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Studies on the local graft versus host reactivity of normal and chronic lymphocytic leukemia lymphocytes
CLINICAL
IMMUNOLOGY
AND
IMMUNOPATHOLOGY
7, 24-35 (1977)
Studies on the Local Graft versus Host Reactivity Normal and Chronic Lymphocytic Leukemia Lymphocytes
of
I. A Comparison of Assays in Rat Kidney and Muscle VILMA D. MOTTIRONI New
York
State
Kidney
AND ANN E. GABRIELSEX
Disease Institute’ and the Department qf Pediatrics, Medical College, Albany. Nen, York 12208
A/ban>
Received May 3. 1976 Many methods have been used to identify T and B cells and their subtypes in terms of both physical attributes and function in a range of in vitro tests. The present report deals with an in viva approach to the character of circulating lymphocytes from 10 patients with chronic lymphocytic leukemia (CLL) as compared to lymphoid cells from normal donors, using the Elkins graft versus host reaction (GVHR) assay in kidney and an assay involving injection of lymphoid cells in the external oblique abdominal muscle of immunosuppressed rats. GVHR in the muscle was assessed by (a) measuring diameter of the lesion, (b) examining histology of the injected site. and (cl determining uptake of [‘=I]deoxyuridine into DNA of proliferating cells. Results show that lymphocytes from normal donors elicit a GVHR in both kidney and muscle, producing characteristic invasion and destruction of the injected tissue, while CLL lymphocytes, even in large numbers, produce no response by any of the criteria in either tissue. CLL cells remain localized at the injection site without undergoing the changes of activation and proliferation. Results in the muscle parallel those in the kidney. The muscle assay requires fewer cells and permits a larger number of assessments per animal. Potentially it can be applied to classification of lymphoreticular disorders and immunodeficiency states.
INTRODUCTION
The lymphoid cells active in the immune system consist of two antigenically and functionally distinct lymphocyte populations: thymus derived cells (T cells) and bone marrow derived cells (B cells) which can be distinguished by their different membrane properties (1). Based on the study of various surface markers on human lymphocytes it has now become possible to define some diseases of the lymphoid system in terms of T or B cell proliferation. Chronic lymphocytic leukemia (CLL) has been studied extensively and been identified as a B cell malignancy (2-6). Phytohemagglutinin (PHA)-induced lymphocyte stimulation and mixed leukocyte culture (MLC) responses are parameters for the presence of functional normal T cell populations. In CLL the PHA response is depressed and delayed (7- 1 I) and the responding capacity of these cells in MLC is markedly diminished (12). It is not clear if this is due to impaired T lymphocyte function or because the T cells are diluted by large numbers of B lymphocytes. Nevertheless, the PHA
r A unit of the New York State Department of Health. 24 Copyright All rights
@ 1977 by Academic Press, Inc of reproduction in any form reserved.
LOCAL
GVHR
ASSAYS
IN
RAT
KIDNEY
AND
MUSCLE
25
and MLC reactivity in these in vitro tests does not necessarily reflect the ability of peripheral lymphocytes to respond in vivo. Induction of graft-versus-host reactions (GVHR) has been considered to reflect immunocompetence of donor T cells (13). Recent work suggests that T cells are essential for the production of a GVHR (14). This phenomenon was tested in a model of local GVHR. If rats are immunosuppressed with cyclophosphamide, purified fresh peripheral lymphocytes from normal human donors produce a transitory GVHR when injected under the kidney capsule (15). The injected kidney enlarges in comparison with the untreated kidney, and the injected cells produce the invasive destructive histologic picture which characterizes the local GVHR in F, hybrid rats receiving parental cells by the same route. In an earlier study (16) we tested purified CLL lymphocytes in the local GVHR assay. Kidney enlargement did not ensue, and histologic assessment suggested minimal invasion by these cells. In the present study we sought to confirm these findings with cells from additional patients with CLL using the kidney assay, and we have developed a new simplified GVHR assay involving injections of lymphoid cells in the external oblique abdominal muscle of immunosuppressed rats. MATERIALS
AND METHODS
Lymphocytes were obtained from the peripheral blood of normal donors and from 10 patients with CLL. The diagnosis of CLL was based on clinical and hematologic criteria. Nine of these patients were not under therapy at the time of the experiments. One was being treated with prednisone, Leukeran (Burroughs-Wellcome Co., Research. Triangle Park, N.C.) and irradiation to the spleen. Lymphocytes were separated from 30-50 ml of heparinized blood by centrifugation on a Ficoll-Hypaque gradient (17), washed twice, and resuspended in McCoy’s 5a medium without serum (Microbiological Associates, Bethesda, Md.). Cell concentration was adjusted to the desired number per unit volume for inoculation. Viability was assessed by Trypan-blue exclusion, and the GVHR test was performed in female outbred Wistar rats weighing lOO- 130 g. Rats were injected intraperitoneally with 150 mg/kg body weight of cyclophosphamide (Cytoxan, Mead Johnson, Evansville, Ind.) 24 hr before the administration of lymphoid cells. GVHR
Rat Kidney Assay
The technique for inducing a local GVHR in rat kidney has been described elsewhere (15,18). The immunosuppressed host received an inoculum of 15-70 x lo6 lymphoid cells in 0.1-O. 15 ml under the left renal capsule. Rats were sacrificed 7 days later, both kidneys were excised and weighed. The GVHR was evaluated by the weight ratio of the injected (left) to the noninjected (right) kidney (L/R ratio) and correlated with their microscopic appearance. The mitotic activity in the GVHR was assessed following intraperitoneal injection of 5 &i of [1251]deoxyuridine ([lz51]UdR) (Amersham-Searle, Arlington Heights, Ill.). The rats were sacrificed 3 hr after isotope injection, and the kidneys were removed, weighed, and washed several times in 95% ethanol and absolute ethanol. The radioactivity of individual kidneys was counted for 10 min in a Nuclear Chicago (Arlington Heights, Ill.) gamma radiation counter.
26
MOTTIRONI
The index of mitotic
GVHR
Rat Muscle
AND
GABKIELSEN
activity was calculated as cpm injected kidney cpm noninjected kidney ’
Assa)
A paravertebral incision was made in the left lumbar region of the rat. and the ventral side of the external oblique abdominal muscle exposed. At several muscle sites, each rat received an injection of a 0.05-ml suspension of (a) normal donor lymphocytes, (b) irradiated normal lymphocytes (2400 rad), (c) CLL lymphocytes. or (d) 0.05 ml of McCoy’s medium. A 27-gauge needle was used, bevel up, and a bleb was formed. Areas injected were coded and marked with small hemoclips (Edward Week and Co.. Inc., Long Island City. N.Y.) at 3-5 mm from the margin of the injected area. Intensity of the response was determined by (a) measuring diameter of the lesion, (b) histologic examination, and (c) incorporation of [1251]UdR into DNA of proliferating cells. Rats were sacrificed 3, 3, 6, 7, and 14 days after the intramuscular injection of lymphoid cells; the marked sites were measured and excised, and the net wet weight was determined. Samples were fixed in 10% formalin for microscopic examination. Five &i of [ 1251]UdR was administered intraperitoneally 3 hr before sacrifice. Tissue to be counted was washed repeatedly in 95% ethanol and absolute ethanol to remove unincorporated isotope. Samples were counted for 10 min each as described for the kidney assay. The proliferative activity of donor cells in the injected muscle site was calculated as cpm/g cell-injected tissue cpm/g medium-injected tissue
KIDNL’I.
WKIGH.IINDWED
BY LVMPHOCYTES
Lymphocytes Cell dose x 106 15
20 30 40 50 70
Weight L/R I .235 1.390 1.416 1.440 1.446 1.605
TABLE I UPI.AI(E RATIOS
ANI) [‘Y]UDR
from
ratios (g) t SD” t 2 2 -t k f
0.162 0.210 0.240 0.160 0.080 0.021
FROM
normal
NORMAL,
donors
i t k 2 + ‘-
ON SEVLN.I,H DONORS
AND
Lymphocytes
[ lz51] UdR (cpm) LIR 2 SD 1.211 1.281 1.316 1.375 1.680 2.536
’
0.106 0.107 0.197 0.150 0.141 0.196
Weight L/R 1.002 0.975 1.070 0.946 1.000 1.070
DA\ or LOYAL. CLL PMIENTS from
ratios tg) ?I SD -t 2 i+ 2 -+
0.043 0.024 0.042 0.039 0.020 0.044
CLL
GVHR
patients
[Y]UdR L/R
(cpm) t SD
0.770 1.020 0.981 1.020 1.000 0.960
e 0.046 _t 0.059 k 0.018 I?- 0.037 k 0.091 t 0.101
a The results are the pooled data of 10 independent experiments. Each value represents and standard deviations of the L/R ratios from an average of 24 rats/group. The differences series (CLL and normal donor) are statistically significant with a p value of < 0.01.
the mean in the two
LOCAL
GVHR
ASSAYS
IN
RAT
KIDNEY
AND
MUSCLE
27
FIG. 1. GVHR in kidney cortex. Invasive-destructive lesions are observed 7 days after subcapsular injection of normal human peripheral lymphocytes in cyclophosphamide treated rat. H.E., x 80.
RESULTS GVHR in the Kidney
Local GVHR of peripheral lymphocytes from 10 CLL patients was evaluated in the rat kidney assay. The pooled data of these experiments are presented in Table 1. Number of cells in the inocula varied from 15 to 70 x 1Og. In each experiment, lo-12 rats were injected with CLL cells; an equal number of rats was injected with the same number of cells from normal donors. Cells from CLL patients failed to react; the L/R weight ratios ranged from 0.94 to 1.07, while the control inocula of normal lymphocytes elicited a reaction. Isotope uptake also shows that CLL cells did not proliferate. The differences between CLL and normal lymphocyte responses were statistically significant with ap value of < 0.01. Histologically, the GVHR produced by normal peripheral lymphocytes was characterized by a high proliferation index, with infiltration and destruction of the renal cortex (Fig. 1). CLL lymphocytes placed under the kidney capsule remained localized at the injection site, and proliferation was not seen. Occasionally cells infiltrated the cortex, but no parenchymal damage was observed (Fig. 2). GVHR in the Muscle
The reactivity of normal lymphocytes and CLL cells was tested in the muscle of immunosuppressed rats. Control injections of normal irradiated lymphocytes and
28
MOTTIRONI
AIVD
GABRIELSt:h
FIG. 2. Renal cortex of cyclophosphamide treated rat 7 days after the subcapsular injection of CL1 lymphocytes. No invasive-destructive lesions are observed in the cortical parenchyma. H.E.. - X0.
McCoy’s medium were done in the same ammal. Rats were sacrificed at various times after intramuscular injection. On Days 2 and 3, areas injected with normal lymphocytes had a diffuse, red, congestive appearance while CLL cell injected sites and control areas showed little or no evidence of cell or medium administration. Histologically. normal lymphocytes had infiltrated the muscle and mononuclear cells surrounded the fibers and blood vessels, but there were few manifestations of muscle damage. CLL cells and irradiated lymphocytes did not infiltrate muscle tissue. Isotope uptake was not significant. At 6-7 days a macroscopic lesion had developed at the site inoculated with normal lymphocytes, consisting of a firm, discrete pale nodule, while CLL cells, irradiated cells, or medium injected sites could be identified only by the position of the markers. Microscopically. areas injected with normal lymphocytes showed infiltration and proliferation of the lymphoid cells and destruction of the muscle fibers. Numerous mitotic and mononuclear cells were observed (Figs. 3a and 3bI. CLL lymphocytes remained at the injection site, showing occasional focal infiltration, but proliferation of the injected cells or muscle damage was not seen (Fig. 4). Most irradiated cells stayed at the inoculated area without causing tissue damage (Fig. 5).
LOCAL
GVHR
ASSAYS
IN
RAT
KIDNEY
AND
MUSCLE
29
These results were corroborated by the isotope uptake of the injected muscle, as diagrammed in Fig. 6 on a scale of adjusted counts per minute. Despite variation among rats, the normal lymphoid cells always showed the highest rate of proliferation, significantly increased over that of CLL cells, irradiated cells, or medium. In both experiments, these results were found to be significant at the 0.05 level. Data analyses from the present and previous experiments, using the Friedman test (19), indicated that DNA synthesis by the injected normal lymphocytes versus controls and CLL lymphocytes were significantly higher on the seventh day. Statistically, similar pairs of observations within experiments were the controls (irradiated lymphocytes versus medium) and CLL cells versus medium. Since a comparison of the control inocula yielded insignificant differences in isotope uptake, we used medium-injected sites as the control in all subsequent experiments. By 14 days lesions regressed. The cellular infiltrate decreased, few mitotic figures or motionuclear cells were observed. When normal lymphocytes were injected into the rat muscle at different dosages, the response obtained could be seen to be dose related. Table 2 summarizes the results. Expression of GVHR was seen at a dose as low as 2.5 x IO6 cells. CLL lymphocytes elicited no response. DISCUSSION
In the present study we have compared the in vivo activity of human lymphocytes on rat kidney and muscle in a model of local GVHR, using normal and CLL cells. The events in the local GVHR in the kidney have been described in detail elsewhere (20,21). It has been shown that in a xenogeneic combination, i.e., normal human lymphocytes injected into immunosuppressed rats, the immunocompetent cells have the capacity to recognize host antigen, and thus, they undergo blastogenic transformation and infiltrate and destroy target tissue. Lymphocyte reactivity in this system depends on the immunologic status of the donor. Patients with CLL may have diminished cell-mediated immunity expressed as depressed cutaneous reactivity and impaired lymphocyte responsiveness to PHA (7,8,11.14). It has been postulated that this unresponsiveness to PHA in vitro may be due to the presence of abnormal T cells (22,23) or to a dilution of normal T cells by a large increase in the number of B cells (24,25). Although the data on the reactivity of CLL cells and their subpopulations to mitogens are still conflicting, our data using the GVHR in rat kidney and muscle indicate that CLL cells lack immunocompetence even when injected in relatively large numbers. These findings correlate with the work of others (26). Normal lymphocytes were capable of inducing local GVHR in both assays. We observed that by the seventh day, normal lymphocytes injected in the muscle produced a reaction that was macroscopically characterized by the presence of a well limited nodule. The histology showed unmistakable evidence of proliferation of the injected cells and muscle fiber destruction. In this respect it is interesting to note that histopathology of the GVHR in the muscle mimics the picture of acute rejection of heart homografts, both having mononuclear cell infiltrates and profound disruption of muscle structure (27,28).
FIL. ?a. GVHR in muscle. Invasive-destructive reaction in abdominal muscle of cyclophosphamide .lrmal human peripheral lymphocytes, H.E.. x80.
treated
rat
7 days after intramuscular injection of
2
E
3 c
LOCAL
GVHR
ASSAYS
IN
RAT
KIDNEY
AND
MUSCLE
31
,.
Frc.. 4. Muscle section Seven day:, postinjection
-
7 days after injection of irradiated normal human peripheral the mononuclear cells remain at the inoculation \ite. L3.F
Flc,. 5. Muscle zection 7 days after injection of CL1 lymphocytes. The maculation site chowing a mild degree of infiltration without over1 proliferation H.E.. x80 3’
*n
Iymphocvtr\ I. X0
in,jected crll~~ sta\ at tht or muscle de\tructl<>r~
LOCAL
-
GVHR
ASSAYS -
IN
RAT
KIDNEY
-
AND -
MUSCLE
0
50-
2 d”:
cl
&I a c .
0
0
30-
-2 rl x -5
i I,dI 0
40-
0
:
.
0
0 .
0
0
0..
E
20-
!
lo-
-
-
0
0
:
0
0 cl* .
0..
O.=
. 09
0
i
:
-
FIG. 6. Incorporation of [‘z ]UdR on seventh day of GVHR in muscle induced by lymphocytes from normal donors and CLL pat ietIts. Each column represents a rat. Cell dose injected 40 x 108. Two experiments are shown. 0, normal lymphocytes; l , CLL lymphocytes; n , medium; 0, irradiated normal lymphocytes.
Isotope uptake was significantly higher in muscle areas inoculated with normal lymphocytes when compared to that of irradiated lymphocytes, CLL cells, or medium injected tissue. The observation of the similarity of isotope uptake by the controls and CLL cell-inoculated areas led us to use medium as a negative control and to conclude that CLL lymphocytes could not be stimulated by rat antigens. TABLE CELL
DOSE
RESPONSE
CLL
LYMPHOCYTES
Normal lymphocytes
Number of rats
Cell dose
7 5 3 3 4 4
40 x 106 20 x 106 10 x 106 5 x 106 2.5 x lo6 1.25~10~
3 3 3 3
OF NORMAL
6.25 3.15 1.56 7.81
x x x x
lo5 105 lo5 104
2
PERIPHERAL
GVHR
IN
CLL lymphocytes
BLOOD
LYMPHOCYTES
AND
IN MUSCLE
Normal lymphocytes
CLL lymphocytes
Mean diameters” (mm)
Mean
SW
Mean
8x3 7 x 2.5 6x2 4x2 2x2 -
2.247 1.714 1.552 1.619 1.480 1.060 0.993 0.747 0.942 0.715
kO.747 ?0.408 +0.469 kO.250 k-o.258 kO.114 20.227 %0.078 kO.264 a0.076
0.952 0.993 0.871 0.990 0.851 0.900 0.970 0.910 0.770 0.810
-
SD 20.083 20.034 eO.223 ?0.017 eO.034 50.020 20.013 40.070 20.023 kO.063
LI Marked injected areas were examined and lesions measured. b Values represent the mean and standard deviations of the ratios of [iz51]UdR uptake in lymphocyte-injected muscle. Counts were taken at seventh day and adjusted to net weights as described in methods.
34
MOT’TIRONI
;\NL)
(;AHKIF:LSt.h
Our studies indicate that parallel results could be obtained in the GVHR using kidney or muscle inoculated with normal lymphocytes or CLL cells. The assay in rat muscle rather than kidney offers a distinct advantage in the study of these cells: (a) CLL cells from several patients. subpopulations of fractionated cells and positive and negative controls can be tested within the same animal. thus. eliminating the considerable variability of the immunosuppressed hosts; (b) the cell dose can be titrated within the same animal and. by multiple injections. the time-course of the cellular infiltrates may be assessed: and cc) obvious practical aspects of the GVHR in the muscle is the lower number of animals required for each assay and the fewer cells needed to induce the reaction. Potentially this assay can be applied to classify the T or B cell origin of lymphoproliferative disorders or immunodeficiency disease as well as a simplified method to test ALG activity LIZ IS&J. ACKNOWLEDGMENTS The excellent technical Rudofsky for his review Department of Hematology blood from their patients.
assistance of Mrs. R. Swift is gratefully acknowledged. We thank Mr. L of the manuscript and Drs. F. Lizzi. ‘4. Tartaglia. and R. Propp from the at Albany Medical Center and V. A. Hospital for providing us with data and We also thank Ms. A. Kondrat for her secretarial assistance,
REFERENCES I. Cooper, M. D.. Peterson, R. D. A., Gabrielsen. A. E.. and Good. R. A.. C’trm,er Rm. 26, 1165. 1966. 2. Johansson. B.. and Klein, E.. Cl&. E.rp. fmmur7d. 6, 421, 1970. 3. Wilson. J. D.. and Nossal. G. J. V.. Luncet 2, 788. 1971. 4. Grey. H. M.. Rabellino, E.. and Pirofsky. B.. J. C/in. b7t~~.sc. 50, 2368, 1971. 5. Pernis. B., Forni. L.. and Amante. L.. Anr7. NY Acad. Sci. 190. 420. 1971. 6. Preud’homme. J. L., and Seligmann. M., Blood 40, 777, 1972. 7. Schrek, R., and Rabinowitz. Y., Proc. Sot. Exp. Biol. Med. 113, 191. 1963. 8. Robbins, H. J.. Experientia 20, 164. 1964. 9. Quaglino. D., and Cowling. D. C.. Brit. J. Htrrmutol. 10, 3.58. 1Y64. 10. Winter, G. C. B.. Osmond, D. G.. Yoffey. J. M.. and Mahy. D. J.. Lancet 2. 563. IY64. I I. Rubin. .4. D., Havemann. K., and Dameshek, W.. Blood 33, 313. 1969. 12. Smith. M. J.. Browne. E.. and Slungaard, A., Blood 41. 505. 1973. 13. Heim. L. R., Yunis. E. J.. and Good, R. A., Int. Arch. AlkerRy 42. 56.5. 1972. 14. Argyris. B. F., Transplantation 17, 387, 1974. 15. Saleh. W., MacLean. L. D., and Gordon. J.. J. /mmrrnol. 103, 114, 1969. 16. Mottironi, V. D., and Meuwissen. H. J.. Trunsplantation 16. 177. 1973. 17. Parker, K. L.. Schreinemachers, D. M.. and Meuwissen. H. J.. Transpluntutior7 14, 135. 1972. 18. Elkins. W.. J. Exp. Med. 120, 329. 1964. 19. Friedman, M., In “Handbook of Statistical Tables” (D. B. Owen. Ed.). pp. 407-419. Addison-Wesley. Reading. Mass./PaloAltolLondon. 1967. 20. Elkins. W. L.. Transplantation 9, 273. 1970. 21. Elkins, W. L.. Trunsplanfation 11, 551. 1971. 22. Rubin, A. D.. In “Proceedings of Fifth Leukocyte Culture Conference” (J. E:. Harris. Ed.). pp. 239-260. Academic Press, New York, 1970. 23. Robbins. J. H., and Levis. J. R., Int. Arch. Allergy 43, 845, 1972.
LOCAL
GVHR
ASSAYS
IN
RAT
KIDNEY
AND
MUSCLE
35
24. Melief, C. J. M., Schweitzer, M., and Eijsvoogel, V. P.. In “Proceedings of Seventh Leukocyte Culture Conference” (F. Daguillard, Ed.), pp. 459-476, Academic Press, New York, 1973. 25. Wybran, J., Chantler, S., and Fudenberg, H. H., Lancer 1, 126, 1973. 26. Shohat. B., Joshua, H., and Ben-Bassat. M., J. Reticuloendothel. Sot. 16, 276, 1974. 27. Abbott. C., Dewitt, C. W., and Creech, 0.. Jr., Transplantation 3, 432, 1965. 28. Kosek, J., Hurley, E. J., and Lower, R. R., Lab. Invest. 19, 97, 1968.
IMMUNOLOGY
AND
IMMUNOPATHOLOGY
7, 24-35 (1977)
Studies on the Local Graft versus Host Reactivity Normal and Chronic Lymphocytic Leukemia Lymphocytes
of
I. A Comparison of Assays in Rat Kidney and Muscle VILMA D. MOTTIRONI New
York
State
Kidney
AND ANN E. GABRIELSEX
Disease Institute’ and the Department qf Pediatrics, Medical College, Albany. Nen, York 12208
A/ban>
Received May 3. 1976 Many methods have been used to identify T and B cells and their subtypes in terms of both physical attributes and function in a range of in vitro tests. The present report deals with an in viva approach to the character of circulating lymphocytes from 10 patients with chronic lymphocytic leukemia (CLL) as compared to lymphoid cells from normal donors, using the Elkins graft versus host reaction (GVHR) assay in kidney and an assay involving injection of lymphoid cells in the external oblique abdominal muscle of immunosuppressed rats. GVHR in the muscle was assessed by (a) measuring diameter of the lesion, (b) examining histology of the injected site. and (cl determining uptake of [‘=I]deoxyuridine into DNA of proliferating cells. Results show that lymphocytes from normal donors elicit a GVHR in both kidney and muscle, producing characteristic invasion and destruction of the injected tissue, while CLL lymphocytes, even in large numbers, produce no response by any of the criteria in either tissue. CLL cells remain localized at the injection site without undergoing the changes of activation and proliferation. Results in the muscle parallel those in the kidney. The muscle assay requires fewer cells and permits a larger number of assessments per animal. Potentially it can be applied to classification of lymphoreticular disorders and immunodeficiency states.
INTRODUCTION
The lymphoid cells active in the immune system consist of two antigenically and functionally distinct lymphocyte populations: thymus derived cells (T cells) and bone marrow derived cells (B cells) which can be distinguished by their different membrane properties (1). Based on the study of various surface markers on human lymphocytes it has now become possible to define some diseases of the lymphoid system in terms of T or B cell proliferation. Chronic lymphocytic leukemia (CLL) has been studied extensively and been identified as a B cell malignancy (2-6). Phytohemagglutinin (PHA)-induced lymphocyte stimulation and mixed leukocyte culture (MLC) responses are parameters for the presence of functional normal T cell populations. In CLL the PHA response is depressed and delayed (7- 1 I) and the responding capacity of these cells in MLC is markedly diminished (12). It is not clear if this is due to impaired T lymphocyte function or because the T cells are diluted by large numbers of B lymphocytes. Nevertheless, the PHA
r A unit of the New York State Department of Health. 24 Copyright All rights
@ 1977 by Academic Press, Inc of reproduction in any form reserved.
LOCAL
GVHR
ASSAYS
IN
RAT
KIDNEY
AND
MUSCLE
25
and MLC reactivity in these in vitro tests does not necessarily reflect the ability of peripheral lymphocytes to respond in vivo. Induction of graft-versus-host reactions (GVHR) has been considered to reflect immunocompetence of donor T cells (13). Recent work suggests that T cells are essential for the production of a GVHR (14). This phenomenon was tested in a model of local GVHR. If rats are immunosuppressed with cyclophosphamide, purified fresh peripheral lymphocytes from normal human donors produce a transitory GVHR when injected under the kidney capsule (15). The injected kidney enlarges in comparison with the untreated kidney, and the injected cells produce the invasive destructive histologic picture which characterizes the local GVHR in F, hybrid rats receiving parental cells by the same route. In an earlier study (16) we tested purified CLL lymphocytes in the local GVHR assay. Kidney enlargement did not ensue, and histologic assessment suggested minimal invasion by these cells. In the present study we sought to confirm these findings with cells from additional patients with CLL using the kidney assay, and we have developed a new simplified GVHR assay involving injections of lymphoid cells in the external oblique abdominal muscle of immunosuppressed rats. MATERIALS
AND METHODS
Lymphocytes were obtained from the peripheral blood of normal donors and from 10 patients with CLL. The diagnosis of CLL was based on clinical and hematologic criteria. Nine of these patients were not under therapy at the time of the experiments. One was being treated with prednisone, Leukeran (Burroughs-Wellcome Co., Research. Triangle Park, N.C.) and irradiation to the spleen. Lymphocytes were separated from 30-50 ml of heparinized blood by centrifugation on a Ficoll-Hypaque gradient (17), washed twice, and resuspended in McCoy’s 5a medium without serum (Microbiological Associates, Bethesda, Md.). Cell concentration was adjusted to the desired number per unit volume for inoculation. Viability was assessed by Trypan-blue exclusion, and the GVHR test was performed in female outbred Wistar rats weighing lOO- 130 g. Rats were injected intraperitoneally with 150 mg/kg body weight of cyclophosphamide (Cytoxan, Mead Johnson, Evansville, Ind.) 24 hr before the administration of lymphoid cells. GVHR
Rat Kidney Assay
The technique for inducing a local GVHR in rat kidney has been described elsewhere (15,18). The immunosuppressed host received an inoculum of 15-70 x lo6 lymphoid cells in 0.1-O. 15 ml under the left renal capsule. Rats were sacrificed 7 days later, both kidneys were excised and weighed. The GVHR was evaluated by the weight ratio of the injected (left) to the noninjected (right) kidney (L/R ratio) and correlated with their microscopic appearance. The mitotic activity in the GVHR was assessed following intraperitoneal injection of 5 &i of [1251]deoxyuridine ([lz51]UdR) (Amersham-Searle, Arlington Heights, Ill.). The rats were sacrificed 3 hr after isotope injection, and the kidneys were removed, weighed, and washed several times in 95% ethanol and absolute ethanol. The radioactivity of individual kidneys was counted for 10 min in a Nuclear Chicago (Arlington Heights, Ill.) gamma radiation counter.
26
MOTTIRONI
The index of mitotic
GVHR
Rat Muscle
AND
GABKIELSEN
activity was calculated as cpm injected kidney cpm noninjected kidney ’
Assa)
A paravertebral incision was made in the left lumbar region of the rat. and the ventral side of the external oblique abdominal muscle exposed. At several muscle sites, each rat received an injection of a 0.05-ml suspension of (a) normal donor lymphocytes, (b) irradiated normal lymphocytes (2400 rad), (c) CLL lymphocytes. or (d) 0.05 ml of McCoy’s medium. A 27-gauge needle was used, bevel up, and a bleb was formed. Areas injected were coded and marked with small hemoclips (Edward Week and Co.. Inc., Long Island City. N.Y.) at 3-5 mm from the margin of the injected area. Intensity of the response was determined by (a) measuring diameter of the lesion, (b) histologic examination, and (c) incorporation of [1251]UdR into DNA of proliferating cells. Rats were sacrificed 3, 3, 6, 7, and 14 days after the intramuscular injection of lymphoid cells; the marked sites were measured and excised, and the net wet weight was determined. Samples were fixed in 10% formalin for microscopic examination. Five &i of [ 1251]UdR was administered intraperitoneally 3 hr before sacrifice. Tissue to be counted was washed repeatedly in 95% ethanol and absolute ethanol to remove unincorporated isotope. Samples were counted for 10 min each as described for the kidney assay. The proliferative activity of donor cells in the injected muscle site was calculated as cpm/g cell-injected tissue cpm/g medium-injected tissue
KIDNL’I.
WKIGH.IINDWED
BY LVMPHOCYTES
Lymphocytes Cell dose x 106 15
20 30 40 50 70
Weight L/R I .235 1.390 1.416 1.440 1.446 1.605
TABLE I UPI.AI(E RATIOS
ANI) [‘Y]UDR
from
ratios (g) t SD” t 2 2 -t k f
0.162 0.210 0.240 0.160 0.080 0.021
FROM
normal
NORMAL,
donors
i t k 2 + ‘-
ON SEVLN.I,H DONORS
AND
Lymphocytes
[ lz51] UdR (cpm) LIR 2 SD 1.211 1.281 1.316 1.375 1.680 2.536
’
0.106 0.107 0.197 0.150 0.141 0.196
Weight L/R 1.002 0.975 1.070 0.946 1.000 1.070
DA\ or LOYAL. CLL PMIENTS from
ratios tg) ?I SD -t 2 i+ 2 -+
0.043 0.024 0.042 0.039 0.020 0.044
CLL
GVHR
patients
[Y]UdR L/R
(cpm) t SD
0.770 1.020 0.981 1.020 1.000 0.960
e 0.046 _t 0.059 k 0.018 I?- 0.037 k 0.091 t 0.101
a The results are the pooled data of 10 independent experiments. Each value represents and standard deviations of the L/R ratios from an average of 24 rats/group. The differences series (CLL and normal donor) are statistically significant with a p value of < 0.01.
the mean in the two
LOCAL
GVHR
ASSAYS
IN
RAT
KIDNEY
AND
MUSCLE
27
FIG. 1. GVHR in kidney cortex. Invasive-destructive lesions are observed 7 days after subcapsular injection of normal human peripheral lymphocytes in cyclophosphamide treated rat. H.E., x 80.
RESULTS GVHR in the Kidney
Local GVHR of peripheral lymphocytes from 10 CLL patients was evaluated in the rat kidney assay. The pooled data of these experiments are presented in Table 1. Number of cells in the inocula varied from 15 to 70 x 1Og. In each experiment, lo-12 rats were injected with CLL cells; an equal number of rats was injected with the same number of cells from normal donors. Cells from CLL patients failed to react; the L/R weight ratios ranged from 0.94 to 1.07, while the control inocula of normal lymphocytes elicited a reaction. Isotope uptake also shows that CLL cells did not proliferate. The differences between CLL and normal lymphocyte responses were statistically significant with ap value of < 0.01. Histologically, the GVHR produced by normal peripheral lymphocytes was characterized by a high proliferation index, with infiltration and destruction of the renal cortex (Fig. 1). CLL lymphocytes placed under the kidney capsule remained localized at the injection site, and proliferation was not seen. Occasionally cells infiltrated the cortex, but no parenchymal damage was observed (Fig. 2). GVHR in the Muscle
The reactivity of normal lymphocytes and CLL cells was tested in the muscle of immunosuppressed rats. Control injections of normal irradiated lymphocytes and
28
MOTTIRONI
AIVD
GABRIELSt:h
FIG. 2. Renal cortex of cyclophosphamide treated rat 7 days after the subcapsular injection of CL1 lymphocytes. No invasive-destructive lesions are observed in the cortical parenchyma. H.E.. - X0.
McCoy’s medium were done in the same ammal. Rats were sacrificed at various times after intramuscular injection. On Days 2 and 3, areas injected with normal lymphocytes had a diffuse, red, congestive appearance while CLL cell injected sites and control areas showed little or no evidence of cell or medium administration. Histologically. normal lymphocytes had infiltrated the muscle and mononuclear cells surrounded the fibers and blood vessels, but there were few manifestations of muscle damage. CLL cells and irradiated lymphocytes did not infiltrate muscle tissue. Isotope uptake was not significant. At 6-7 days a macroscopic lesion had developed at the site inoculated with normal lymphocytes, consisting of a firm, discrete pale nodule, while CLL cells, irradiated cells, or medium injected sites could be identified only by the position of the markers. Microscopically. areas injected with normal lymphocytes showed infiltration and proliferation of the lymphoid cells and destruction of the muscle fibers. Numerous mitotic and mononuclear cells were observed (Figs. 3a and 3bI. CLL lymphocytes remained at the injection site, showing occasional focal infiltration, but proliferation of the injected cells or muscle damage was not seen (Fig. 4). Most irradiated cells stayed at the inoculated area without causing tissue damage (Fig. 5).
LOCAL
GVHR
ASSAYS
IN
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KIDNEY
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29
These results were corroborated by the isotope uptake of the injected muscle, as diagrammed in Fig. 6 on a scale of adjusted counts per minute. Despite variation among rats, the normal lymphoid cells always showed the highest rate of proliferation, significantly increased over that of CLL cells, irradiated cells, or medium. In both experiments, these results were found to be significant at the 0.05 level. Data analyses from the present and previous experiments, using the Friedman test (19), indicated that DNA synthesis by the injected normal lymphocytes versus controls and CLL lymphocytes were significantly higher on the seventh day. Statistically, similar pairs of observations within experiments were the controls (irradiated lymphocytes versus medium) and CLL cells versus medium. Since a comparison of the control inocula yielded insignificant differences in isotope uptake, we used medium-injected sites as the control in all subsequent experiments. By 14 days lesions regressed. The cellular infiltrate decreased, few mitotic figures or motionuclear cells were observed. When normal lymphocytes were injected into the rat muscle at different dosages, the response obtained could be seen to be dose related. Table 2 summarizes the results. Expression of GVHR was seen at a dose as low as 2.5 x IO6 cells. CLL lymphocytes elicited no response. DISCUSSION
In the present study we have compared the in vivo activity of human lymphocytes on rat kidney and muscle in a model of local GVHR, using normal and CLL cells. The events in the local GVHR in the kidney have been described in detail elsewhere (20,21). It has been shown that in a xenogeneic combination, i.e., normal human lymphocytes injected into immunosuppressed rats, the immunocompetent cells have the capacity to recognize host antigen, and thus, they undergo blastogenic transformation and infiltrate and destroy target tissue. Lymphocyte reactivity in this system depends on the immunologic status of the donor. Patients with CLL may have diminished cell-mediated immunity expressed as depressed cutaneous reactivity and impaired lymphocyte responsiveness to PHA (7,8,11.14). It has been postulated that this unresponsiveness to PHA in vitro may be due to the presence of abnormal T cells (22,23) or to a dilution of normal T cells by a large increase in the number of B cells (24,25). Although the data on the reactivity of CLL cells and their subpopulations to mitogens are still conflicting, our data using the GVHR in rat kidney and muscle indicate that CLL cells lack immunocompetence even when injected in relatively large numbers. These findings correlate with the work of others (26). Normal lymphocytes were capable of inducing local GVHR in both assays. We observed that by the seventh day, normal lymphocytes injected in the muscle produced a reaction that was macroscopically characterized by the presence of a well limited nodule. The histology showed unmistakable evidence of proliferation of the injected cells and muscle fiber destruction. In this respect it is interesting to note that histopathology of the GVHR in the muscle mimics the picture of acute rejection of heart homografts, both having mononuclear cell infiltrates and profound disruption of muscle structure (27,28).
FIL. ?a. GVHR in muscle. Invasive-destructive reaction in abdominal muscle of cyclophosphamide .lrmal human peripheral lymphocytes, H.E.. x80.
treated
rat
7 days after intramuscular injection of
2
E
3 c
LOCAL
GVHR
ASSAYS
IN
RAT
KIDNEY
AND
MUSCLE
31
,.
Frc.. 4. Muscle section Seven day:, postinjection
-
7 days after injection of irradiated normal human peripheral the mononuclear cells remain at the inoculation \ite. L3.F
Flc,. 5. Muscle zection 7 days after injection of CL1 lymphocytes. The maculation site chowing a mild degree of infiltration without over1 proliferation H.E.. x80 3’
*n
Iymphocvtr\ I. X0
in,jected crll~~ sta\ at tht or muscle de\tructl<>r~
LOCAL
-
GVHR
ASSAYS -
IN
RAT
KIDNEY
-
AND -
MUSCLE
0
50-
2 d”:
cl
&I a c .
0
0
30-
-2 rl x -5
i I,dI 0
40-
0
:
.
0
0 .
0
0
0..
E
20-
!
lo-
-
-
0
0
:
0
0 cl* .
0..
O.=
. 09
0
i
:
-
FIG. 6. Incorporation of [‘z ]UdR on seventh day of GVHR in muscle induced by lymphocytes from normal donors and CLL pat ietIts. Each column represents a rat. Cell dose injected 40 x 108. Two experiments are shown. 0, normal lymphocytes; l , CLL lymphocytes; n , medium; 0, irradiated normal lymphocytes.
Isotope uptake was significantly higher in muscle areas inoculated with normal lymphocytes when compared to that of irradiated lymphocytes, CLL cells, or medium injected tissue. The observation of the similarity of isotope uptake by the controls and CLL cell-inoculated areas led us to use medium as a negative control and to conclude that CLL lymphocytes could not be stimulated by rat antigens. TABLE CELL
DOSE
RESPONSE
CLL
LYMPHOCYTES
Normal lymphocytes
Number of rats
Cell dose
7 5 3 3 4 4
40 x 106 20 x 106 10 x 106 5 x 106 2.5 x lo6 1.25~10~
3 3 3 3
OF NORMAL
6.25 3.15 1.56 7.81
x x x x
lo5 105 lo5 104
2
PERIPHERAL
GVHR
IN
CLL lymphocytes
BLOOD
LYMPHOCYTES
AND
IN MUSCLE
Normal lymphocytes
CLL lymphocytes
Mean diameters” (mm)
Mean
SW
Mean
8x3 7 x 2.5 6x2 4x2 2x2 -
2.247 1.714 1.552 1.619 1.480 1.060 0.993 0.747 0.942 0.715
kO.747 ?0.408 +0.469 kO.250 k-o.258 kO.114 20.227 %0.078 kO.264 a0.076
0.952 0.993 0.871 0.990 0.851 0.900 0.970 0.910 0.770 0.810
-
SD 20.083 20.034 eO.223 ?0.017 eO.034 50.020 20.013 40.070 20.023 kO.063
LI Marked injected areas were examined and lesions measured. b Values represent the mean and standard deviations of the ratios of [iz51]UdR uptake in lymphocyte-injected muscle. Counts were taken at seventh day and adjusted to net weights as described in methods.
34
MOT’TIRONI
;\NL)
(;AHKIF:LSt.h
Our studies indicate that parallel results could be obtained in the GVHR using kidney or muscle inoculated with normal lymphocytes or CLL cells. The assay in rat muscle rather than kidney offers a distinct advantage in the study of these cells: (a) CLL cells from several patients. subpopulations of fractionated cells and positive and negative controls can be tested within the same animal. thus. eliminating the considerable variability of the immunosuppressed hosts; (b) the cell dose can be titrated within the same animal and. by multiple injections. the time-course of the cellular infiltrates may be assessed: and cc) obvious practical aspects of the GVHR in the muscle is the lower number of animals required for each assay and the fewer cells needed to induce the reaction. Potentially this assay can be applied to classify the T or B cell origin of lymphoproliferative disorders or immunodeficiency disease as well as a simplified method to test ALG activity LIZ IS&J. ACKNOWLEDGMENTS The excellent technical Rudofsky for his review Department of Hematology blood from their patients.
assistance of Mrs. R. Swift is gratefully acknowledged. We thank Mr. L of the manuscript and Drs. F. Lizzi. ‘4. Tartaglia. and R. Propp from the at Albany Medical Center and V. A. Hospital for providing us with data and We also thank Ms. A. Kondrat for her secretarial assistance,
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24. Melief, C. J. M., Schweitzer, M., and Eijsvoogel, V. P.. In “Proceedings of Seventh Leukocyte Culture Conference” (F. Daguillard, Ed.), pp. 459-476, Academic Press, New York, 1973. 25. Wybran, J., Chantler, S., and Fudenberg, H. H., Lancer 1, 126, 1973. 26. Shohat. B., Joshua, H., and Ben-Bassat. M., J. Reticuloendothel. Sot. 16, 276, 1974. 27. Abbott. C., Dewitt, C. W., and Creech, 0.. Jr., Transplantation 3, 432, 1965. 28. Kosek, J., Hurley, E. J., and Lower, R. R., Lab. Invest. 19, 97, 1968.