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A reliable method for reconstituting thymectomized, lethally irradiated guinea pigs with bone marrow cells
Journal of Immunological Methods, 70 (1984) 221-231
221
© 1984 Elsevier JIM03084
A Reliable Method for Reconstituting Thymectomized, Lethally Irradiated Guinea Pigs with Bone Marrow Cells N. Terata, Y. Tanio and B. Zbar 1 Laboratory of Immunobiology, National Cancer Institute, Frederick Cancer Research Facility, Frederick, MD 21701, U.S.A.
(Received 9 December 1983, accepted 30 January 1984)
We developed a reliable method for reconstituting thymectomized, lethally irradiated guinea pigs. Injection of 2.5-10x 107 syngeneic bone marrow cells into adult thymectomized, lethally irradiated guinea pigs produced survival of 46-100% of treated animals. Gentamycin sulfate (5 mg/kg of body weight) for 10 days was required for optimal results. Acidified drinking water (pH 2.5) appeared to be required for optimal results. Thymectomized, lethally irradiated, bone marrow reconstituted ('B') guinea pigs had impaired ability to develop delayed cutaneous hypersensitivity to mycobacterial antigens and cutaneous basophil hypersensitivity to keyhole limpet hemocyanin; proliferative responses to phytohemagglutinin were impaired. Key words: T cell depletion - guinea pigs
Introduction Studies of mice, depleted of T cells by adult thymectomy, lethal irradiation and b o n e m a r r o w reconstitution, have contributed to understanding of cellular i m m u n i t y (Zinkernagel, 1978). Although guinea pigs have been used in the study of delayed cutaneous hypersensitivity (Turk, 1980), a u t o i m m u n e diseases (Turk, 1980) and t u m o r i m m u n i t y (Rapp, 1973), there have been few reports of experiments in thymectomized, lethally irradiated, bone m a r r o w reconstituted animals. Our initial attempts to apply published methods ( C a m p o s - N e t o et al., 1978) for reconstitution of adult thymectomized, lethally irradiated guinea pigs were unsuccessful; between 80-100% of the guinea pigs that received 108 bone m a r r o w cells died within 14 days of irradiation. Here we report information on the influence of bone m a r r o w cell dose, route of administration and antibiotic treatment on survival after b o n e m a r r o w reconstitution. U n d e r optimal conditions 76% of guinea pigs survived. These animals i To whom reprint requests should be addressed.
222 were tested for ability to develop delayed cutaneous hypersensitivity to mycobacterial antigens and cutaneous basophil hypersensitivity reactions to keyhole limpet hemocyanin. Proliferative responses of peripheral white blood cells to phytohemagglutin were also measured.
Materials and Methods Animals
Male Sewall-Wright strain 2 guinea pigs were obtained from the Experimental Animal Breeding Facility of the National Cancer Institute, Frederick Cancer Research Facility, Frederick, MD and the Laboratory Aids Branch, Division of Research Services, National Institutes of Health. Animals, housed 3 to a cage, were given N I H guinea pig chow and drinking water, acidified by addition of 6 ml of 1 N HCI to each liter of filtered water (pH 2.5). An animal room was reserved for experiments with thymectomized, lethally irradiated, bone marrow reconstituted ('B') guinea pigs. Thymectomy
Guinea pigs were anesthetized by inhalation of methoxyflurane (Metofane; Pitman Moore, Washington Crossing, N J) and oxygen delivered by a veterinary anesthesia machine (Model 970; Ohio Medical, Madison, WI). The thymus, located between the platysma and the anterior cervical muscles, was removed with scissors and forceps. Extreme care was used to avoid damage to the jugular veins; the incision was closed with autoclips (Clay Adams, Parsippany, N J). Irradiation
Guinea pigs were treated in a Mark I irradiator (Model 68; J.L. Shepherd and Assoc., Glendale, CA) containing a 137cesium source. Animals were restrained without anesthesia in close-fitting individual 72 mm diameter plastic holders which were attached to a plastic platform. Guinea pigs received 1000 rads. at a rate of 144 rads./min (6.94 min). Animals were irradiated 2 - 9 weeks after thymectomy. Bone marrow reconstitution
Bone marrow cells were obtained from normal 7-10-week-old sex-matched, syngeneic donor guinea pigs and injected into recipients within 8 h after irradiation. In selected experiments, as indicated in the results, 33-week-old animals were used as donors. Long bones (humeri, femora and tibia) were cracked with bone cutters and marrow was flushed with Hanks' balanced salt solution (HBBS) using 50 ml syringes and 19-gauge needles. Bone marrow suspensions were forced through 60 mesh stainless steel wire screen tubes. After centrifuging at 250 x g for 10 min, the suspensions were filtered through 100 mesh stainless steel screen tubes. About 8 x 108 viable bone marrow cells were obtained from each guinea pig. Bone marrow cells were injected i.v. through the penile vein or into the heart.
223
Intracardiac injection With anesthetized guinea pigs in the supine position, 2 ml of marrow suspension was injected through a 23 gauge, 1 inch needle attached to a 3 ml syringe. The puncture was made horizontally at a point located in the left thorax about 1 cm lateral to the midline and about 3.5 cm cephalad to the tip of the xyphoid process. Death from this procedure was less than 1% (1/142). Antibiotic treatment Beginning 5 days before irradiation polymyxin B sulfate (100,000 U/liter) (Sigma Chemical Company, St. Louis, MO) dissolved in drinking water was given to guinea pigs for 16 days. Beginning on the day of irradiation, gentamycin sulfate (5 mg/kg of body weight) (Sigma Chemical Company, St. Louis, MO) dissolved in 0.9% saline was injected i.p. daily for 10 days. Evaluation of T-cell function Guinea pigs were immunized to Mycobacterium boris strain Bacillus CalmetteGubrin and to keyhole limpet hemocyanin 21-30 days after whole body irradiation. Animals were immunized i.d. with 100/~g of BCG cell walls (Ribi Immunochem, Hamilton, MT) in an oil-in-water emulsion (Ashley et al., 1980). Two to 3 weeks after immunization, delayed cutaneous hypersensitivity was evaluated by i.d. injection of 10 #g of purified protein derivative of tuberculin (PPD, Connaught Laboratories, Toronto) in Dulbecco's phosphate buffered saline (DPBS). Animals were immunized to keyhole limpet hemocyanin (Calbiochem-Behring Corp., La Jolla, CA) by i.d. injection of 200 #g of keyhole limpet hemocyanin in DPBS (Leonard et al., 1979). Seven days later, cutanous basophil hypersensitivity was evaluated by i.d. challenge with 20 #g of keyhole limpet hemocyanin. Skin reactions were measured 24 h after injection of antigen by determining the diameter of erythema and induration. In vitro lymphocyte proliferation The technique of whole blood culture of Merikanto et al. (1979) was used. Blood was collected by cardiac puncture and anticoagulated with heparin (20 U / m l of blood) (preservative-free heparin; O'Neil, Junes and Feldman, St. Louis, MO). The concentration of leukocytes was determined. Blood was diluted 1 : 1 with RPMI 1640 and 40 #1 was added per well to test plates containing 96 U-shaped wells (cat. no. 76-013-05; Linbro, Flow Laboratories, McLean, VA). PHA (50 #g/ml), diluted with RPMI 1640, was added in a volume of 50 #1; control cultures received an equal volume of RPMI 1640 without mitogen. Cultures were incubated for 3 days in a 95% air-5% CO z humid atmosphere at 37°C. Sixteen hours before harvesting, 20 #1 of RPMI containing 1 #Ci of [methyl-3H]thymidine (New England Nuclear, Boston, MA) specific activity 2.0 Ci/mmol, were added to each well. Cells were harvested on glass fiber filters using a Multiple Automated Sample Harvester (Mash II; Microbiological Associates, Bethesda, MD). Radioactivity was counted in liquid scintillation spectrophotometer (Model LS-333; Beckman Instruments, Irvine, CA). All tests were performed in triplicate. Results were expressed as stimulation index (S.I.): the
224 ratio of the mean cpm of experimental cultures to the mean cpm of control cultures. Phytohemagglutinin M (PHA) was purchased from Difco Laboratories, Detroit, MI. Results in Table IV represent the proliferative response of 5 individual guinea pigs for each group of 'B' guinea pigs and of 10 conventional guinea pigs. Statistical analysis Results were compared with Student's t-test. Results with a P > 0.05 are indicated by, NS.
Results
In preliminary experiments between 80 and 100% of thymectomized, irradiated guinea pigs that received 10 x 107 bone marrow cells died within 14 days of reconstitution. To simplify analysis, guinea pigs were lethally irradiated (but not thymectomized) and conditions were determined for survival of treated animals. Effect of bone marrow cell dose, route of bone marrow cell injection and antibiotic treatment on survival of lethally irradiated guinea pigs To determine the number of bone marrow cells required for survival of lethally irradiated guinea pigs, 1 x 108, 3 x 108 or 9 x 108 bone marrow cells were injected i.c. after 1000 rads. whole body irradiation. Animals that did not receive bone marrow cells died with sepsis, hemorrhage and dehydration between 6 and 14 days after irradiation. Injection of 1 x 108 bone marrow cells gave almost complete protection from radiation-induced death (Table I, experiment A). Higher doses of bone marrow cells resulted in faster recovery from leucopenia and weight loss (data not shown). Congdon et al. (1952) found that the intracardiac route of injection of bone marrow cells was significantly more effective than the intravenous route in promoting survival of lethally irradiated guinea pigs. In the next experiment, performed to evaluate this observation in our guinea pigs and to examine again the relationship between dose of bone marrow cells and survival, lethally irradiated guinea pigs were reconstituted with bone marrow cells (5 or 20 X 10 7) by the i.c. or i.v. routes (Table I, experiment B). The results show no differences in survival between guinea pigs that received comparable doses of bone marrow cells by the i.c. compared to the i.v. routes. A dose of 20 x 10 7 bone marrow cells was more effective in promoting survival than a dose of 5 x 107 bone marrow cells. Thymectomized, lethally irradiated, reconstituted guinea pigs may be more susceptible to the lethal effects of infection than non-thymectomized, lethally irradiated guinea pigs. Appropriate antibiotic administration may improve the survival of thymectomized, lethally irradiated, bone marrow reconstituted guinea pigs. Unfortunately, guinea pigs are susceptible to the toxic effects of many antibiotics (Wagner, 1976) and few antibiotic regimens have been studied in the guinea pig. In preliminary experiments the drug dose and schedule chosen were based on a regimen effective in improving survival of lethally irradiated, bone marrow reconstituted rats
225 TABLE I EFFECT OF BONE MARROW CELL DOSE, ROUTE OF INOCULATION AND ANTIBIOTIC TREATMENT ON SURVIVAL OF LETHALLY IRRADIATED GUINEA PIGS Experiment
Group
Bone marrow cell dose a X10 -7
Route of inoculation
Antibiotic treatment
Survivors/total no. animals treated r
A
1 2 3 4
0 10 30 90
i.c. i.c. i.c.
_ b ----
0/6 5/6 c 5/5 d 5/6 c
B
1 2 3 4 5
0 5 5 20 20
i.v. i.c. i.v. i.c.
-
0/8 3/7 4/7 e 7/7 t 6/7 g
C
1 2 3 4
0 0 10 10
_ i.c. i.c.
_~ h +
0/6 0/6 5/6 i 5/6 i
a Bone marrow cells were injected by the i.v. (intravenous) or i.c. (intracardiac) routes into lethally irradiated (1000 rads.) guinea pigs on day 0. Survivors were determined at 30 days after irradiation. b No antibiotic treatment. c Significantly different from group 1 (P < 0.01 by Fisher exact test). d Significantly different from group 1 (P < 0.001 by Fisher exact test). e Significantly different from group 1 (P < 0.05 by Fisher exact test). f Significantly different from group 2 (P < 0.05 by Fisher exact test). g Significantly different from group 1 (P < 0.005 by Fisher exact test). h Polymyxin B (100,000 U/liter) was given in the drinking water from day - 5 to day 9; gentamycin sulfate (5 mg/kg of body weight) was injected. i Significantly different from groups 1 and 3 (P < 0.01 by Fisher exact test).
(Tutschka, personal communication). This regimen, consisting of polymyxin B (100,000 U / h t e r ) , n e o m y c i n (525 m g / h t e r ) , g e n t a m y c i n (200 m g / k g o f b o d y w e i g h t ) a n d b a c t r i m (0.7 t a b l e t s / l i t e r ) p r o v e d l e t h a l to n o r m a l g u i n e a pigs ( d a t a n o t s h o w n ) . A n o t h e r a n t i b i o t i c r e g i m e n c o n s i s t i n g o f g e n t a m y c i n (200 m g / k g o f b o d y w e i g h t ) a n d t e r r a m y c i n (1.24 g / l i t e r ) also p r o v e d t o x i c ( d a t a n o t shown). T h e f o l l o w i n g e x p e r i m e n t was p e r f o r m e d to e v a l u a t e t h e s a f e t y o f a n a n t i b i o t i c r e g i m e n c o n t a i n i n g p o l y m y x i n B (100,000 U / l i t e r ) a n d g e n t a m y c i n s u l f a t e (5 m g / k g o f b o d y w e i g h t ) ( T a b l e I, e x p e r i m e n t C). L e t h a l l y i r r a d i a t e d g u i n e a pigs r e c e i v e d a n t i b i o t i c treatment alone, bone marrow reconstitution alone or combination of antibiotic treatment and bone marrow reconstitution. Antibiotic treatment alone did not i m p r o v e t h e s u r v i v a l o f i r r a d i a t e d a n i m a l s n o r d i d it i m p r o v e t h e s u r v i v a l o f i r r a d i a t e d a n i m a l s t h a t r e c e i v e d b o n e m a r r o w cells. T h e a n t i b i o t i c r e g i m e n c o n t a i n i n g p o l y m y x i n B (100,000 U / l i t e r ) a n d g e n t a m y c i n (5 m g / k g b o d y w e i g h t ) w a s t o x i c b e c a u s e t h e a v e r a g e w e i g h t g a i n in t h e g u i n e a p i g s t h a t r e c e i v e d b o n e m a r r o w cells
226 with a n t i b i o t i c was less than the weight gain in g u i n e a pigs that received b o n e m a r r o w cells without a n t i b i o t i c t r e a t m e n t ( d a t a n o t shown).
Effect of bone marrow cell dose on the survival of thymectomized, lethally irradiated guinea pigs H a v i n g e s t a b l i s h e d c o n d i t i o n s for p r o m o t i n g survival of lethally i r r a d i a t e d , n o n - t h y m e c t o m i z e d guinea pigs we e x t e n d e d the w o r k to t h y m e c t o m i z e d animals. L e t h a l l y i r r a d i a t e d , t h y m e c t o m i z e d guinea pigs were r e c o n s t i t u t e d with 108 b o n e m a r r o w cells with a n d without g e n t a m y c i n sulfate (5 m g / k g b o d y weight). A n a d d i t i o n a l g r o u p of a n i m a l s received 3 × 108 b o n e m a r r o w cells. T h e d a t a (Table II, e x p e r i m e n t A ) i n d i c a t e that g e n t a m y c i n sulfate did not i m p r o v e the survival rate after r e c o n s t i t u t i o n with 108 syngeneic b o n e m a r r o w cells. The n u m b e r of a n i m a l s (9) in each g r o u p was small. E x a m i n a t i o n of the results in a larger n u m b e r of a n i m a l s f r o m the a c c u m u l a t e d d a t a of a series of 4 e x p e r i m e n t s showed a statistically significant difference in survival b e t w e e n a n i m a l s treated with g e n t a m y c i n plus 108 b o n e m a r r o w cells ( 3 7 / 4 9 ) a n d 108 b o n e m a r r o w cells alone ( 1 8 / 3 3 ) ( P < 0.05 b y chi square analysis). Since b o n e m a r r o w cells m a y c o n t a i n a small n u m b e r of T cells ( D o e n h o f f et al., 1970), it w o u l d b e desirable to reconstitute a n i m a l s with the m i n i m a l n u m b e r of b o n e m a r r o w cells n e e d e d for survival. E x p e r i m e n t B (Table II) was p e r f o r m e d to test the effect of v a r y i n g the b o n e m a r r o w dose in t h y m e c t o m i z e d , i r r a d i a t e d guinea pigs receiving g e n t a m y c i n sulfate. R e d u c t i o n of the b o n e m a r r o w cell dose f r o m 108 to 2.5 × 107 r e d u c e d survival f r o m 80% to 46%.
TABLE II EFFECT OF BONE MARROW CELL DOSE AND GENTAMYCIN TREATMENT ON SURVIVAL OF LETHALLY IRRADIATED, THYMECTOMIZED GUINEA PIGS Experiment
A
Group
Bone marrow cell dose a x l 0 -7
Gentamycin b
1
10
-
2 3
10 30
+ -
1
2.5
+
2 3 4
5.0 7.5 10
+ + +
Survivors/total no. animals tested 5/9 8/9 8/9 7/15 10/14 14/14 c • 12/14 d
Bone marrow cells were injected 5 weeks after thymectomy in experiment A and 2 weeks after thymectomy in experiment B. b Gentamycin sulfate (5 mg/kg of body weight) was injected i.p. from day 0 to day 9 to those groups indicated with a ( + ). Survival was assessed 28 days after irradiation. ¢ Significantly different from group 1 (P < 0.005 by Fisher exact test). d Significantly different from group 1 (P < 0.05 by Fisher exact test).
2 3
1
I m m u n i z e d , nonthymectomized, irradiated, b o n e marrow reconstituted I m m u n i z e d conv. U n t r e a t e d conv.
U n t r e a t e d conv.
4
-
10
-
10 30 -
-
2.5 5.0 7.5 10 _
Bone marrow cell dose
-
-
-
0,0,8 0,2.5 9,9,11
-
0,0,0 0,0,0 0,0,0 0,0,0 10,10,11
0.001 0.001 0.001 0.001
NS < 0.01
< < < <
17,19.5,20 0,0,0
13,13, 13,14,16
0,0,0
6,6,7.5 9,11 12,14,17
15.5 0,0,0
0,0,0 0,0,9 0,0,6 0,4,6 14,15,
Diameter mm
Diameter mm
P
DCH b to PPD
Primary a response to K L H
0.001 0.02 0.01 0.01
< 0.01
< 0.01 NS
< < < <
P
-
-
13.5 0,0,0
7,7,8 9,12 9,12,
15 0,0,0
0,0,0 0,0,5 0,0,0 0,0,3 13,14,
Diameter mm
CBH c to KLH
0.001 0.01 0.001 0.001
< 0.05 NS
< < < <
P
a The initial injection of K L H elicited a n i n f l a m m a t o r y reaction; the size of the response to the p r i m a r y K L H injection was m e a s u r e d on d a y 7. b Three weeks after i m m u n i z a t i o n with BCG, 10 # g of P P D was challenged i n t r a d e r m a l l y . Twenty-four hours after P P D inoculation, skin reaction was measured. Statistical c o m p a r i s o n s were m a d e between i m m u n i z e d treated a n i m a l s a n d i m m u n i z e d c o n v e n t i o n a l animals. c One week after i m m u n i z a t i o n with K L H , 20 ~tg of K L H was injected intradermally. Twenty-four hours after K L H inoculation, skin reaction against these secondary injection sites was measured. The n u m b e r s show the d i a m e t e r s of i n d u r a t i o n and erythema. d Thymectomized, lethally irradiated a n d b o n e m a r r o w r e c o n s t i t u t e d g u i n e a pigs which were i m m u n i z e d with B C G and K L H 3 - 4 weeks after irradiation. ¢ C o n v e n t i o n a l a n i m a l s i m m u n i z e d with B C G and K L H . f U n t r e a t e d c o n v e n t i o n a l animals.
C
Immunized 'B' I m m u n i z e d ' B' I m m u n i z e d conv.
1 2 3
U n t r e a t e d conv. f
6
B
I m m u n i z e d ' B' d I m m u n i z e d ' B' Immunized 'B' I m m u n i z e d 'B' I m m u n i z e d conv. e
1 2 3 4 5
A
Animal status
Group
Experiment
EVALUATION OF DELAYED CUTANEOUS HYPERSENSITIVITY AND CUTANEOUS BASOPHIL HYPERSENSITIVITY IN CONVENTIONAL A N D 'B' G U I N E A P I G S
T A B L E III
228
Effect of age of bone marrow cell donor and "double transfer" on survival of lethally irradiated, thymectomized guinea pigs Adult thymectomized, lethally irradiated recipients received 108 bone marrow cells harvested from 7- or 33-week-old normal guinea pigs or 21-week-old 'B' guinea pigs. All animals received gentamycin 5 m g / k g body weight between day 0 and day 9 after irradiation. Animals that received bone marrow cells from 'B' animals ('double transfer') showed significantly decreased survival (2/13) when compared to thymectomized, irradiated animals that received bone marrow cells from conventional animals. There was no significant difference in efficacy of bone marrow cells from 7-week-old compared to 33-week-old donors in promoting survival of lethally irradiated thymectomized guinea pigs. Ten of 13 recipients of bone marrow from 7-week-old donors survived; 7 of 13 recipients of bone marrow from 33-week-old donors survived. T cell function in thymectomized, lethally irradiated, bone marrow reconstituted guinea pigs To determine if these 'B' guinea pigs had impaired T cell function, we measured delayed cutaneous hypersensitivity, cutaneous basophil hypersensitivity and proliferation of cells to the T cell mitogen, phytohemagglutinin (Doenhoff et al., 1970). Guinea pigs immunized with BCG CW and keyhole limpet hemocyanin were tested for DCH or CBH reactivity by injection of PPD or KLH. The results (Table III) indicate that D C H reactions to PPD and CBH reactions to K L H were reduced in thymectomized, irradiated, bone marrow reconstituted guinea pigs. 'B' guinea pigs showed either no detectable skin reactions or reactions which were smaller in diameter (Table III), paler in color and thinner than skin reactions in conventional animals (data not shown). D C H to PPD was measured in 'B' guinea pigs reconstituted with varying doses of bone marrow cells. The results suggest that bone marrow doses of 2.5 × 10 7 cells were associated with lesser D C H than bone marrow doses of 3 x 108 cells. Two thymectomized, lethally irradiated guinea pigs survived reconstitution with bone marrow obtained from 'B' guinea pigs; these animals were immunized to Mycobacterium boris strain Bacillus Calmette-Gukrin and to keyhole limpet hemocyanin; no skin reactions were detected after challenge with antigen. The ability of non-thymectomized, lethally irradiated, bone marrow reconstituted guinea pigs to develop D C H reactions to PPD was evaluated (Table III, experiment C). Non-thymectomized, lethally irradiated, bone marrow reconstituted guinea pigs developed D C H reactions to PPD. The size of the D C H reactions (13.8 + 0.5 mm) in these animals was significantly smaller than D C H reactions in conventional animals (18.8 + 0.8) but greater than the size of DCH reactions in thymectomized, lethally irradiated, bone marrow reconstituted animals (mean skin reaction size in 17 'B' guinea pigs than received 108 bone marrow cells, 5.5 + 0.8). 'B' guinea pigs had markedly reduced proliferative response to phytohemagglutin; there was no significant difference in proliferative response in guinea pigs reconstituted with different bone marrow doses (Table IV).
229 T A B L E IV PROLIFERATIVE RESPONSE TO PHYTOHEMAGGLUT1NIN OF PERIPHERAL WHITE BLOOD CELLS OBTAINED FROM 'B' AND CONVENTIONAL GUINEA PIGS Animal number
A n i m a l status
N u m b e r of b o n e m a r r o w cells transferred xl0 -7
c p m stimulus None
PHA
SI
5 839 5833 5 835 5 838 5 834 5 842 5 840 5 836 5 837 5 841
C a C C C C C C C C C
_ -
273 102 245 153 221 242 174 170 164 129
3 743 2014 3 662 7308 4266 5 990 3410 4 378 1 365 2 355
13.7 19.7 14.9 47.8 19.3 24.8 19.6 25.8 8.3 18.3
5447 5 448 5450 5 453 5 454
'B' ' B' 'B' ' B' ' B'
2.5 2.5 2.5 2.5 2.5
215 145 193 180 202
1424 425 1003 876 911
6.6 2.9 5.2 4.9 4.5
5 429 5 430 5431 5 433 5 434
' B' ' B' 'B' ' B' ' B'
5.0 5.0 5.0 5.0 5.0
199 128 87 165 169
1 260 773 177 ] 299 899
6.3 6.0 2.0 7.9 5.3
5417 5 832 5419 5 420 5421
'B' ' B' 'B' ' B' 'B'
7.5 7.5 7.5 7.5 7.5
182 83 174 181 115
929 243 1 408 730 1 132
5.1 2.9 8.1 4.0 9.8
5 895 5 896 5 403 5405 5 407
' B' 'B' ' B' 'B' ' B'
10.0 10.0 10.0 10.0 10.0
192 140 140 125 88
1 159 1 218 362 1685 246
6.0 8,7 2.6 13.5 2.8
a The white b l o o d cell c o u n t s for c o n v e n t i o n a l a n i m a l s was 10,540+ 1800; for g u i n e a pigs that received 2.5 x 107 b o n e m a r r o w cells, 4 8 6 0 + 7 2 8 ; for g u i n e a pigs that received 5 . 0 × 107 b o n e m a r r o w cells, 6 433 5:411; for g u i n e a pigs that received 7.5 x 107 b o n e m a r r o w cells, 6 580 + 1417; for g u i n e a pigs that received 10 × 107 b o n e m a r r o w cells, 8 440 _+ 1116. The proliferative responses of the g r o u p s of g u i n e a pigs that received 2 . 5 - 1 0 x 107 b o n e m a r r o w cells was significantly different from c o n v e n t i o n a l a n i m a l s P < 0.01.
230 Discussion
We have defined conditions required for reliable preparation of thymectomized, lethally irradiated, bone marrow reconstituted guinea pigs. Under optimal conditions, 76% of thymectomized, irradiated guinea pigs were successfully reconstituted with syngeneic bone marrow cells. Several factors were essential in promoting survival: (1) appropriate antibiotic and antibiotic dose, (2) adequate dose of bone marrow cells, and (3) acidified drinking water. Several antibiotic regimens were evaluated for toxicity in the guinea pig. Multidrug regimens containing polymyxin B, neomycin, bactrim and gentamycin proved to be toxic. Gentamycin (5 mg/kg body weight) i.p. for 10 days proved to be safe and effective in promoting survival of thymectomized, lethally irradiated, bone marrow reconstituted guinea pigs. Seventysix percent of gentamycin-treated, thymectomized guinea pigs survived lethal irradiation after reconstitution with 108 bone marrow cells; without gentamycin treatment the survival percentage dropped to 57%. Dose response studies performed in thymectomized, lethally irradiated guinea pigs treated with gentamycin showed that 2.5 X 107 bone marrow ceils led to survival of 7 of 15 treated guinea pigs. Using Pegg's (1966) figure for the total number of bone marrow cells per guinea pig (16.0 x 10 9 cells), 0.2-0.6% of total bone marrow cells was required to reconstitute a thymectomized, irradiated, gentamycin-treated guinea pig. Acidified drinking water was not studied as an independent variable. However, in preliminary experiments, mortality was 80-100% when thymectomized, irradiated animals receiving non-acidified drinking water were reconstituted with 108 bone marrow cells. Delayed cutaneous hypersensitivity reactions to PPD and cutaneous basophil hypersensitivity reactions to KLH were decreased or absent in 'B' guinea pigs; proliferative responses to phytohemagglutinin were reduced in 'B' guinea pigs. The stimulation index in treated animals was 2.6-13.5 compared to 8.3-47.8 in conventional animals. Possibly the reduced proliferative response of leukocytes from 'B' animals reflects the lower white blood cell counts of these animals. Against this possibility is the observation that 5 of 30 untreated normal guinea pigs with white blood cell counts between 3000-5000/mm 3 had stimulation indices ranging from 14.6 to 91.6. The demonstration that 'B' animals had detectable DCH and CBH reactions and proliferative responses to PHA indicates that T cell functions were not entirely eliminated in some 'B' guinea pigs. There are 2 sources of T ceils in these pigs: the bone marrow used for reconstitution and accessory portions of the thymus not removed surgically. The proportion of T cells in adult murine bone marrow has been estimated to be 2 to 4% (Komuro and Boyse, 1973). Comparable figures for the number of T cells in guinea pig bone marrow are not available. Parks (1917) reported that because of the presence of accessory thymus nodules, some in association with the parathyroid gland, it was not possible to remove the thymus entirely from guinea pigs. We have described a method for preparing T cell depleted guinea pigs. These guinea pigs vary in degree of functional T cell depletion. Experiments in progress indicate that these animals may be useful in studying tumor rejection.
231
Acknowledgements T h e a u t h o r s t h a n k J o h n N . H a t g i for e x c e l l e n t a n i m a l care, K e n G r e e n for t e c h n i c a l assistance, a n d Dr. R. R u s s e l l for a d v i c e o n a n t i b i o t i c t h e r a p y in g u i n e a pigs.
References Ashley, M.P., B. Zbar, J.T. Hunter, H.J. Rapp and T. Sugimoto, 1980, Cancer Res. 40, 4197. Campos-Neto, A., H. Levine and S.F. Schlossman, 1978, J. Immunol. 121, 2235. Congdon, C.C., D. Uphoff and E. Lorenz, 1952, J. Natl. Cancer Inst. 13, 73. Doenhoff, M.J., A.J.S. Davies, E. Leuchars and V. Wallis, 1970, Proc. Roy. Soc. Lond. B. 176, 69. Komuro, K. and E.A. Boyse, 1973, J. Exp. Med. 138, 479. Leonard, E.J., M.A. Lett-Brown and P.W. Askenase, 1979, Int. Arch. Allergy Appl. Immunol. 58, 460. Merikanto, J., E. Soppi and O. Ruuskanen, 1979, Cell. Immunol. 47, 227. Parks, E.A., 1917, J. Exp. Med. 25, 129. Pegg, D.E., 1966, in: Bone Marrow Transplantation, ed. D.E. Pegg (Lloyd-Luke Medical Books, London) p. 41. Rapp, H.J., 1973, lsr. J. Med. Sci. 9, 366. Turk, J.L., 1980, Delayed Hypersensitivity (Elsevier, Amsterdam). Wagner, J.E., 1976, in: The Biology of the Guinea Pig, eds. J.E. Wagner and P.J. Manning (Academic Press, New York) p. 229. Zinkernagel, R.M., 1978, Immunol. Rev. 42, 224.
221
© 1984 Elsevier JIM03084
A Reliable Method for Reconstituting Thymectomized, Lethally Irradiated Guinea Pigs with Bone Marrow Cells N. Terata, Y. Tanio and B. Zbar 1 Laboratory of Immunobiology, National Cancer Institute, Frederick Cancer Research Facility, Frederick, MD 21701, U.S.A.
(Received 9 December 1983, accepted 30 January 1984)
We developed a reliable method for reconstituting thymectomized, lethally irradiated guinea pigs. Injection of 2.5-10x 107 syngeneic bone marrow cells into adult thymectomized, lethally irradiated guinea pigs produced survival of 46-100% of treated animals. Gentamycin sulfate (5 mg/kg of body weight) for 10 days was required for optimal results. Acidified drinking water (pH 2.5) appeared to be required for optimal results. Thymectomized, lethally irradiated, bone marrow reconstituted ('B') guinea pigs had impaired ability to develop delayed cutaneous hypersensitivity to mycobacterial antigens and cutaneous basophil hypersensitivity to keyhole limpet hemocyanin; proliferative responses to phytohemagglutinin were impaired. Key words: T cell depletion - guinea pigs
Introduction Studies of mice, depleted of T cells by adult thymectomy, lethal irradiation and b o n e m a r r o w reconstitution, have contributed to understanding of cellular i m m u n i t y (Zinkernagel, 1978). Although guinea pigs have been used in the study of delayed cutaneous hypersensitivity (Turk, 1980), a u t o i m m u n e diseases (Turk, 1980) and t u m o r i m m u n i t y (Rapp, 1973), there have been few reports of experiments in thymectomized, lethally irradiated, bone m a r r o w reconstituted animals. Our initial attempts to apply published methods ( C a m p o s - N e t o et al., 1978) for reconstitution of adult thymectomized, lethally irradiated guinea pigs were unsuccessful; between 80-100% of the guinea pigs that received 108 bone m a r r o w cells died within 14 days of irradiation. Here we report information on the influence of bone m a r r o w cell dose, route of administration and antibiotic treatment on survival after b o n e m a r r o w reconstitution. U n d e r optimal conditions 76% of guinea pigs survived. These animals i To whom reprint requests should be addressed.
222 were tested for ability to develop delayed cutaneous hypersensitivity to mycobacterial antigens and cutaneous basophil hypersensitivity reactions to keyhole limpet hemocyanin. Proliferative responses of peripheral white blood cells to phytohemagglutin were also measured.
Materials and Methods Animals
Male Sewall-Wright strain 2 guinea pigs were obtained from the Experimental Animal Breeding Facility of the National Cancer Institute, Frederick Cancer Research Facility, Frederick, MD and the Laboratory Aids Branch, Division of Research Services, National Institutes of Health. Animals, housed 3 to a cage, were given N I H guinea pig chow and drinking water, acidified by addition of 6 ml of 1 N HCI to each liter of filtered water (pH 2.5). An animal room was reserved for experiments with thymectomized, lethally irradiated, bone marrow reconstituted ('B') guinea pigs. Thymectomy
Guinea pigs were anesthetized by inhalation of methoxyflurane (Metofane; Pitman Moore, Washington Crossing, N J) and oxygen delivered by a veterinary anesthesia machine (Model 970; Ohio Medical, Madison, WI). The thymus, located between the platysma and the anterior cervical muscles, was removed with scissors and forceps. Extreme care was used to avoid damage to the jugular veins; the incision was closed with autoclips (Clay Adams, Parsippany, N J). Irradiation
Guinea pigs were treated in a Mark I irradiator (Model 68; J.L. Shepherd and Assoc., Glendale, CA) containing a 137cesium source. Animals were restrained without anesthesia in close-fitting individual 72 mm diameter plastic holders which were attached to a plastic platform. Guinea pigs received 1000 rads. at a rate of 144 rads./min (6.94 min). Animals were irradiated 2 - 9 weeks after thymectomy. Bone marrow reconstitution
Bone marrow cells were obtained from normal 7-10-week-old sex-matched, syngeneic donor guinea pigs and injected into recipients within 8 h after irradiation. In selected experiments, as indicated in the results, 33-week-old animals were used as donors. Long bones (humeri, femora and tibia) were cracked with bone cutters and marrow was flushed with Hanks' balanced salt solution (HBBS) using 50 ml syringes and 19-gauge needles. Bone marrow suspensions were forced through 60 mesh stainless steel wire screen tubes. After centrifuging at 250 x g for 10 min, the suspensions were filtered through 100 mesh stainless steel screen tubes. About 8 x 108 viable bone marrow cells were obtained from each guinea pig. Bone marrow cells were injected i.v. through the penile vein or into the heart.
223
Intracardiac injection With anesthetized guinea pigs in the supine position, 2 ml of marrow suspension was injected through a 23 gauge, 1 inch needle attached to a 3 ml syringe. The puncture was made horizontally at a point located in the left thorax about 1 cm lateral to the midline and about 3.5 cm cephalad to the tip of the xyphoid process. Death from this procedure was less than 1% (1/142). Antibiotic treatment Beginning 5 days before irradiation polymyxin B sulfate (100,000 U/liter) (Sigma Chemical Company, St. Louis, MO) dissolved in drinking water was given to guinea pigs for 16 days. Beginning on the day of irradiation, gentamycin sulfate (5 mg/kg of body weight) (Sigma Chemical Company, St. Louis, MO) dissolved in 0.9% saline was injected i.p. daily for 10 days. Evaluation of T-cell function Guinea pigs were immunized to Mycobacterium boris strain Bacillus CalmetteGubrin and to keyhole limpet hemocyanin 21-30 days after whole body irradiation. Animals were immunized i.d. with 100/~g of BCG cell walls (Ribi Immunochem, Hamilton, MT) in an oil-in-water emulsion (Ashley et al., 1980). Two to 3 weeks after immunization, delayed cutaneous hypersensitivity was evaluated by i.d. injection of 10 #g of purified protein derivative of tuberculin (PPD, Connaught Laboratories, Toronto) in Dulbecco's phosphate buffered saline (DPBS). Animals were immunized to keyhole limpet hemocyanin (Calbiochem-Behring Corp., La Jolla, CA) by i.d. injection of 200 #g of keyhole limpet hemocyanin in DPBS (Leonard et al., 1979). Seven days later, cutanous basophil hypersensitivity was evaluated by i.d. challenge with 20 #g of keyhole limpet hemocyanin. Skin reactions were measured 24 h after injection of antigen by determining the diameter of erythema and induration. In vitro lymphocyte proliferation The technique of whole blood culture of Merikanto et al. (1979) was used. Blood was collected by cardiac puncture and anticoagulated with heparin (20 U / m l of blood) (preservative-free heparin; O'Neil, Junes and Feldman, St. Louis, MO). The concentration of leukocytes was determined. Blood was diluted 1 : 1 with RPMI 1640 and 40 #1 was added per well to test plates containing 96 U-shaped wells (cat. no. 76-013-05; Linbro, Flow Laboratories, McLean, VA). PHA (50 #g/ml), diluted with RPMI 1640, was added in a volume of 50 #1; control cultures received an equal volume of RPMI 1640 without mitogen. Cultures were incubated for 3 days in a 95% air-5% CO z humid atmosphere at 37°C. Sixteen hours before harvesting, 20 #1 of RPMI containing 1 #Ci of [methyl-3H]thymidine (New England Nuclear, Boston, MA) specific activity 2.0 Ci/mmol, were added to each well. Cells were harvested on glass fiber filters using a Multiple Automated Sample Harvester (Mash II; Microbiological Associates, Bethesda, MD). Radioactivity was counted in liquid scintillation spectrophotometer (Model LS-333; Beckman Instruments, Irvine, CA). All tests were performed in triplicate. Results were expressed as stimulation index (S.I.): the
224 ratio of the mean cpm of experimental cultures to the mean cpm of control cultures. Phytohemagglutinin M (PHA) was purchased from Difco Laboratories, Detroit, MI. Results in Table IV represent the proliferative response of 5 individual guinea pigs for each group of 'B' guinea pigs and of 10 conventional guinea pigs. Statistical analysis Results were compared with Student's t-test. Results with a P > 0.05 are indicated by, NS.
Results
In preliminary experiments between 80 and 100% of thymectomized, irradiated guinea pigs that received 10 x 107 bone marrow cells died within 14 days of reconstitution. To simplify analysis, guinea pigs were lethally irradiated (but not thymectomized) and conditions were determined for survival of treated animals. Effect of bone marrow cell dose, route of bone marrow cell injection and antibiotic treatment on survival of lethally irradiated guinea pigs To determine the number of bone marrow cells required for survival of lethally irradiated guinea pigs, 1 x 108, 3 x 108 or 9 x 108 bone marrow cells were injected i.c. after 1000 rads. whole body irradiation. Animals that did not receive bone marrow cells died with sepsis, hemorrhage and dehydration between 6 and 14 days after irradiation. Injection of 1 x 108 bone marrow cells gave almost complete protection from radiation-induced death (Table I, experiment A). Higher doses of bone marrow cells resulted in faster recovery from leucopenia and weight loss (data not shown). Congdon et al. (1952) found that the intracardiac route of injection of bone marrow cells was significantly more effective than the intravenous route in promoting survival of lethally irradiated guinea pigs. In the next experiment, performed to evaluate this observation in our guinea pigs and to examine again the relationship between dose of bone marrow cells and survival, lethally irradiated guinea pigs were reconstituted with bone marrow cells (5 or 20 X 10 7) by the i.c. or i.v. routes (Table I, experiment B). The results show no differences in survival between guinea pigs that received comparable doses of bone marrow cells by the i.c. compared to the i.v. routes. A dose of 20 x 10 7 bone marrow cells was more effective in promoting survival than a dose of 5 x 107 bone marrow cells. Thymectomized, lethally irradiated, reconstituted guinea pigs may be more susceptible to the lethal effects of infection than non-thymectomized, lethally irradiated guinea pigs. Appropriate antibiotic administration may improve the survival of thymectomized, lethally irradiated, bone marrow reconstituted guinea pigs. Unfortunately, guinea pigs are susceptible to the toxic effects of many antibiotics (Wagner, 1976) and few antibiotic regimens have been studied in the guinea pig. In preliminary experiments the drug dose and schedule chosen were based on a regimen effective in improving survival of lethally irradiated, bone marrow reconstituted rats
225 TABLE I EFFECT OF BONE MARROW CELL DOSE, ROUTE OF INOCULATION AND ANTIBIOTIC TREATMENT ON SURVIVAL OF LETHALLY IRRADIATED GUINEA PIGS Experiment
Group
Bone marrow cell dose a X10 -7
Route of inoculation
Antibiotic treatment
Survivors/total no. animals treated r
A
1 2 3 4
0 10 30 90
i.c. i.c. i.c.
_ b ----
0/6 5/6 c 5/5 d 5/6 c
B
1 2 3 4 5
0 5 5 20 20
i.v. i.c. i.v. i.c.
-
0/8 3/7 4/7 e 7/7 t 6/7 g
C
1 2 3 4
0 0 10 10
_ i.c. i.c.
_~ h +
0/6 0/6 5/6 i 5/6 i
a Bone marrow cells were injected by the i.v. (intravenous) or i.c. (intracardiac) routes into lethally irradiated (1000 rads.) guinea pigs on day 0. Survivors were determined at 30 days after irradiation. b No antibiotic treatment. c Significantly different from group 1 (P < 0.01 by Fisher exact test). d Significantly different from group 1 (P < 0.001 by Fisher exact test). e Significantly different from group 1 (P < 0.05 by Fisher exact test). f Significantly different from group 2 (P < 0.05 by Fisher exact test). g Significantly different from group 1 (P < 0.005 by Fisher exact test). h Polymyxin B (100,000 U/liter) was given in the drinking water from day - 5 to day 9; gentamycin sulfate (5 mg/kg of body weight) was injected. i Significantly different from groups 1 and 3 (P < 0.01 by Fisher exact test).
(Tutschka, personal communication). This regimen, consisting of polymyxin B (100,000 U / h t e r ) , n e o m y c i n (525 m g / h t e r ) , g e n t a m y c i n (200 m g / k g o f b o d y w e i g h t ) a n d b a c t r i m (0.7 t a b l e t s / l i t e r ) p r o v e d l e t h a l to n o r m a l g u i n e a pigs ( d a t a n o t s h o w n ) . A n o t h e r a n t i b i o t i c r e g i m e n c o n s i s t i n g o f g e n t a m y c i n (200 m g / k g o f b o d y w e i g h t ) a n d t e r r a m y c i n (1.24 g / l i t e r ) also p r o v e d t o x i c ( d a t a n o t shown). T h e f o l l o w i n g e x p e r i m e n t was p e r f o r m e d to e v a l u a t e t h e s a f e t y o f a n a n t i b i o t i c r e g i m e n c o n t a i n i n g p o l y m y x i n B (100,000 U / l i t e r ) a n d g e n t a m y c i n s u l f a t e (5 m g / k g o f b o d y w e i g h t ) ( T a b l e I, e x p e r i m e n t C). L e t h a l l y i r r a d i a t e d g u i n e a pigs r e c e i v e d a n t i b i o t i c treatment alone, bone marrow reconstitution alone or combination of antibiotic treatment and bone marrow reconstitution. Antibiotic treatment alone did not i m p r o v e t h e s u r v i v a l o f i r r a d i a t e d a n i m a l s n o r d i d it i m p r o v e t h e s u r v i v a l o f i r r a d i a t e d a n i m a l s t h a t r e c e i v e d b o n e m a r r o w cells. T h e a n t i b i o t i c r e g i m e n c o n t a i n i n g p o l y m y x i n B (100,000 U / l i t e r ) a n d g e n t a m y c i n (5 m g / k g b o d y w e i g h t ) w a s t o x i c b e c a u s e t h e a v e r a g e w e i g h t g a i n in t h e g u i n e a p i g s t h a t r e c e i v e d b o n e m a r r o w cells
226 with a n t i b i o t i c was less than the weight gain in g u i n e a pigs that received b o n e m a r r o w cells without a n t i b i o t i c t r e a t m e n t ( d a t a n o t shown).
Effect of bone marrow cell dose on the survival of thymectomized, lethally irradiated guinea pigs H a v i n g e s t a b l i s h e d c o n d i t i o n s for p r o m o t i n g survival of lethally i r r a d i a t e d , n o n - t h y m e c t o m i z e d guinea pigs we e x t e n d e d the w o r k to t h y m e c t o m i z e d animals. L e t h a l l y i r r a d i a t e d , t h y m e c t o m i z e d guinea pigs were r e c o n s t i t u t e d with 108 b o n e m a r r o w cells with a n d without g e n t a m y c i n sulfate (5 m g / k g b o d y weight). A n a d d i t i o n a l g r o u p of a n i m a l s received 3 × 108 b o n e m a r r o w cells. T h e d a t a (Table II, e x p e r i m e n t A ) i n d i c a t e that g e n t a m y c i n sulfate did not i m p r o v e the survival rate after r e c o n s t i t u t i o n with 108 syngeneic b o n e m a r r o w cells. The n u m b e r of a n i m a l s (9) in each g r o u p was small. E x a m i n a t i o n of the results in a larger n u m b e r of a n i m a l s f r o m the a c c u m u l a t e d d a t a of a series of 4 e x p e r i m e n t s showed a statistically significant difference in survival b e t w e e n a n i m a l s treated with g e n t a m y c i n plus 108 b o n e m a r r o w cells ( 3 7 / 4 9 ) a n d 108 b o n e m a r r o w cells alone ( 1 8 / 3 3 ) ( P < 0.05 b y chi square analysis). Since b o n e m a r r o w cells m a y c o n t a i n a small n u m b e r of T cells ( D o e n h o f f et al., 1970), it w o u l d b e desirable to reconstitute a n i m a l s with the m i n i m a l n u m b e r of b o n e m a r r o w cells n e e d e d for survival. E x p e r i m e n t B (Table II) was p e r f o r m e d to test the effect of v a r y i n g the b o n e m a r r o w dose in t h y m e c t o m i z e d , i r r a d i a t e d guinea pigs receiving g e n t a m y c i n sulfate. R e d u c t i o n of the b o n e m a r r o w cell dose f r o m 108 to 2.5 × 107 r e d u c e d survival f r o m 80% to 46%.
TABLE II EFFECT OF BONE MARROW CELL DOSE AND GENTAMYCIN TREATMENT ON SURVIVAL OF LETHALLY IRRADIATED, THYMECTOMIZED GUINEA PIGS Experiment
A
Group
Bone marrow cell dose a x l 0 -7
Gentamycin b
1
10
-
2 3
10 30
+ -
1
2.5
+
2 3 4
5.0 7.5 10
+ + +
Survivors/total no. animals tested 5/9 8/9 8/9 7/15 10/14 14/14 c • 12/14 d
Bone marrow cells were injected 5 weeks after thymectomy in experiment A and 2 weeks after thymectomy in experiment B. b Gentamycin sulfate (5 mg/kg of body weight) was injected i.p. from day 0 to day 9 to those groups indicated with a ( + ). Survival was assessed 28 days after irradiation. ¢ Significantly different from group 1 (P < 0.005 by Fisher exact test). d Significantly different from group 1 (P < 0.05 by Fisher exact test).
2 3
1
I m m u n i z e d , nonthymectomized, irradiated, b o n e marrow reconstituted I m m u n i z e d conv. U n t r e a t e d conv.
U n t r e a t e d conv.
4
-
10
-
10 30 -
-
2.5 5.0 7.5 10 _
Bone marrow cell dose
-
-
-
0,0,8 0,2.5 9,9,11
-
0,0,0 0,0,0 0,0,0 0,0,0 10,10,11
0.001 0.001 0.001 0.001
NS < 0.01
< < < <
17,19.5,20 0,0,0
13,13, 13,14,16
0,0,0
6,6,7.5 9,11 12,14,17
15.5 0,0,0
0,0,0 0,0,9 0,0,6 0,4,6 14,15,
Diameter mm
Diameter mm
P
DCH b to PPD
Primary a response to K L H
0.001 0.02 0.01 0.01
< 0.01
< 0.01 NS
< < < <
P
-
-
13.5 0,0,0
7,7,8 9,12 9,12,
15 0,0,0
0,0,0 0,0,5 0,0,0 0,0,3 13,14,
Diameter mm
CBH c to KLH
0.001 0.01 0.001 0.001
< 0.05 NS
< < < <
P
a The initial injection of K L H elicited a n i n f l a m m a t o r y reaction; the size of the response to the p r i m a r y K L H injection was m e a s u r e d on d a y 7. b Three weeks after i m m u n i z a t i o n with BCG, 10 # g of P P D was challenged i n t r a d e r m a l l y . Twenty-four hours after P P D inoculation, skin reaction was measured. Statistical c o m p a r i s o n s were m a d e between i m m u n i z e d treated a n i m a l s a n d i m m u n i z e d c o n v e n t i o n a l animals. c One week after i m m u n i z a t i o n with K L H , 20 ~tg of K L H was injected intradermally. Twenty-four hours after K L H inoculation, skin reaction against these secondary injection sites was measured. The n u m b e r s show the d i a m e t e r s of i n d u r a t i o n and erythema. d Thymectomized, lethally irradiated a n d b o n e m a r r o w r e c o n s t i t u t e d g u i n e a pigs which were i m m u n i z e d with B C G and K L H 3 - 4 weeks after irradiation. ¢ C o n v e n t i o n a l a n i m a l s i m m u n i z e d with B C G and K L H . f U n t r e a t e d c o n v e n t i o n a l animals.
C
Immunized 'B' I m m u n i z e d ' B' I m m u n i z e d conv.
1 2 3
U n t r e a t e d conv. f
6
B
I m m u n i z e d ' B' d I m m u n i z e d ' B' Immunized 'B' I m m u n i z e d 'B' I m m u n i z e d conv. e
1 2 3 4 5
A
Animal status
Group
Experiment
EVALUATION OF DELAYED CUTANEOUS HYPERSENSITIVITY AND CUTANEOUS BASOPHIL HYPERSENSITIVITY IN CONVENTIONAL A N D 'B' G U I N E A P I G S
T A B L E III
228
Effect of age of bone marrow cell donor and "double transfer" on survival of lethally irradiated, thymectomized guinea pigs Adult thymectomized, lethally irradiated recipients received 108 bone marrow cells harvested from 7- or 33-week-old normal guinea pigs or 21-week-old 'B' guinea pigs. All animals received gentamycin 5 m g / k g body weight between day 0 and day 9 after irradiation. Animals that received bone marrow cells from 'B' animals ('double transfer') showed significantly decreased survival (2/13) when compared to thymectomized, irradiated animals that received bone marrow cells from conventional animals. There was no significant difference in efficacy of bone marrow cells from 7-week-old compared to 33-week-old donors in promoting survival of lethally irradiated thymectomized guinea pigs. Ten of 13 recipients of bone marrow from 7-week-old donors survived; 7 of 13 recipients of bone marrow from 33-week-old donors survived. T cell function in thymectomized, lethally irradiated, bone marrow reconstituted guinea pigs To determine if these 'B' guinea pigs had impaired T cell function, we measured delayed cutaneous hypersensitivity, cutaneous basophil hypersensitivity and proliferation of cells to the T cell mitogen, phytohemagglutinin (Doenhoff et al., 1970). Guinea pigs immunized with BCG CW and keyhole limpet hemocyanin were tested for DCH or CBH reactivity by injection of PPD or KLH. The results (Table III) indicate that D C H reactions to PPD and CBH reactions to K L H were reduced in thymectomized, irradiated, bone marrow reconstituted guinea pigs. 'B' guinea pigs showed either no detectable skin reactions or reactions which were smaller in diameter (Table III), paler in color and thinner than skin reactions in conventional animals (data not shown). D C H to PPD was measured in 'B' guinea pigs reconstituted with varying doses of bone marrow cells. The results suggest that bone marrow doses of 2.5 × 10 7 cells were associated with lesser D C H than bone marrow doses of 3 x 108 cells. Two thymectomized, lethally irradiated guinea pigs survived reconstitution with bone marrow obtained from 'B' guinea pigs; these animals were immunized to Mycobacterium boris strain Bacillus Calmette-Gukrin and to keyhole limpet hemocyanin; no skin reactions were detected after challenge with antigen. The ability of non-thymectomized, lethally irradiated, bone marrow reconstituted guinea pigs to develop D C H reactions to PPD was evaluated (Table III, experiment C). Non-thymectomized, lethally irradiated, bone marrow reconstituted guinea pigs developed D C H reactions to PPD. The size of the D C H reactions (13.8 + 0.5 mm) in these animals was significantly smaller than D C H reactions in conventional animals (18.8 + 0.8) but greater than the size of DCH reactions in thymectomized, lethally irradiated, bone marrow reconstituted animals (mean skin reaction size in 17 'B' guinea pigs than received 108 bone marrow cells, 5.5 + 0.8). 'B' guinea pigs had markedly reduced proliferative response to phytohemagglutin; there was no significant difference in proliferative response in guinea pigs reconstituted with different bone marrow doses (Table IV).
229 T A B L E IV PROLIFERATIVE RESPONSE TO PHYTOHEMAGGLUT1NIN OF PERIPHERAL WHITE BLOOD CELLS OBTAINED FROM 'B' AND CONVENTIONAL GUINEA PIGS Animal number
A n i m a l status
N u m b e r of b o n e m a r r o w cells transferred xl0 -7
c p m stimulus None
PHA
SI
5 839 5833 5 835 5 838 5 834 5 842 5 840 5 836 5 837 5 841
C a C C C C C C C C C
_ -
273 102 245 153 221 242 174 170 164 129
3 743 2014 3 662 7308 4266 5 990 3410 4 378 1 365 2 355
13.7 19.7 14.9 47.8 19.3 24.8 19.6 25.8 8.3 18.3
5447 5 448 5450 5 453 5 454
'B' ' B' 'B' ' B' ' B'
2.5 2.5 2.5 2.5 2.5
215 145 193 180 202
1424 425 1003 876 911
6.6 2.9 5.2 4.9 4.5
5 429 5 430 5431 5 433 5 434
' B' ' B' 'B' ' B' ' B'
5.0 5.0 5.0 5.0 5.0
199 128 87 165 169
1 260 773 177 ] 299 899
6.3 6.0 2.0 7.9 5.3
5417 5 832 5419 5 420 5421
'B' ' B' 'B' ' B' 'B'
7.5 7.5 7.5 7.5 7.5
182 83 174 181 115
929 243 1 408 730 1 132
5.1 2.9 8.1 4.0 9.8
5 895 5 896 5 403 5405 5 407
' B' 'B' ' B' 'B' ' B'
10.0 10.0 10.0 10.0 10.0
192 140 140 125 88
1 159 1 218 362 1685 246
6.0 8,7 2.6 13.5 2.8
a The white b l o o d cell c o u n t s for c o n v e n t i o n a l a n i m a l s was 10,540+ 1800; for g u i n e a pigs that received 2.5 x 107 b o n e m a r r o w cells, 4 8 6 0 + 7 2 8 ; for g u i n e a pigs that received 5 . 0 × 107 b o n e m a r r o w cells, 6 433 5:411; for g u i n e a pigs that received 7.5 x 107 b o n e m a r r o w cells, 6 580 + 1417; for g u i n e a pigs that received 10 × 107 b o n e m a r r o w cells, 8 440 _+ 1116. The proliferative responses of the g r o u p s of g u i n e a pigs that received 2 . 5 - 1 0 x 107 b o n e m a r r o w cells was significantly different from c o n v e n t i o n a l a n i m a l s P < 0.01.
230 Discussion
We have defined conditions required for reliable preparation of thymectomized, lethally irradiated, bone marrow reconstituted guinea pigs. Under optimal conditions, 76% of thymectomized, irradiated guinea pigs were successfully reconstituted with syngeneic bone marrow cells. Several factors were essential in promoting survival: (1) appropriate antibiotic and antibiotic dose, (2) adequate dose of bone marrow cells, and (3) acidified drinking water. Several antibiotic regimens were evaluated for toxicity in the guinea pig. Multidrug regimens containing polymyxin B, neomycin, bactrim and gentamycin proved to be toxic. Gentamycin (5 mg/kg body weight) i.p. for 10 days proved to be safe and effective in promoting survival of thymectomized, lethally irradiated, bone marrow reconstituted guinea pigs. Seventysix percent of gentamycin-treated, thymectomized guinea pigs survived lethal irradiation after reconstitution with 108 bone marrow cells; without gentamycin treatment the survival percentage dropped to 57%. Dose response studies performed in thymectomized, lethally irradiated guinea pigs treated with gentamycin showed that 2.5 X 107 bone marrow ceils led to survival of 7 of 15 treated guinea pigs. Using Pegg's (1966) figure for the total number of bone marrow cells per guinea pig (16.0 x 10 9 cells), 0.2-0.6% of total bone marrow cells was required to reconstitute a thymectomized, irradiated, gentamycin-treated guinea pig. Acidified drinking water was not studied as an independent variable. However, in preliminary experiments, mortality was 80-100% when thymectomized, irradiated animals receiving non-acidified drinking water were reconstituted with 108 bone marrow cells. Delayed cutaneous hypersensitivity reactions to PPD and cutaneous basophil hypersensitivity reactions to KLH were decreased or absent in 'B' guinea pigs; proliferative responses to phytohemagglutinin were reduced in 'B' guinea pigs. The stimulation index in treated animals was 2.6-13.5 compared to 8.3-47.8 in conventional animals. Possibly the reduced proliferative response of leukocytes from 'B' animals reflects the lower white blood cell counts of these animals. Against this possibility is the observation that 5 of 30 untreated normal guinea pigs with white blood cell counts between 3000-5000/mm 3 had stimulation indices ranging from 14.6 to 91.6. The demonstration that 'B' animals had detectable DCH and CBH reactions and proliferative responses to PHA indicates that T cell functions were not entirely eliminated in some 'B' guinea pigs. There are 2 sources of T ceils in these pigs: the bone marrow used for reconstitution and accessory portions of the thymus not removed surgically. The proportion of T cells in adult murine bone marrow has been estimated to be 2 to 4% (Komuro and Boyse, 1973). Comparable figures for the number of T cells in guinea pig bone marrow are not available. Parks (1917) reported that because of the presence of accessory thymus nodules, some in association with the parathyroid gland, it was not possible to remove the thymus entirely from guinea pigs. We have described a method for preparing T cell depleted guinea pigs. These guinea pigs vary in degree of functional T cell depletion. Experiments in progress indicate that these animals may be useful in studying tumor rejection.
231
Acknowledgements T h e a u t h o r s t h a n k J o h n N . H a t g i for e x c e l l e n t a n i m a l care, K e n G r e e n for t e c h n i c a l assistance, a n d Dr. R. R u s s e l l for a d v i c e o n a n t i b i o t i c t h e r a p y in g u i n e a pigs.
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