANN P., PERLMANN H. & WIZZELL H. (1972) Lymphocyte mediated cytotoxicity in vitro induction and
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inhibition by humoral antibody and nature of effector cells. Transplant. Rev. 13, 91. PERRIN L.H. & OLDSTONE M.B.A. (1977) The formation and fate of virus antigen-antibody complexes. J. Immunol. 118, 316. PERRIN L.H., TISHON A. & OLDSTONE M.B.A. (1977) Immunologic injury in measles virus infection. II Presence and characterization of human cytotoxic lymphocytes. J. Immunol. 118, 282. RAMSHAW L.A. & PARISH C.P. (1976) Surface properties of cells involved in antibody-dependent cytotoxicity. Cell Immunol. 21, 276. ROUSE B.T., WARDLEY R.C. & BABIUK L.A. (1976a) Antibody dependent cell-mediated cytotoxicity in cows: comparison of effector cell activity against heterologous erythrocyte and herpesvirus infected bovine target cells. Infect. Immun. 13, 1433. ROUSE B.T., WARDLEY R.C. & BABIUK L.A. (1967b) The role of antibody dependent cytotoxicity in recovery from herpesvirus infections. Cell Immunol. 22, 182. ROUSE B.T. & BABIUK L.A. (1977) The direct antiviral cytotoxicity of bovine lymphocytes is not restricted by genetic incompatibility of lymphocytes and target cells. J. Immunol. 188, 618. ROUSE B.T., GREWAL A.S., BABIUK L.A. & FUJIMIYA Y. (1977a) Enhancement of antibody dependent cell cytotoxicity of herpesvirus infected cells by complement. Infect. Immun. 18, 660. ROUSE B.T., WARDLEY R.C., BABIUK L.A. & MUKKUR T.K.S. (1977b) The role of neutrophils in antiviral defense: in vitro studies on the mechanism of antiviral inhibition. J. Immunol. 118, 1957. STEELE R.W., FUCILLO D.A., HENSEN S.A., VINCENT M.M. & BELLANTI J.A. (1976) Specific inhibition factors of cellular immunity in children with subacute sclerosing panencephalitis. J. Pediatr. 88, 56. ZIGELBOIM J., BONAVIDA B. & FAHEY J.K. (1973) Evidence for several cell populations active in antibody dependent cellular cytotoxicity. J. Immunol. 111, 1737.
Immune mechanisms against canine distemper I.
IDENTIFICATION OF K CELL AGAINST CANINE DISTEMPER VIRUS INFECTED TARGET CELLS IN VITRO
CHI KUAN HO I LORNE A. BABIUK Department of Veterinary Microbiology, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada
Received 2 October 1978; acceptedfor publication 26 October 1978
in the recovery from, as well as resistance to, viral infections (Allison, 1974; Notkins, 1974; Rouse, Wardley & Babuik 1976a,b). Because of its potentially important role in defence, considerable interest has been generated to determine the particular cell type(s) involved in this activity. These studies have utilized cell separation techniques, surface marker studies and functional analysis in attempts to define the properties of the effector cells against various target cells. In all the studies conducted to date the effector cell in ADCC has always been found to possess Fc receptors for IgG regardless of other surface markers present. Since the Fc receptor is of crucial importance it is not surprising that neutrophils (Gale & Zighelboim 1975; Rouse et al., 1976b; Rouse, Grewal, Babiuk & Fujimiya, 1977a; Rouse, Wardley, Babiuk & Mukkur, 1977b), mononuclear phagocytic cells (Dennert & Lennox, 1972), B lymphocytes (Perlmann, Perlmann & Wizzell, 1972; Zighelboim, Bonavida & Fahey, 1973) as well as the recently described null (Greenberg, Hudson, Shen & Roitt, 1973, Melewicz, Shore, Ades & Phillips, 1977) and presumably natural killer (NK) cells (Kay, Bonnard, West & Herberman, 1977; Oehler, Lindsay, Nunn, Holden & Herberman, 1978) have been shown to be cytotoxic to sensitized target cells in at least some systems. Although it appears that the most effective cells in mediating ADCC differ among animal species, the effectiveness of different cell types may also vary with the target cells. Thus, in cattle the neutrophil is very effective against both virus
Summary. Canine peripheral blood lymphocytes, polymorphonuclear leucocytes (PMN) and monocytes (macrophages) were obtained by various cell separation techniques and were tested for their cytotoxic capacity against antibody-sensitized canine distemper virus (CDV) infected Vero cells by an in vitro chromium release assay. Canine lymphocytes were found to destroy CDV infected target cells effectively, while neither PMN nor monocytes (macrophages) could do so. The active lymphocyte was characterized by various rosetting techniques to be a non-T and a non-B lymphocyte. These cells bear no surface immunoglobulin (Slg-) but possessed both Fc receptors (Fc+) and complement receptors (EAC+) suggesting that these cells are neither classical T nor B cells. The possible roles of this K cell in the resistance against canine distemper are discussed. INTRODUCTION Antibody-dependent cell-mediated cytotoxicity (ADCC) represents an in vitro model of antiviral activity which may be an important immune mechanism Correspondence: Dr Lorne A. Babiuk, Department of Veterinary Microbiology, W.C.V.M., University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N OWO. 00 1 9-2805/79/0500-0231$02.00 ) 1979 Blackwell Scientific Publications
231
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Chi Kuan Ho & Lorne A. Babiuk
infected cells and heterologous erythrocytes whereas the peripheral blood lymphocyte can kill erythrocytes but not virus infected cells (Rouse et al., 1976a). Despite the fact that dogs are often used as experimental transplantation models very little is known about the cytotoxic potential of canine leucocytes. We have recently initiated studies in this regard to demonstrate the ability of canine T lymphocytes to mediate direct cytotoxic activity against vaccinia virus infected cells in vitro (Ho, Babiuk & Rouse, 1978). The present communication reports the extension of these studies of canine effector cell function to ADCC and the characterization of the specific cell type(s) involved in this activity. MATERIALS AND METHODS
Animals Six healthy male and female mongrel dogs (2-6 months old) were immunized with a single subcutaneous injection of live canine distemper virus (Nordon Laboratories, Lincoln, Neb.) to guard against natural infection with virulent virus. These dogs were then used as blood donors 4 months post-vaccination. Virus Vero cell adapted canine distemper virus (Green strain) kindly provided by Dr D. J. Shen (Washington State University, Pullman, Washington) was used
throughout this study. Subconfluent monolayers of Vero cells (African Green Monkey kidney cells) were infected with canine distemper virus (5 x 105 TCID50/ml) at a multiplicity of infection (MOI) of 0-1 by incubation at 350 for 4 h with occasional shaking. The virus was removed and replaced by Eagle's minimum essential medium (MEM) supplemented with 0-1 mM glutamine, 1% non-essential amino acids, 5% foetal calf serum (FCS) and 50 ig/ml of gentamycin. The infected cells were incubated at 350 in a humidified 5% CO2 incubator. After 48 h of incubation, when extensive cell degeneration occurred, the culture supernatant was harvested, cell debris was removed by centrifugation at 200 g for 10 min and the virus suspension was dispensed into small vials which were kept at - 700 until used. Due to the labile nature of the virus, a fresh batch of virus was prepared every 2-3 months.
Preparation of F(ab')2 fragment of rabbit anti-dog immunoglobulin (IgG) Rabbit IgG F(ab')2 fragment was prepared by a method similar to that described by Campbell, Gar-
vey, Cremer & Sussdorf (1977). Rabbit IgG from pooled antisera was precipitated by ammonium sulphate (50% (NH4)2SO4, pH 7-8) followed by passage through a DEAE-cellulose column equilibrated with 0-01 M phosphate buffer, pH 7-5+005 M sodium chloride (NaCl). The eluted protein (IgG fraction) was concentrated by pervaporation to a concentration of 10 mg/ml and dialysed against 0-07 M acetate buffer+ 0-05 M sodium chloride, pH 4 0. Crystallized pepsin (Sigma Chem. Company, Lot No. 83C-8080, St Louis, Mo.) was added to the dialysate at a concentration of 0 3 mg/ml and the mixture was incubated at 370 for 18 h. The F(ab')2 fragments in the digest were separated from the intact IgG and Fc molecules by Sephadex G-100 column filtration. Antibody activity of the purified F(ab')2 was tested by immunodiffusion. Preparation of lymphocyte effector cells Dog peripheral blood was collected from the cephalic vein into syringes containing preservative free heparin (15 i.u./ml of blood). The blood was diluted 1: I with Hanks's balanced salt solution (HBSS), layered onto Ficoll-Hypaque (specific gravity 1-077 at 250) and centrifuged at 400 g for 30 min as described elsewhere (Ho et al., 1978). Interface peripheral blood leucocytes (PBL) were harvested and contaminating monocytes were removed by incubating the PBL in plastic culture dishes for 30 min at 37°. To remove possible cytophilic antibody, the nonadherent PBL were washed four times in HBSS, prewarmed at 370, and resuspended in MEM + 5% FCS. Viability of this cell preparation was > 99% as assessed by trypan blue exclusion. These cells contained about 83% lymphocytes and were used as effector cells (PBL) or employed for the following cell separations.
Removal of surface immunoglobulin (SIg+) bearing cells. Sheep red blood cells (SRBC), coated with rabbit anti-dog Ig F(ab')2 were prepared by the method of Parish & Hayward (1974) with slight modification. Briefly, a suspension of 5% SRBC in 0 9 NaCl containing 0-2 mg/ml of F(ab')2 and 0-00 I% chromium chloride was incubated at room temperature for 5 min. The Ig-SRBC were then washed once in 0-1 M phosphate buffered saline, pH 7-2 and twice in HBSS prior to being resuspended to a 2% solution in HBSS+2% FCS. To remove SIg-SRBC rosetting cells, equal volumes of PBL (3 x 106 cells/ml) and Ig-SRBC were gently mixed, incubated for 30 min at 40 and centrifuged at 200g for 10 min. The pelleted cells were gently resuspended to half the original volume in MEM + 2%
Canine K-cell activity
FCS, layered onto Ficoll-Hypaque (specific gravity 1-082 g/ml at 250) and centrifuged for 20 min at 400 g. The interface cells were harvested, washed once, and resuspended at a concentration of 3 x 106 cells/ml in MEM + 5% FCS. These cells represented the surface immunoglobulin depleted (SIg-) population. Depletion of lymphocytes bearing Fc receptors (Fc depleted populations). The preparation of antibody coated SRBC (EA) and the depletion of Fc receptor bearing lymphocytes has been described in detail previously (Ho & Babiuk, 1978b). Briefly, washed 5% SRBC were coated with a sub-haemagglutinating level of canine anti-SRBC hyperimmune serum by incubation at 370 for 15 min. The EA was washed and incubated with canine leucocytes at 370 for 20 min, the EA and leucocyte suspension was then layered onto Ficoll-Hypaque and centrifuged for 20 min at 400 g as described above. The cells present at the interface were referred to as the Fc depleted (Fc-) cells. Depletion of lymphocytes bearing complement receptors (EAC+-depletedpopulations). The preparation of antibody-complement coated SRBC (EAC) was similar to that described elsewhere (Ho & Babiuk, 1978b). Briefly, SRBC were sensitized with a sub-haemagglutinating level of rabbit anti-SRBC IgM (Cordis No. 768-990, Miami, Fl.) followed by reaction with fresh AKR mouse serum which served as a source of complement. Following sensitization of the SRBC, equal volumes of lymphocytes (3 x 106 cells/ml) and 2% EAC were mixed and incubated for 20 min at 37°. Non-rosetting lymphocytes were removed from the rosetting cells by Ficoll-Hypaque flotation (specific gravity 1-082 g/ml) as described above. The interface non-rosetting cells were harvested and were referred to as complement depleted (EAC-) cells.
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viously (Ho & Babiuk, 1978a). Erythrocytes were sedimented from canine peripheral blood by mixing with a 1/5 volume of 6% dextran in Tris-EDTA (0 01 M Tris, 0.5% EDTA) at room temperature for 30 min. The few contaminating erythrocytes present in the cell rich plasma were removed by washing the cells with 5 volumes of cold 0.87% ammonium chloride. The contaminating B cells were removed by sedimentation of SIg rosetting cells as described above. The nonrosetting cells were washed four times in HBSS, and resuspended in MEM + 5% FCS. These cells contained 70-80% neutrophils and constituted the PMN population.
Preparation of monocytes Peripheral blood leucocytes were incubated in plastic petri dishes at a concentration of 5-8 x 106 cells/ml for 30 min at 370. The non-adherent cells were removed at the end of the incubation period. The adherent cells were washed three times with 0.025% trypsin in 0-01% EDTA+0 85 M NaCl (prewarmed at 370) and then removed from the petri dishes by gentle scraping with a rubber policeman. The dislodged cells were washed four times in HBSS prewarmed at 370, resuspended in MEM + 15% FCS and were kept at 40 until used. In some cases, 0-2 ml of PBL were incubated (10 x 106 cells/ml) in wells of microtitre plates and adherent cells were used directly as effector cells. The number of effector cells present was estimated from the percentage of mononuclear phagocytic cells (latex ingesting) in the PBL population and ratio of effector to target cells were adjusted by varying the number of target cells. Viability of the dislodged cells was about 80% as assessed by trypan blue exclusion and > 80% of the adherent cells were identified as monocytes.
T-lymphocyte enriched populations. Peripheral blood leucocytes (PBL) were passed through a nylon wool column which had been equilibrated at 370 with HBSS+10% autologous plasma as described previously (Ho & Babiuk, 1978a). The effluent cells were further depleted of complement receptor (EAC) bearing cells by rosetting and removal of rosetted cells as described above. The non-rosetting cells were referred to as enriched T lymphocytes.
Preparation of macrophages Pure canine macrophage cultures were established from peripheral blood leucocytes as described in detail elsewhere (Ho & Babiuk, 1978a, b). Canine peripheral blood leucocytes were incubated in plastic tissue culture dishes in a humidified 5% CO2 incubator. Culture fluid was MEM + 20% horse serum supplemented with 1% autologous red cell lysate. Following 10 days of incubation, when purity of the macrophages reached > 99% as judged by various criteria, these cells were mechanically dislodged as above and were used as effector cells.
Preparation ofpolymorphonuclear leucocytes Polymorphonuclear leucocytes (PMN) were obtained by slight modification of the method described pre-
Immunofluorescent staining of surface immunoglobulin (SIg) bearing cells Surface Ig bearing cells were detected by a modifi-
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Chi Kuan Ho & Lorne A. Babiuk
cation of an indirect immunofluorescence technique described elsewhere (Ho & Babiuk, 1978b). Washed lymphocytes (3 x 106 cells/ml) were incubated at 40 for 30 min with 200 yig/ml of rabbit anti-dog F(ab')2 fragments. The cells were washed and further incubated with FITC-conjugated goat anti-rabbit IgG (heavy and light chains specific, Cappel, Downington, Pa.) under the same conditions. After three washings in HBSS the cells were examined with a phase fluorescent microscope. Target cells Subconfluent monolayers of Vero cells grown in Eagle's minimum essential medium (MEM) supplemented with 5% heat inactivated FCS were infected with canine distemper virus (Green strain) at a MOI of 1. After 16-24 h, the infected cells were removed by trypsinization, washed, resuspended in 0-9 ml of medium and 100 pCi of sodium 5"chromate (New England Nuclear, Cat. No. NEZ-030, Boston, Mass.) prior to incubation at 370 for 1 h in a humidified 5% CO2 incubator. Following labelling, the cells were washed three times in HBSS, resuspended in MEM + 5% FCS and were used as target cells. Noninfected Vero cells were labelled with chromium in the same manner to serve as controls. Antibody dependent cell-mediated cytotoxicity assays (ADCC) Cytotoxicity assays were performed in ninety-six-well microtitre plates as described previously (Ho & Babiuk, 1978a), 51chromium labelled CDV virusinfected Vero cells were first incubated with varying amounts of pooled dog anti-CDV antiserum (50% serum neutralizing titre approximately 1250) for 1 h at 4°. The antibody coated target cells were washed twice in HBSS and suspended in MEM + 5% FCS at a concentration of 5 x 104 cells/ml and 0- 1 ml aliquots were dispensed into wells of the microtitre plates. In some cases, antiserum was added directly to the wells. Equal volumes of effector cell preparations were then added to the target cells at different effector to target cells ratios and the plates were incubated at 37° in a humidified 5% CO2 incubator. At various time intervals following incubation, halfthe supernatant from each well was harvested and the isotopic release was measured with the aid of a gamma counter. The amount of cytotoxicity was calculated from the following formula: Percentage specific release= (CPM test - CPM control) v 100 Total releasible assay -CPM control
Controls included effector cells + target cells, effector cells + target cells in the presence of normal dog serum, effector cells + uninfected Vero cells in the presence or absence of antibody. All sera were heat inactivated at 560 for 30 min and absorbed twice with Vero cells (40 for 1 h) before use. Normal serum was obtained from gnotobiotic dogs and had no detectable neutralizing antibody against CDV. In experiments designed to measure the inhibition of ADCC by Staphylococcus aureus protein A, rabbit anti-dog Ig F(ab')2 or heat aggregated (630 for 10 min) normal dog gammaglobulin inhibitor was added at various concentrations to an ADCC assay and harvested as described above.
RESULTS Evidence and conditionsfor demonstrating ADCC in the dog Canine leucocytes were found to destroy antibody sensitized canine distemper virus-infected Vero cells. In the presence of specific anti-CDV serum, the leucocytes were capable of killing infected target cells but not uninfected cells. Normal serum, containing no CDV antibody activity could not sensitize infected cells so that they could be lysed by the effector cells
40[r Ne
301-
aJ
0
0)
20H 1-
a
In
lo0 .1I
io-5
lo
10-3d
10-2
lo-,
Antiserumn dilution
Figure 1. The effect of antiserum concentration on ADCC. Antiserum was added directly to the assay system with an effector (PBL) to target cell ratio of 50:1 and an incubation period of 20 h in the presence of canine distemper antiserum (-) or normal serum (A).
Canine K-cell activity
suggesting that the lytic activity observed was specific antibody dependent especially since the activity was directly proportional to the concentration of antibody (Fig. 1). This mechanism of destruction was extremely sensitive in that only low levels of antibody were required. Furthermore, the number of effector cells required for detecting destruction was very low (Fig. 2), suggesting that this mechanism could possibly be important in preventing CDV spread at an early stage of the disease when antibody levels were low and 50
40
c 30
20 o 20 _
/
0
1
I 0
25
50
100
Effector/target cell ratio
Figure 2. ADCC in the presence of varying effector to CDV infected target cell ratios. Incubation period was 20 h in the presence of a 1/20 dilution of anti-CDV serum (A) and normal serum (-). Uninfected target cells incubated in the presence of effector cells and anti-CDV serum served as controls (o).
40 -
S
30 -
20
ci,10
0
5
10 15 Hours of incubation
20
25
Figure 3. Effect of incubation period on ADCC. PBL effector cells were used at an effector to target cell ratio of 50: 1, in the presence of a 1/20 dilution of anti CDV serum (&), normal serum (o) and with anti-CDV serum in the absence of effector cells (-).
235
leucocyte levels were low and leucocyte infiltration was not maximal. For specific lysis to occur the effector and target cells had to be incubated for relatively long periods of time (Fig. 3). Thus, specific 5'Cr release was barely detectable during the first 10 h of incubation but increased rapidly thereafter such that over the next 5-10 h specific release reached maximum levels. If the mechanism of killing was due to ADCC then the addition of heat aggregated dog IgG, Staphylococcal protein A or rabbit anti-dog F(ab')2 might be expected to interfere with the binding of the leucocyte Fc receptor and sensitizing antibody and thereby reduce killing. This was shown to be the case in all three instances (Table 1) suggesting that killing was due to ADCC. Characterization of cells active in ADCC Various populations of leucocytes, obtained from the same animal were assessed for their cytotoxic potential against CDV infected targets. As illustrated in Table 2, lymphocytes were the most effective cells in mediating ADCC with the polymorphonuclear and adherent population being less effective. Further enrichment of macrophages by continued cultivation resulted in death of lymphocytes and a further decrease in cytotoxicity suggesting that macrophages were very inefficient in mediating ADCC as were the polymorphonuclear cells even though both cell types possess Fc receptors (Ho & Babiuk, 1978a). To characterize further the lymphocyte subpopulations capable of mediating ADCC, lymphocytes were separated into various subpopulations by rosetting techniques and nylon wool filtration. Depletion of surface immunoglobulin bearing cells from the PBL population did not alter the degree of cytotoxicity (Table 3) suggesting that a classical B cell was not responsible for the majority of the cytotoxicity observed. The effector cell did, however, possess Fc receptors as one would expect for ADCC to occur, since removal of EA rosetting cells dramatically reduced the degree of cytotoxicity. Further evidence that suggested the K cell was not a B cell was obtained from nylon wool fractionation experiments where the activity was found in the nylon effluent population (Table 3), a population previously shown to be depleted of B cells and enriched in T cells. The active cell was probably also not a classical T cell since the canine K cells expressed C receptors but T cells of most species do not generally express C receptors. Thus, the K cell probably represents an Fc and C receptor, surface immunoglobulin negative nylon
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Chi Kuan Ho & Lorne A. Babiuk
Table 1. Inhibition of antibody dependent cell mediated cytotoxicity by rabbit anti-dog F(ab')2 fragments, staphylococcal protein A and heat aggregated dog immunoglobulin*
Inhibitor None F(ab')2
ProteinA Hagg.
Igt
Concentration (pug) Specific releaset (%) Inhibition (%) 1.0 10 0 100.0 1.0 100 1000 50 500 5000
28 29 5 22 16 271 18 7 28 4 182 126
20 42 -
40 75
35 56
* ADCC assay was performed as described in Materials and Methods with 1/20 dilution of antiserum with effector to target cell ratio of 50:1 and an incubation time of 20 h. Infected Vero cells in the presence ofantiserum and/or inhibitors were used as control spontaneous releases. t Calculated by formula: percentage inhibition= counts per minute in the presence of inhibitors/counts per minute specific release x 100. t Heat aggregated normal dog gammaglobulin (630 for 10 min).
Table 2. Antibody dependent cytotoxic action expressed by different leucocyte
preparations Specific 5 1Cr release + SDt (%)
Cell preparation
Major cell type ± SD* (%)
PBL 85 + 7 lymphocytes Dextran sedimentation 77 + 5 PMN Plastic adherent cells 84 + 6 monocytes > 99 macrophages Macrophage culture
6h
20 h
3.7 + 1 8 4 + 27 3+2 4 2+2
32 + 7 5 11 + 3 4 8.6 + 3 4 + 1.5
* Based on morphological observation by phase contrast microscopy. Cells were stained with 0 5% methylene blue in the absence of serum. SD, standard deviation. t ADCC was performed with PBL as effector cells at an effector to target cell ratio of 25:1; target cells were preabsorbed with 1/20 antiserum and incubation period is 20 h. Spontaneous release (21 %) was subtracted during the calculation of specific release.
237
Canine K-cell activity Table 3. Surface markers of lymphocyte effector cells and their activity against CDV infected targets.
Cells with specific surface markers* + SD (%) Cell preparations PBL
SIg depleted PBL Nylon wool effluent EAC depleted Nylon wool effluent EAdepletedPBL EAC depleted PBL
Fc
EAC
SIg
Specific 5ICr release + SD§ (%)
25.1+4 2 215 +4-5 10+3
23 6+3 22-3+5 7 5+2 5
21 +6t 4.3 + 24
26 5+3 255 +4-5 19+2 2
10+3
7 5+2 5
6 5+3.7
1-3+1-5 2 4+ 17
26+2 18 + 27
3+1.5 52 + 34
6 5 + 3-7
19+2-2 43+3 28 + 27
* Based on the percentage of rosetting cells with EA, EAC and SIg respectively. SD, standard deviation. t The same cell preparation had 24 3 + 3.5% SIg+ cells based on immunofluorescence.
tN.D., not done. § ADCC was performed under the same conditions as described in Table 2.
wool non-adherent cell belonging to neither the T- nor B-cell lineage.
DISCUSSION
Antibody-dependent cell-mediated cytotoxicity (ADCC) has been shown to occur in a large number of species by a variety of different cell types all of which bear Fc receptors. Its actual biological role in antiviral defence in vivo, however, remains speculative. In the systems studied to date ADCC appears to be a very effective method of killing virus infected cells in vitro in that cytotoxicity can be detected very rapidly in the presence of low levels of antibody suggesting that it may be an important mechanism in the early stages of disease when antibody levels are low. To date, no reports have been published to suggest that a similar mechanism exists in dogs and could be of any relevance to recovery from canine distemper infections or be involved in post-distemper encephalitis. The present study has demonstrated that such a mechanism may be functional in the dog and that the canine cell type most effective in mediating ADCC against canine distemper virus-infected cells was a lymphocyte while the polymorphonuclear and monocyte, which also express Fc receptors (Ho & Babiuk, 1978a,b) are ineffective. In fact, if it were possible to remove all contaminating lymphocytes from the polymorphonuclear and monocyte populations these cells would probably
be totally incapable of mediating cytotoxicity (Table 1). The reason why these cells are ineffective, even though they possess the required Fc receptor, is not understood but it could be that the number of Fc receptors, the tenacity of binding of immunoglobulin to both target and effector cells as well as the type of antibody and target cell may influence the ability to mediate ADCC. Some evidence for such possibilities has been reported recently (Lustig & Bianco, 1976) which shows that ADCC was influenced by the type of antibody and/or the presence of complement. Some types of antibody and effector cells normally unable to participate in ADCC could do so if low levels of complement were added. These results suggested that possibly the presence of a second bond may increase the tenacity of the binding and also the efficiency of killing. It could be argued that since our effector cells were from immune animals and because the activity was found in T-cell enriched populations we were measuring direct cytotoxicity and not ADCC. This is unlikely for a number of reasons. Firstly, it has been shown that T-cell activity against virus infected cells is transient and short lived in all species studied to date (Doherty, Zinkernagel & Ramshaw, 1974; Ho et al., 1978; Rouse & Babiuk, 1977) including the canine species. Thus, after an initial sensitization, direct T-cell killing is maximal at 1-2 weeks, then declines rapidly to background levels. Since our effector cells were taken 4 months post-distemper immunization one
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Chi Kuan Ho & Lorne A. Babiuk
would expect T-cell cytotoxicity to be minimal. Secondly, cytotoxicity was not detectable unless specific antibody was added. Thirdly, blockage of the Fc receptor by various agents (Table 3) dramatically reduced cytotoxicity. Since most T cells do not normally express Fc receptors (Chiao, Ascensao, Pahwa, Pahwa & Good, 1976; Gy6ngyossy, Arnaiz-Villena, Soteriades-Viachos & Playfair, 1975) we felt that the T cell was not responsible for the activity observed. Unfortunately, it is not yet possible to prove this definitely by depleting T cells with rosetting techniques since other cell types as well as canine T cells form spontaneous E rosettes (Esser, Cosgrove, Scott & Cosimi, 1977; Gyongyossy et al., 1975; Ho & Babiuk, 1978a, b). Furthermore, specific anti-canine T-cell serum is not presently available to eliminate all T cells. The effector cell was also shown not to be a B cell since depletion of surface immunoglobulin bearing lymphocytes has little effect on the level of cytotoxicity. From these observations we are led to conclude that the canine effector cell responsible for cytotoxicity against canine distemper infected cells is a nylon nonadherent or weakly adherent non-T or B lymphocyte which bears both Fc and complement receptors. These cells are probably equivalent to the null cells reported to be the K cells in humans (Perrin, Tishon & Oldstone, 1977) and mice (Ramshaw & Parish, 1976). Although we have shown evidence for the existence of a K cell in the dog it still does not demonstrate that this cell is of any relevance in preventing canine distemper in vivo. Ideally, for any defense mechanism to be effective in this regard the K cell must be present at the site of viral replication and must be able to kill the infected cell prior to viral dissemination. If killing occurs after cell to cell spread of the virus then this mechanism cannot prevent spread even though it may reduce the virus load in the case of enveloped viruses where release occurs over extended periods. The present study demonstrates that cytotoxicity takes a relatively long time (- 10 h) before killing is detectable. Preliminary results with the Green strain of CDV have demonstrated that during a lytic infection, virus maturation and spread occurs at approximately 8 h post-infection (Ho & Babiuk, unpublished-results), therefore, spread would have occurred before the cell was killed. Kinetic experiments with other strains have shown similar results, therefore one would suspect that K cells may not totally inhibit viral spread unless they are cytostatic as well as cytotoxic. It is tempting to speculate that since CDV replicates in canine lymphocytes the K cell does play an im-
portant role in both the early stages of the infection as well as in areas such as the brain where antibody levels are generally low. If the immune response does not eliminate the virus at a critical time in an infection, possibly due to virus replication in lymphocytes and suppression of CMI, the virus may evade the animal's immune responses. Under these conditions, virus persistence may be established and antibody levels increase to such a level that there is antigenic modulation and elimination of the viral antigens on the cell membrane. Once this occurs the K cell can no longer kill the virus infected cell thus permitting continued persistence which may lead to post-distemper encephalitis as has been suggested in measles infected cells (Perrin et al., 1977). If these virus-antigen antibody complexes are shed into the circulation they could block ADCC in a manner similar to blocking by aggregated immunoglobulin (Table 3). This possibility is reinforced by the observation that Ag-Ab complexes are shed from measles virus-infected cells (Perrin & Oldstone, 1977) and that high levels of antibody do block ADCC in SSPE (Steele, Fucillo, Hensen, Vincent & Bellanti, 1976) and CDV infected cell systems (Ho & Babiuk, unpublished results).
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