Veterinary Immunology and lmmunopathology, 28 ( 1991 ) 67-80
67
Elsevier Science Publishers B.V., Amsterdam
In vitro lymphocyte stimulation by Concanavaiin A and with histamine as a co-mitogen in dogs with atopic dermatitis J.S. Nimmo Wilkie Department of Pathology, Ontario Veterinary College. Universityof Guelph, Guelph, Ont.. NIG 2WI, Canada (Accepted l May 1990)
ABSTRACT Nimmo Wilkie, J.S., 1991. In vitro lymphocyte stimulation by ConcaPavalin A and with histamine as a co-mitogen in dogs with atopic dermatitis. Vet. Immunol. lmmunopathoL, 28: 67-80. Cell-mediated immune function was assessed in a group of dogs with atopic dermatitis by measuring the responses of peripheral-blood lymphocytes (PBL) to various concentralions of Concanavalin A (Con A) and comparing them to those of normal dogs. No difference from normal was found in any of the stimulation indi~:es neither was spontaneous tritium uptake of unstimulated cells different between the groups. We also measured the response to Con A stimulation in vitro of PBL preincubated for 24 h, either in cell-culture medium at 37°C, or in whole blood containing EDTA at room temperature, as an indirect measure of function of a subgroup of suppressor cells. Preincubation caused enhancement of mitogenesis for normal dog lymphocytes but not for the atopic dog cells, particularly for suboptimal concentrations of Con A. No differences were found in the responsiveness following incubatmn in cell-culture medium between normal and atopie dog cells but for both groups the cells preincubated in whole blood were generally more responsive. Histamine, which is one of the mediators of type l hypersensitivities such as atopy, can modulate lymphocyte function. At l0 -4 and l0 -s M histamine, when added simultaneously with Con A, enhanced mitogenesis of normal dog PBL but suppressed mitogenesis of atopic dog PBL. By using histamine H~ and H~ antagonists, we concluded that histamine enhanced mitogenesis via H~, receptors and suppressed it via H2 receptors. Our results suggest that there are abnormalities in lymphocyte function in dogs with atopic dermatitis which may be important in the pathogenesis of the disease.
INTRODUCTION
The lymphocyte stimulation test is frequently used in man and animals to evaluate cell-mediated immune responses in vitro (Barta and Oyekan, 198 l; Kristensen et al., 1982a). It has been used to assess immunosuppression in dogs with a variety of diseases, including viral and bacterial infections and ectoparasitism (Knox and Shifrine, 1980; Barta and Oyekan, 1981 ), but to our knowledge not in dogs with atopic dermatitis. 0165-2427/91/$03.50
© 1991 - - Elsevier Science Publishers B.V.
68
J.s. NIMMO WILKIE
Atopic dermatitis in people has long been associated with defective cellmediated immune responses (Krafchik, 1983). The response to stimulation by some mitogens and antigens oflymphocytes from human atopic dermatitis patients has been found, by some investigators, to be deficient (Strannegard et al., 1976; Elliott ~nd Hanifin, 1979b; Horsmanheimo et al., 1979; Rola-Pleszcynski and Blanchard, 1981 ) but not by all investigators or for all mitogens (Strannegard et al., 1976; Horsmanheimo et al., 1979; Valverde et al., 1983; Schultz Larsen and Grunnet, 1985 ). Others have found depressed lymphocyte responses only in people with severe atopic dermatitis (Elliott and Hanifin, 1979a; Ogden et al., 1979). Whilst the lymphocyte stimulation test is used as a measure of immune responsiveness and has been shown to correlate well with in vivo immune responses (Thilsted et al., 1979), the same test done on lymphocytes preincubated for 24 h has been used to test the function of a suppressor cell subset. Bresnihan and Jasin (1977) observed that preincubation for 24 h of normal human peripheral blood mononuclear cells increased the subsequent lymphocyte stimulation response to Concanavalin A (Con A), and that this enhanced response was not present for a group of patients with systemic lupus erythematosus. They suggested that the enhanced response in normal persons is due to the loss of a short-lived suppressor cell during preincubation and that patients with systemic lupus erythematosus lacked these cells. Subsequently, several investigators examined lymphocyte stimulation, after a period of preincubation, in people with atopic dermatitis. The results have been conflicting, indicating decreased (Rola-Pleszcynski and Blanchard~ 1981 ), increased (El!iott ~ d Hanifin, 1979b) or the same enhancement of proliferation as normal (Ogden et al., 1979; Schuster et al., 1979). More consistent results have been reported on the effect of histamine as a co-mitogen for lymphocytes from human atopics. Since histamine is one of the major mediators of the type I hypersensitivity reactions, which occur in the atopic diseases, modulation of lymphocyte function by histamine is a possible mechanism for altered lymphocyte reactivity in these diseases. It is known that histamine can affect many lymphocyte functions (Rocldin, 1983), enhancing or suppressing mitogenesis depending upon its concentration, or by acting via H~ or H2 histamine receptors respectively (Wang and Zweiman, 1978; Ogden and Hill, 1980; Suzuki and Huchet, 1981; Badger et al., 1983). Most investigators found that lymphocytes from human atopics were even more susceptible than normal to histamine-induced suppression of mitogenesis (Strannegard and Strannegard, 1977; Wang and Zweiman, 1978; Ogden et al., 1979; Boot et al., 1980; Brostoffet al., 1980; Hall et al., 1983) with one dissenter (Martinez et al., 1979 ), who found the atopic cells to be less susceptible than normal to histamine. In the present study, the responsiveness of canine peripheral blood mononuclear cells (PBMC) to various doses of Con A, using either freshly sepa-
LYMPHOCYTE STIMULATION IN DOGS WITH ATOPIC DERMATITIS
69
rated cells or cells preincubated for 24 h in either cell-culture medium at 37 °C or whole blood at room temperature, was measured. We also examined the effect of a high or low dose of histamine on the optimal response to Con A of the fresh cells and whether this effect could be blocked by a Hi or H2 histamine antagonist. By comparing the responses of lymphocytes from atopic dermatitis dogs and those of normal dogs we have tried to illuminate differences in lymphocyte reactivity that may be involved in the pathogenesis of atopic dermatitis in dogs. MATERIALS AND METHODS
Animals There were 17 dogs with atopic dermatitis, including dogs of both sexes, and of various ages and breeds including mixed breeds. The dogs were accepted as having atopic dermatitis if they met all the following criteria: ( 1 ) a clinical history and clinical signs appropriate to atopy (Scott, 1981 ); (2) absence of any other dermatological disease; (3) a skin biopsy with histopathological findings typical of atopic dermatitis (Scott, 1981 ); (4) a positive skin reaction to at least one allergen, other than allergens from biting insects, oa intradermal testing with fifteen standard allergens (Hollister-Stier Canada, Mississauga, Ont. ). A positive reaction was defined as a wheal greater in diameter than one half of the sum of the diameters of the negative and positive controls, which were saline and 1 : 100 000 histamine respectively (Scott, 1981 ). Except for the five dogs used to examine the effect of prior corticosteroid treatment, the clogs were required to have been free of any medication for at least 4 weeks previously.
Lymphocyte separation Peripheral blood was obtained by venipuncture into evacuated tubes containing liquid EDTA (Vacutainer, Becton Dickinson Canada, Mississauga, Ont. ). One half of the blood was stored at room temperature in these tubes. The remainder was centrifuged twice at 8 0 0 x g for 3 min and the plateletrich plasma removed and discarded. The volume was replenished with calcium and magnesium free Hank's buffered salt solution (HBSS-Ca, Mg free) and centrifuged for 10 min at 800×g and the buffy coat harvested into an equivalent volume of HBSS-Ca, Mg free. This was layered onto half its volume of Ficoll-Hypaque (Pharmacia, Uppsala, Sweden ) at room temperature and centrifuged at 400 × g for 30 rain. The interface cells were harvested and washed three times in HBSS-Ca, Mg free before being resuspended in RPMI1640 containing 0.5 units penicillin and 0.25 gg streptomycin/ml, 15% fetal calf serum, 1% sodium pyruvate and 1% L-glutamine, The viability of the cells was checked using trypan blue exclusion and was consistently greater than 92%.
70
J.S. NIMMO WILKIE
Lymphocyte stimulation Separated cells were adjusted to 1 × 106 cells/ml and dispensed in quadruplicates at 200 gl/well into 96-well flat-bottomed microtitre plates (Linbro, Flow Labs, McLean, Virginia). To each well was added one of the following doses 0, 0.5, l, 5, 10 or 100 gg/ml of Concanavalin A (type IV, Sigma, St. Louis, MO). The plates were incubated for 72 h at 37°C in 5% humidified CO, atmosphere. Tritiated thymidine (Amersham, Oakville, Ont.), at 0.5 gCi per well, was added and the plates were incubated a further 18-20 h. The cells were then harvested on glass-wool filter paper (Titre Tek, Flow Labs Rockville, Maryland) using a cell harvester (Titre Tek, Flow labs, Rockville, MD) and incorporated tritium was quantified using a Beckman LS-3133 liquid scintillation counter. The stimulation index (SI) was calculated as follows SI = cpm Con A stimulated cells cpm unstimulated cells where cpm=counts per minute for tritium minus background counts per minute.
24 hour preincubated cells Cells at 1 X 106 cells/ml were dispensed in quadruplicates at 200/d/well into 96-well flat-bottomed microtitre plates and incubated at 37 °C in humidified 5% CO2 atmosphere for 24 h before being stimulated, incubated and harvested exactly as for the freshly separated cells. In addition, the whole blood containing EDTA, after 24 h at room temperature, was separated as before, stimulated with Con A, incubated and harvested as already described. The percentage enhancement w ~ calculatad a~ fnl!ows % enhancement = 100 × [cpm Con A stimulated 24 hour preincubated cells cpm Con A stimulated fresh cells
] 1
Histamine co-mitogenesis Freshly separated PBMC were dispensed at 200 gl/well, in quadruplicates into microtitre plates as already described. Histamine dihydrochlofide (Sigma, St. Louis, MO) in RPMI- 1640, that had been previously prepared and stored in aliquots at - 3 0 ° C , was added at 10 -4 and 10 - 6 M concentrations. An optimal concentration of Con A (5 gg/ml) was added to each well before incubating and harvesting the cultures as before. For thirteen experiments a ten-fold greater concentration of an Hrhistamine antagonist, pyrilamine (Sigma, St. Louis, MO ) or an H2-histamine antagonist, cimetidine (gift from Dr. P. Eyre, University of Guelph), was added to the cells before adding the histamine and the mitogen and incubating and harvesting as previously described. The percentage enhancement due to the addition of histamine was calculated as follows, for each concentration of histamine
71
LYMPHOCYTE STIMULATION IN DOGS WITH ATOPIC DERMATITIS
% histamine enhancement= 100 × lepta5/zg/ml Con A plus histamine stimulated cells cpm 5/,g/ml Con A stimulated fresh cells L
] !J
Effect of Hi and He histamine antagonists The percentage enhancement due to the addition of histamine was calculated as above. The percentage enhancement in the presence of histamine HI antagonist was calculated similarly as follows % HI blocked enhancement = 100 X
[cpm 5/tg/ml Con A and histamine + HI antagonist stimulated cells cpm 5/tg/ml Con A stimulated fresh cells L
,]
The H2 blocked enhancement was calculated similarly. The difference in percentages was calculated as % change due to HI receptor = % H2 blocked enhancement- % histamine enhancement
Responsiveness after incubation in cell culture medium A comparison of the lymphocyte responsiveness following 24 h in cell culture medium or in whole blood containing EDTA was effected by calculating the percentage change in cpm at the same dose of Con A 1 00 ×
["1 cpm Con A stimulated cells preincubated in cell culture medium 1 cpm Con A stimulated cells preincubated in ~ b l - 0 - 0 - d J
L
Statistical analyses The means for each test for the groups ofatopic and normal dogs were compared using Student's t test. The means were considered statistically different if the probability (P) was less than 0.05.
RESULTS
Lymphocyte stimulation by Con A Freshly separated cells. From preliminary experiments with PBMC from normal dogs it was established that 5/tg/ml of Con A gave optimum lymphocyte stimulation in our system. At 100/zg/ml Con A the cpm from incorporated tritiated thymidine were always similar to cpm from the unstimulated cells.
72
J.S. NIMMO WILKIE 10 ug/ml
5 ug/ml
1 ug/ml 5o
40 x o lo _¢ c o
30 :/
"5
¢o
:E
lO-
o = 28.05 n=21
= 27.86
i ffi 34.77
s x = 14.45 s x = 2 8 . 8 0 n=15
x ffi 39.21
S x = 12.82 Sx = 29.32 n=21
~--~7:~ Normals
n=15
= 30.04 ~ = 32.65 sx = 14.71 SX = 30.10 n=2!
n=15
~;~ Atopics
Fig. 1. Concanavalin A-induced blastogenesis of PBMC from normal dogs and from dogs with atopic dermatitis incubated for 72 h with 1 sg/ml, 5 sg/ml or 10 #g/ml of Con A.
Fig. 1 shows stimulation indices (SI) for the normal and atopic dog cells at 1, 5 and 10/zg/ml of Con A. No significant difference was detectable between means for normal and for atopic dogs at any of the dose rates. Fig. 2 shows a • . . . '" . . U. .l U. S , which represents companson ,.,1 ..r the •t -. . u•:"-m c p m #^loa unstimulated UUII, the spontaneous uptake of tritiated thymidine for atopic and normal dogs PBMC. There was no significant difference between the means of the groups•
24 hour preincubated cells For both the normal and the atopic dogs the response to Con A stimulation was greater for cells stored as whole blood rather than stored in cell culture medium. Table 1 gives a typical comparison using the 5/~g/ml Con A level. There was no significant difference between the groups with respect to the decrease in responsiveness of cells stored in cell culture medium, and for both groups, the number of dogs which showed decreased responsiveness of cells stored in cell culture medium was very similar. Preincubation enhanced Con A mitogenesis of normal dog PBMC. Fig. 3 shows the percentage enhancement of mitogenesis due to preincubation in whole blood, of PBMC from normal and from atopic dogs at optimal (5/zg/ ml) and suboptimal ( 1/lg/ml) levels of Con A. At suboptimal levels the mean percentage enhancement is negative for the atopic group and is significantly different from the normal group (P~< 0.01 ). At optimal Con A level the mean
LYMPHOCYTESTIMULATIONIN DOGSWITHATOPICDERMATITIS
73
2 1.9 1.8 1.7 1.6 1.5 1,4
1.1
~
o.5
-, ~
o.5
X
0.?
0,6 0.5 °
0.4 03 02
x
0,1 0
= 478.7 SX = 3 1 6 . 8 n=21
x
Normals
x = 428.0 sx = 4 8 7 . 9 n=15
Atopics
Fig. 2. Spontaneous uptake o f tritiated thymidine by unstimulated, 72-h cultures o f PBMC from normal dogs and from dogs with atopic dermatitis. TABLE 1 Comparison of the response to 5/zg/ml Con A of PBMC from normal dogs and dogs with atopic dermatitis, preincubated in cell culture medium, expressed as the percentage decrease in cpm from incorporated tritiated thymidine following stimulation by 5/zg/mi of Con A, for cells preincubated in cell culture medium compared with those preincubated in whole blood for 24 h
Normal Atopic
Total no. of dogs
% decrease in cpm mean + SD
No. of dogs with decreased cpm after preincubation in cell culture medium
13 12
- 3.78 + 121.5 0.78+ 115.6
9 (30.8%) 8 (33.3%)
ISD = standard deviation.
for the atopics is lower than that of the normals but not significantly so. The percentage of dogs for which there was no enhancement, (that is zero or negative percentage enhancement) is higher for the atopic group, especially at suboptimal levels of Con A (Table 2 ).
Histamine co-mitogenesis Both the low ( 10 -6) and high ( 10 -4 M) doses of histamine enhanced optimal Con A lymphocyte stimulation for normal canine PBMC. This was not so for cells from atopic dogs which, on average, showed a slight depression of
74
J.S. NIMMOWILKIE 1 ug/ml Con A
5 ug/ml Con A
2OO
lao -t X
140
X X
120 --4
X
o
E
8C
6O
X
t
X X
40
X
C
N
o O O O
0
0
x
X
x x
O
X
X
o
×
X
g ×
-11111
= 3238 sx = 63.44 n=21 ×
x = -25.22 sx = 43.57 n=13
x = 40.91 sx = 76.24 n=21
Normals
x = 7.38 sx = 63.15 n=13
o Atopics
Fig. 3. E n h a n c e m e n t o f C o n A - i n d u c e d blastogenesis o f P B M C f r o m n o r m a l dogs a n d dogs w i t h
atopic dermatitis due to preincubation o f cells for 24 h prior to stimulation with 1 p g / m l or 5 pg/ml of Con A. TABLE 2 Enhancement of Con A mitogenesis of normal and of atopic dog PBMC by 24 h preincubation in whole blood containing EDTA Dogs
Total no.
% enhancement of mitogenesis 1 #g/ml Con A (me~:n _+SF I )
No. of dogs with - ve % enhancement
% enhancement of mitogenesis 5 pg/ml Con A (mean ± SD }
No. of dogs with - ve % enhancement
Normal Atopic
21 i3
32.8+63.4 -25.22_+43.6%
6 (28.6%) 9 (69.2%)
40.91._+76.2 7.38+63.15
7 (33.3%) 7 (53.9%)
'SD = standard deviation.
optimal Con A lymphocyte stimulation, as is shown in Fig. 4. The means of the normal and atopic groups were significantly different for 10 -8 M histamine (P~0.01) and for 10 - 4 histamine (P~
67
Elsevier Science Publishers B.V., Amsterdam
In vitro lymphocyte stimulation by Concanavaiin A and with histamine as a co-mitogen in dogs with atopic dermatitis J.S. Nimmo Wilkie Department of Pathology, Ontario Veterinary College. Universityof Guelph, Guelph, Ont.. NIG 2WI, Canada (Accepted l May 1990)
ABSTRACT Nimmo Wilkie, J.S., 1991. In vitro lymphocyte stimulation by ConcaPavalin A and with histamine as a co-mitogen in dogs with atopic dermatitis. Vet. Immunol. lmmunopathoL, 28: 67-80. Cell-mediated immune function was assessed in a group of dogs with atopic dermatitis by measuring the responses of peripheral-blood lymphocytes (PBL) to various concentralions of Concanavalin A (Con A) and comparing them to those of normal dogs. No difference from normal was found in any of the stimulation indi~:es neither was spontaneous tritium uptake of unstimulated cells different between the groups. We also measured the response to Con A stimulation in vitro of PBL preincubated for 24 h, either in cell-culture medium at 37°C, or in whole blood containing EDTA at room temperature, as an indirect measure of function of a subgroup of suppressor cells. Preincubation caused enhancement of mitogenesis for normal dog lymphocytes but not for the atopic dog cells, particularly for suboptimal concentrations of Con A. No differences were found in the responsiveness following incubatmn in cell-culture medium between normal and atopie dog cells but for both groups the cells preincubated in whole blood were generally more responsive. Histamine, which is one of the mediators of type l hypersensitivities such as atopy, can modulate lymphocyte function. At l0 -4 and l0 -s M histamine, when added simultaneously with Con A, enhanced mitogenesis of normal dog PBL but suppressed mitogenesis of atopic dog PBL. By using histamine H~ and H~ antagonists, we concluded that histamine enhanced mitogenesis via H~, receptors and suppressed it via H2 receptors. Our results suggest that there are abnormalities in lymphocyte function in dogs with atopic dermatitis which may be important in the pathogenesis of the disease.
INTRODUCTION
The lymphocyte stimulation test is frequently used in man and animals to evaluate cell-mediated immune responses in vitro (Barta and Oyekan, 198 l; Kristensen et al., 1982a). It has been used to assess immunosuppression in dogs with a variety of diseases, including viral and bacterial infections and ectoparasitism (Knox and Shifrine, 1980; Barta and Oyekan, 1981 ), but to our knowledge not in dogs with atopic dermatitis. 0165-2427/91/$03.50
© 1991 - - Elsevier Science Publishers B.V.
68
J.s. NIMMO WILKIE
Atopic dermatitis in people has long been associated with defective cellmediated immune responses (Krafchik, 1983). The response to stimulation by some mitogens and antigens oflymphocytes from human atopic dermatitis patients has been found, by some investigators, to be deficient (Strannegard et al., 1976; Elliott ~nd Hanifin, 1979b; Horsmanheimo et al., 1979; Rola-Pleszcynski and Blanchard, 1981 ) but not by all investigators or for all mitogens (Strannegard et al., 1976; Horsmanheimo et al., 1979; Valverde et al., 1983; Schultz Larsen and Grunnet, 1985 ). Others have found depressed lymphocyte responses only in people with severe atopic dermatitis (Elliott and Hanifin, 1979a; Ogden et al., 1979). Whilst the lymphocyte stimulation test is used as a measure of immune responsiveness and has been shown to correlate well with in vivo immune responses (Thilsted et al., 1979), the same test done on lymphocytes preincubated for 24 h has been used to test the function of a suppressor cell subset. Bresnihan and Jasin (1977) observed that preincubation for 24 h of normal human peripheral blood mononuclear cells increased the subsequent lymphocyte stimulation response to Concanavalin A (Con A), and that this enhanced response was not present for a group of patients with systemic lupus erythematosus. They suggested that the enhanced response in normal persons is due to the loss of a short-lived suppressor cell during preincubation and that patients with systemic lupus erythematosus lacked these cells. Subsequently, several investigators examined lymphocyte stimulation, after a period of preincubation, in people with atopic dermatitis. The results have been conflicting, indicating decreased (Rola-Pleszcynski and Blanchard~ 1981 ), increased (El!iott ~ d Hanifin, 1979b) or the same enhancement of proliferation as normal (Ogden et al., 1979; Schuster et al., 1979). More consistent results have been reported on the effect of histamine as a co-mitogen for lymphocytes from human atopics. Since histamine is one of the major mediators of the type I hypersensitivity reactions, which occur in the atopic diseases, modulation of lymphocyte function by histamine is a possible mechanism for altered lymphocyte reactivity in these diseases. It is known that histamine can affect many lymphocyte functions (Rocldin, 1983), enhancing or suppressing mitogenesis depending upon its concentration, or by acting via H~ or H2 histamine receptors respectively (Wang and Zweiman, 1978; Ogden and Hill, 1980; Suzuki and Huchet, 1981; Badger et al., 1983). Most investigators found that lymphocytes from human atopics were even more susceptible than normal to histamine-induced suppression of mitogenesis (Strannegard and Strannegard, 1977; Wang and Zweiman, 1978; Ogden et al., 1979; Boot et al., 1980; Brostoffet al., 1980; Hall et al., 1983) with one dissenter (Martinez et al., 1979 ), who found the atopic cells to be less susceptible than normal to histamine. In the present study, the responsiveness of canine peripheral blood mononuclear cells (PBMC) to various doses of Con A, using either freshly sepa-
LYMPHOCYTE STIMULATION IN DOGS WITH ATOPIC DERMATITIS
69
rated cells or cells preincubated for 24 h in either cell-culture medium at 37 °C or whole blood at room temperature, was measured. We also examined the effect of a high or low dose of histamine on the optimal response to Con A of the fresh cells and whether this effect could be blocked by a Hi or H2 histamine antagonist. By comparing the responses of lymphocytes from atopic dermatitis dogs and those of normal dogs we have tried to illuminate differences in lymphocyte reactivity that may be involved in the pathogenesis of atopic dermatitis in dogs. MATERIALS AND METHODS
Animals There were 17 dogs with atopic dermatitis, including dogs of both sexes, and of various ages and breeds including mixed breeds. The dogs were accepted as having atopic dermatitis if they met all the following criteria: ( 1 ) a clinical history and clinical signs appropriate to atopy (Scott, 1981 ); (2) absence of any other dermatological disease; (3) a skin biopsy with histopathological findings typical of atopic dermatitis (Scott, 1981 ); (4) a positive skin reaction to at least one allergen, other than allergens from biting insects, oa intradermal testing with fifteen standard allergens (Hollister-Stier Canada, Mississauga, Ont. ). A positive reaction was defined as a wheal greater in diameter than one half of the sum of the diameters of the negative and positive controls, which were saline and 1 : 100 000 histamine respectively (Scott, 1981 ). Except for the five dogs used to examine the effect of prior corticosteroid treatment, the clogs were required to have been free of any medication for at least 4 weeks previously.
Lymphocyte separation Peripheral blood was obtained by venipuncture into evacuated tubes containing liquid EDTA (Vacutainer, Becton Dickinson Canada, Mississauga, Ont. ). One half of the blood was stored at room temperature in these tubes. The remainder was centrifuged twice at 8 0 0 x g for 3 min and the plateletrich plasma removed and discarded. The volume was replenished with calcium and magnesium free Hank's buffered salt solution (HBSS-Ca, Mg free) and centrifuged for 10 min at 800×g and the buffy coat harvested into an equivalent volume of HBSS-Ca, Mg free. This was layered onto half its volume of Ficoll-Hypaque (Pharmacia, Uppsala, Sweden ) at room temperature and centrifuged at 400 × g for 30 rain. The interface cells were harvested and washed three times in HBSS-Ca, Mg free before being resuspended in RPMI1640 containing 0.5 units penicillin and 0.25 gg streptomycin/ml, 15% fetal calf serum, 1% sodium pyruvate and 1% L-glutamine, The viability of the cells was checked using trypan blue exclusion and was consistently greater than 92%.
70
J.S. NIMMO WILKIE
Lymphocyte stimulation Separated cells were adjusted to 1 × 106 cells/ml and dispensed in quadruplicates at 200 gl/well into 96-well flat-bottomed microtitre plates (Linbro, Flow Labs, McLean, Virginia). To each well was added one of the following doses 0, 0.5, l, 5, 10 or 100 gg/ml of Concanavalin A (type IV, Sigma, St. Louis, MO). The plates were incubated for 72 h at 37°C in 5% humidified CO, atmosphere. Tritiated thymidine (Amersham, Oakville, Ont.), at 0.5 gCi per well, was added and the plates were incubated a further 18-20 h. The cells were then harvested on glass-wool filter paper (Titre Tek, Flow Labs Rockville, Maryland) using a cell harvester (Titre Tek, Flow labs, Rockville, MD) and incorporated tritium was quantified using a Beckman LS-3133 liquid scintillation counter. The stimulation index (SI) was calculated as follows SI = cpm Con A stimulated cells cpm unstimulated cells where cpm=counts per minute for tritium minus background counts per minute.
24 hour preincubated cells Cells at 1 X 106 cells/ml were dispensed in quadruplicates at 200/d/well into 96-well flat-bottomed microtitre plates and incubated at 37 °C in humidified 5% CO2 atmosphere for 24 h before being stimulated, incubated and harvested exactly as for the freshly separated cells. In addition, the whole blood containing EDTA, after 24 h at room temperature, was separated as before, stimulated with Con A, incubated and harvested as already described. The percentage enhancement w ~ calculatad a~ fnl!ows % enhancement = 100 × [cpm Con A stimulated 24 hour preincubated cells cpm Con A stimulated fresh cells
] 1
Histamine co-mitogenesis Freshly separated PBMC were dispensed at 200 gl/well, in quadruplicates into microtitre plates as already described. Histamine dihydrochlofide (Sigma, St. Louis, MO) in RPMI- 1640, that had been previously prepared and stored in aliquots at - 3 0 ° C , was added at 10 -4 and 10 - 6 M concentrations. An optimal concentration of Con A (5 gg/ml) was added to each well before incubating and harvesting the cultures as before. For thirteen experiments a ten-fold greater concentration of an Hrhistamine antagonist, pyrilamine (Sigma, St. Louis, MO ) or an H2-histamine antagonist, cimetidine (gift from Dr. P. Eyre, University of Guelph), was added to the cells before adding the histamine and the mitogen and incubating and harvesting as previously described. The percentage enhancement due to the addition of histamine was calculated as follows, for each concentration of histamine
71
LYMPHOCYTE STIMULATION IN DOGS WITH ATOPIC DERMATITIS
% histamine enhancement= 100 × lepta5/zg/ml Con A plus histamine stimulated cells cpm 5/,g/ml Con A stimulated fresh cells L
] !J
Effect of Hi and He histamine antagonists The percentage enhancement due to the addition of histamine was calculated as above. The percentage enhancement in the presence of histamine HI antagonist was calculated similarly as follows % HI blocked enhancement = 100 X
[cpm 5/tg/ml Con A and histamine + HI antagonist stimulated cells cpm 5/tg/ml Con A stimulated fresh cells L
,]
The H2 blocked enhancement was calculated similarly. The difference in percentages was calculated as % change due to HI receptor = % H2 blocked enhancement- % histamine enhancement
Responsiveness after incubation in cell culture medium A comparison of the lymphocyte responsiveness following 24 h in cell culture medium or in whole blood containing EDTA was effected by calculating the percentage change in cpm at the same dose of Con A 1 00 ×
["1 cpm Con A stimulated cells preincubated in cell culture medium 1 cpm Con A stimulated cells preincubated in ~ b l - 0 - 0 - d J
L
Statistical analyses The means for each test for the groups ofatopic and normal dogs were compared using Student's t test. The means were considered statistically different if the probability (P) was less than 0.05.
RESULTS
Lymphocyte stimulation by Con A Freshly separated cells. From preliminary experiments with PBMC from normal dogs it was established that 5/tg/ml of Con A gave optimum lymphocyte stimulation in our system. At 100/zg/ml Con A the cpm from incorporated tritiated thymidine were always similar to cpm from the unstimulated cells.
72
J.S. NIMMO WILKIE 10 ug/ml
5 ug/ml
1 ug/ml 5o
40 x o lo _¢ c o
30 :/
"5
¢o
:E
lO-
o = 28.05 n=21
= 27.86
i ffi 34.77
s x = 14.45 s x = 2 8 . 8 0 n=15
x ffi 39.21
S x = 12.82 Sx = 29.32 n=21
~--~7:~ Normals
n=15
= 30.04 ~ = 32.65 sx = 14.71 SX = 30.10 n=2!
n=15
~;~ Atopics
Fig. 1. Concanavalin A-induced blastogenesis of PBMC from normal dogs and from dogs with atopic dermatitis incubated for 72 h with 1 sg/ml, 5 sg/ml or 10 #g/ml of Con A.
Fig. 1 shows stimulation indices (SI) for the normal and atopic dog cells at 1, 5 and 10/zg/ml of Con A. No significant difference was detectable between means for normal and for atopic dogs at any of the dose rates. Fig. 2 shows a • . . . '" . . U. .l U. S , which represents companson ,.,1 ..r the •t -. . u•:"-m c p m #^loa unstimulated UUII, the spontaneous uptake of tritiated thymidine for atopic and normal dogs PBMC. There was no significant difference between the means of the groups•
24 hour preincubated cells For both the normal and the atopic dogs the response to Con A stimulation was greater for cells stored as whole blood rather than stored in cell culture medium. Table 1 gives a typical comparison using the 5/~g/ml Con A level. There was no significant difference between the groups with respect to the decrease in responsiveness of cells stored in cell culture medium, and for both groups, the number of dogs which showed decreased responsiveness of cells stored in cell culture medium was very similar. Preincubation enhanced Con A mitogenesis of normal dog PBMC. Fig. 3 shows the percentage enhancement of mitogenesis due to preincubation in whole blood, of PBMC from normal and from atopic dogs at optimal (5/zg/ ml) and suboptimal ( 1/lg/ml) levels of Con A. At suboptimal levels the mean percentage enhancement is negative for the atopic group and is significantly different from the normal group (P~< 0.01 ). At optimal Con A level the mean
LYMPHOCYTESTIMULATIONIN DOGSWITHATOPICDERMATITIS
73
2 1.9 1.8 1.7 1.6 1.5 1,4
1.1
~
o.5
-, ~
o.5
X
0.?
0,6 0.5 °
0.4 03 02
x
0,1 0
= 478.7 SX = 3 1 6 . 8 n=21
x
Normals
x = 428.0 sx = 4 8 7 . 9 n=15
Atopics
Fig. 2. Spontaneous uptake o f tritiated thymidine by unstimulated, 72-h cultures o f PBMC from normal dogs and from dogs with atopic dermatitis. TABLE 1 Comparison of the response to 5/zg/ml Con A of PBMC from normal dogs and dogs with atopic dermatitis, preincubated in cell culture medium, expressed as the percentage decrease in cpm from incorporated tritiated thymidine following stimulation by 5/zg/mi of Con A, for cells preincubated in cell culture medium compared with those preincubated in whole blood for 24 h
Normal Atopic
Total no. of dogs
% decrease in cpm mean + SD
No. of dogs with decreased cpm after preincubation in cell culture medium
13 12
- 3.78 + 121.5 0.78+ 115.6
9 (30.8%) 8 (33.3%)
ISD = standard deviation.
for the atopics is lower than that of the normals but not significantly so. The percentage of dogs for which there was no enhancement, (that is zero or negative percentage enhancement) is higher for the atopic group, especially at suboptimal levels of Con A (Table 2 ).
Histamine co-mitogenesis Both the low ( 10 -6) and high ( 10 -4 M) doses of histamine enhanced optimal Con A lymphocyte stimulation for normal canine PBMC. This was not so for cells from atopic dogs which, on average, showed a slight depression of
74
J.S. NIMMOWILKIE 1 ug/ml Con A
5 ug/ml Con A
2OO
lao -t X
140
X X
120 --4
X
o
E
8C
6O
X
t
X X
40
X
C
N
o O O O
0
0
x
X
x x
O
X
X
o
×
X
g ×
-11111
= 3238 sx = 63.44 n=21 ×
x = -25.22 sx = 43.57 n=13
x = 40.91 sx = 76.24 n=21
Normals
x = 7.38 sx = 63.15 n=13
o Atopics
Fig. 3. E n h a n c e m e n t o f C o n A - i n d u c e d blastogenesis o f P B M C f r o m n o r m a l dogs a n d dogs w i t h
atopic dermatitis due to preincubation o f cells for 24 h prior to stimulation with 1 p g / m l or 5 pg/ml of Con A. TABLE 2 Enhancement of Con A mitogenesis of normal and of atopic dog PBMC by 24 h preincubation in whole blood containing EDTA Dogs
Total no.
% enhancement of mitogenesis 1 #g/ml Con A (me~:n _+SF I )
No. of dogs with - ve % enhancement
% enhancement of mitogenesis 5 pg/ml Con A (mean ± SD }
No. of dogs with - ve % enhancement
Normal Atopic
21 i3
32.8+63.4 -25.22_+43.6%
6 (28.6%) 9 (69.2%)
40.91._+76.2 7.38+63.15
7 (33.3%) 7 (53.9%)
'SD = standard deviation.
optimal Con A lymphocyte stimulation, as is shown in Fig. 4. The means of the normal and atopic groups were significantly different for 10 -8 M histamine (P~0.01) and for 10 - 4 histamine (P~
