3h-deoxythymidine incorporation in graft-versus-host disease in the norway rat

Virchows Arch. B Cell Path. 22, 341 - 352 (1976) V' AM B ~�9 by Springer-Verlag 1976

3H-Deoxythymidine Incorporation in Graft-Versus-Host Disease in the Norway Rat

II. Autoradiographic Studies*

John Clancy, Jr., Patrick Chan, and Rodica Schurath Department of Anatomy, University of Kansas Medical Center, Kansas City, Kansas 66103, USA

and University of Manitoba, Winnipeg, Canada R3EOW3

Summary. A sequential analysis was made of various areas within the lymph nodes and spleen of newborn Brown Norway (BN) rats suffering from graft- versus-host disease (GVHD) subsequent to an allogeneic injection of adult Lewis (L) lymph node cells (experimental). One micron thick autoradiographs were compared between such experimental and control littermates having received the same number of syngeneic adult BN cells. Both experimental and control animals received tritiated deoxythymidine (3HdT) one hour before killing. The autoradiographs revealed a 2.25 and 2.50 times higher thymidine labeling index of lymphocytes in the deep cortex of mesenteric lymph nodes and white pulp of the spleen, respectively, for experimental animals. The experimental effect occurred within one day. The majority of the labeled cells in experimental animals were large lymphoblasts with prominent nucleoli. The labeling index within these areas remained significantly higher than control values until day 8 in the spleen and through day 14 within the lymph nodes. However, differences in labeled cells present in high powered microscopic fields reached a peak on day 3 within compartments in experimental animals but fell significantly below control values by day 9 owing to a pronounced disappearance of both small and large lymphocytes from these areas, and a decreased intensity of individual cell labeling as the reaction progressed. In contradistinction the concentration of labeled cells present in high powered microscopic fields of lymph nodes' medulla became 3.13 times controls by day 4. Most of these labeled cells contained a more basophilic cytoplasm than those found in the deep cortex and some were distinctly plasma cell precursors. In contrast to the deep cortex their concentration remained approximately three times control values until death.

The data indicates that the major proliferative events within the spleen and lymph nodes in neonatal rat GVHD are initially restricted to donor cell localization areas of these tissue compartments. Subsequently the GVHD-

* This work was supported by the American Cancer Society-Kansas Division, The Wilkerson Endowment Fund, and The Medical Research Council of Canada

342 J. Clancy, Jr. et al.

related events may be attributed to other areas and possibly cell types. Thus any proliferation contributing to splenomegaly in the latter stages of GVHD appears to occur in the red pulp and that contributing to lymph node enlargement a medullary response.

Key words: Graft-Versus-Host Disease - Neonatal - Cytokinetics - Tri- tiated deoxythymidine autoradiography - White pulp spleen - Deep Cortex and medulla lymph nodes.

Introduction

Results presented in the accompanying paper (Clancy et al., 1976b) document very significant relative as well as absolute differences in tritiated deoxythymidine (3HdT) incorporation developing within the spleen and lymph nodes of neonatal BN rats suffering from graft-versus-host disease (GVHD) as compared to their control littermates. Previous autoradiographic studies have demonstrated that when allogeneic or syngeneic lymphocytes are administered intravenously to newborn or adult animals, a certain percentage of these cells localize rapidly in the deep cortex of lymph nodes and the white pulp of the spleen. Here the allogeneic cells almost immediately enlarge and divide (Clancy and Reike, unpublished observations; Ford et al., 1975; Gowans, 1962; Porter and Cooper, 1962). The present autoradiographic study was thus conducted to compare 3HdT incorporation within the whole spleen and lymph node with that occurring within the white pulp and deep cortex, respectively, throughout the course of lethal GVHD.

The data indicates that although the initial proliferative events within the spleen and lymph nodes during neonatal rat GVHD do indeed occur in the white pulp and deep cortex, respectively, they subsequently subside here as these areas become depleted of lymphocytes. Thus any proliferation leading to splenomegaly appears to be present in the red pulp and any contributing to lymph node enlargement the medulla.

Materials and Methods

Animals. Rats of both sexes of domestically maintained Lewis (L) (Ag-B1) and Brown Norway (BN) (Ag-B3) strains were employed in these studies. Newborn BN rats served as experimental and control animals. Two to four littermate pairs were assessed for each experimental situation reported,

Cell Suspensions and Injections. Thirty million adult L or BN lymph node cells were injected iv into newborn BN rats as described previously (Clancy et al., 1976b).

Systemic Radioactive Labeling. Four hours to 14 days after the cellular inoculation 1 p,C/g body weight of tritiated deoxythymidine (~HdT) was administered iv to experimental and control neonatal animals as described in the previous paper (Clancy et al., 1976b). The animals were killed after 1 h; their spleens and mesenteric lymph nodes were processed for autoradiography.

Autoradiography. Tissues were fixed in Davidson's solution, embedded in glycol methacrylate and one micron sections were cut on a Sorvall JB-4 microtome and mounted on precleaned glass

aHdT Autoradiography of Spleen and Lymph Nodes during GVHD 343

slides. The sections were dipped in Eastman Kodak liquid emulsion, exposed in the cold for 10 days, and the developed and fixed autoradiographs were stained with 1% Buffered Toluidine Blue. A thymidine labeling index was determined by counting the number of labeled small and large lymphocytes in at least 1,000 lymphocytes per animal present in the deeper cortical areas and medulla of the mesenteric nodes as well as the white pulp of the spleen (Figs. 1, 2, 8). Stromal reticular cells (Caffrey et al., 1966) and other cell types found in these areas were ignored. Because of the small size of white pulp areas in the age of animals employed (particularly those of 1 to 4 days old) four to five concentric layers of lymphocytes were scored surrounding randomly selected central arterioles in the spleen. Also, because of the relatively low level of background labeling obtained by employing the above method and exposure time, (0-2 as opposed to 4-5 for 5 micron paraffin sections from the same animal) any small or large lymphocytes in which both nucleus and cytoplasm was readily discernable and exhibited three or more silver grains were considered to be labeled. Cell counts were performed on tissues from two to three animals for each interval (Table 1). Mean grain counts were determined by counting those over 201Y300 labeled cells for each interval (Table 2).

Statistical Analysis. Calculations were performed as described in the previous paper (Clancy et al., 1976b).

Results

Autoradiographic Quantitation of Proliferation in Donor Cell Localization Areas of Spleen and Mesenteric Nodes

Previous studies have shown that when donor allogeneic L or syngeneic BN adult lymphocytes are administered intravenously to newborn BN hosts, some localize rapidly in the deep cortex of lymph nodes and the white pulp of the spleen where a high percentage of allogeneic cells almost immediately enlarge and divide (Clancy and Reike, unpublished observations: Ford et al., 1975; Gowans, 1962). Thus, it was decided to investigate the proliferation of lymphocytes within these areas as compared to that occurring within the whole tissue compartment (Clancy et al., 1976b) at various stages of GVHD. Examination of the deep cortex of mesenteric lymph nodes and white pulp of the spleen revealed a 2.25 and 2.50 times higher labeling index within these areas in experimental compared to control animals even within one day (Figs. 1 and 2). Most labeled cells in experimental animals were lymphoblasts with prominent nucleoli (Fig. 3) and larger than those cells labeled in control animals. This difference appeared to peak on day 3 in the deep cortex of mesenteric nodes and day 6 in the white pulp of the spleen. In the deep cortex of mesenteric nodes there was a persistent higher labeling index than the controls throughout, however, the white pulp of the spleen by day 11-12 exhibited an even lower labeling index than controls which persisted until day 14.

Enumeration of the number of small and large lymphocytes present within 3,112 ~tm 2 microscopic fields of the deep cortex of mesenteric nodes and white pulp of the spleen at various stages of the response revealed a greater number of cells present in the control white pulp of the spleen even after day 1 (Table 1). There was a lower number of cells present in the deep cortex of mesenteric


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3HdT Autoradiography of Spleen and Lymph Nodes during GVHD 345

Fig. 3. Deep cortex of a mesenteric node from a 2 day old neonatal rat injected 24 h previously with 30 x 106 adult allogeneic L lymph node cells. Notice the large labeled cells with prominent nucleoli present throughout the field, x 1,250

Table 1. Mean number of lymphocytes within 3,112 lam 2 microscopic fields of deep cortex mesenteric lymph nodes and white pulp of spleen in GVHD

Day Cells field

Spleen Mesenteric node

Exper. Control p Exper. Control p

1 5.2+11.2 90.2_+ 8.3 NS 47.2_+5.3 50.6_+ 4.1 2 85.4_+ 2.1 101.0_+ 3.4 <0.05 58.7_+6.1 68.4+ 5.9 3 82.7_+ 6.0 105.2_+ 4.1 <0.05 66.2_+4.7 81.3_+ 8.8 4 71.0_+ 6.3 95.3_+ 6.2 <0.05 67.0_+5.4 88.5+ 4.3 5 51.2-+ 5.9 103.4-+ 8.0 <0.01 55.6-+6.0 90.6_+ 8.4 6 36.0_+ 4.1 108.2_+ 9.3 <0.001 51.4_+5.2 100.1_+ 6.2 7 3,2.3_+ 2.9 110.3_+ 8.9 <0.001 40.3_+8.4 105.7+ 7.9 8 26.2-+ 2.1 105.1_+10.4 <0.001 33.5_+7.1 118.0_+ 8.4 9 21.4_+ 3.0 100.3_+ 6.5 <0.001 36.9_+5.0 130.6_+ 5.2

10 20.1_+ 5.2 98.4_+ 5.2 <0.001 33.4+4.6 128.4+ 9.3 11 19.6-+ 6.1 105.5-+ 9.4 <0.001 32.2-+3.7 121.9-+10.0 12 15.1_+ 3.2 98.0_+ 8.8 <0.001 30.0+6.9 123.2_+10.6 13 14.2_+ 2.6 100.0_+11.1 <0.001 28.1_+4.2 128.0+ 4.2 14 9.8-+ 1.9 97.9-+ 7.2 <0.001 25.6-+3.0 120.6_+ 8.8

NS NS NS <0.05 <0.05 <0.05 <0.01 <0.001 <0.001 <0.001 <0.001 _<_0.001 <0.001 <0.001


Fig. 4. Deep cortex of a mesenteric lymph node from a 12 day old neonatal BN rat injected 11 days previously with adult allogeneic L lymph node cells. Note the almost complete depletion of small lymphocytes from the field with only occassional large lymphocytes, and stromal reticular cells (large arrows) remaining. Tissue edema as well as phagocytosed pyknotic debris (small arrow) are evident, x 500

Fig. 5. Cortex of a mesenteric lymph node from a 12 day old neonatal BN rat injected at birth with syngeneic BN lymph node cells. Notice the diffuse collection of mainly small lymphocytes throughout the field compared to Figure 4 as well as the post-capillary venule at the bottom of the field, x 500


Table 2. Mean large lymphocyte labeling intensity within the deep cortex mesenteric lymph nodes and white pulp of Spleen in GVHD

Day Tissue (Grains/Labeled lymphocyte)

Spleen Mesenteric node

Exper. Control p Exper. Control p

l 23,4• 21.9• NS 24.9• 23.4• NS 4 17.8• 24.4• ~0.05 19.0• 25.5• ~0.05 8 11.3• 26.9• ~0.01 14.2• 25.2• G0.01

12 4.8• 20,1• ~0.001 12.1• 21.1• ~0.0l

nodes of experimental animals by day 2 with this difference reaching 21.2% of control density by day 14 (Figs. 4 and 5). However, there was an even more rapid decrease in cell content within the white pulp of the spleen with only 29.2% of those present within the controls by day 7 and 10,0% by day 14 (Fig. 7).

Also, enumeration of the number of labeled cells remaining within these areas at different intervals, revealed a peak in both the deep cortex of mesenteric nodes and white pulp of the spleen on day 3 (Figs. 1 and 2). However, both areas exhibited a lower number labeled of even those few remaining cells within these areas by day 6 with the difference staying about the same until day 14. Thus it appears that although there was a continual decrease in the number of cells present within the deep cortex of mesenteric nodes and white pulp of the spleen of experimental animals, a small persisting population of replicating cells remained in the deep cortex of mesenteric nodes.

Finally, in examining the intensity of labeled cells at different stages of the response, the mean number of grains over experimental and control cells at day 1 as well as control cells at any stage was 20.1-26.9 (Table 2). However, the labeled cells within the deep cortex of mesenteric nodes and white pulp of the spleen on day 12 exhibited only 12.1 and 4.8 grains, respectively (Figs. 6 and 7).

Autoradiographic Quantitation of Proliferation in the Medulla of Lymph Nodes

The medulla of control animals exhibited a level of 5-9% labeled cells throughout (Fig. 8). Most of these cells were medium to large lymphocytes. Plasmablasts were rarely encountered.

However, experimental animals evinced 22% labeling by day 4. This in- creased to approximately 28% by day 7 and although declined to 12.5% by day 11 remained approximately three times control values until death (Fig. 8). Most of these labeled cells exhibited a more basophilic cytoplasm than those found in the deep cortex. In addition, contrary to the deep cortex, the concentra-


Fig. 6. Deep cortex of a mesenteric lymph node from the same animal as Figure 5. Note the large labeled cells in the center of the field, x 1,600

Fig. 7. White pulp of a spleen from an 11 day old neonatal BN rat injected 10 days previously with 30 x 10 6 adult allogeneic L lymph node cells. Notice the marked depletion of lymphocytes from this area. Although two large cells with prominent nucleoli are present next to a central arteriole (arrow), neither is labeled, x 1,250


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tion of these labeled cells per high powered field also remained approximately three times higher in experimental versus control medullas until death (Fig. 8).

Discussion

The results of the present study clearly indicate that incorporation of 3HdT within the spleen and lymph nodes in neonatal rat GVHD occurs sequentially within different areas of these organs. Initially proliferation occurs within donor cell localization areas (deep cortex of lymph nodes and white pulp of the spleen). After cessation of proliferation, there is a gradual depletion of both proliferating and nonproliferating lymphocytes from these areas. Thus subsequent incorporation of 3HdT and organ enlargement must be attributed to other areas of these tissue compartments.

Porter and Cooper (1962) have reported that 30% of 3HdT pre-labeled donor cells had undergone blastic transformation within the white pulp of the host neonatal rat spleens 24 h after their intracardiac administration. Ford et al. (1975) have recently demonstrated that 19% of splenic lymphocytes within previously irradiated F~ hybrid adult rats incorporate a l h pulse of 3HdT 24 h after their inoculation. The lower value of 17% labeling within the white pulp of the spleen obtained in the present study possibly resulted from the use of 1 micron plastic sections rather than 5 micron paraffin sections employed in the other reports. Also, while the present sequential study differs from these


reports in that it detected a peak labeling index difference on day 3 and 6 respectively in the deep cortex of mesenteric nodes and white pulp of the spleen, it is compatible with the extremely high number of L (AgB1) cells which are able to respond to host BN (AgB3) histocompatibility antigens. Whether addi- tional cells are actually proliferating on days 3 and 6 or these intervals represent the peak interval for generation of progeny from those proliferating ceils detected on day 1 cannot be determined from the present data. Although the present study, as well as those mentioned above, do not specifically explain the basic significance of such a massive response by presumably donor T cells in GVH reactions as opposed to normal antibody responses, such a response appears to be necessary in order for systemic GVHD to occur.

The present study when taken together with the previous one compares the proliferative events occurring within donor cell localization areas (deep cortex of mesenteric nodes and white pulp of the spleen) with those occurring within the whole spleen or lymph node compartment throughout the course of lethal GVHD. The data shows that the peak incorporation of aHdT within the lymph node compartment reflects proliferation within the deep cortex. Initial peak incorporation of 3HdT within the spleen probably indicates white pulp proliferation but a secondary peak within the spleen compartment must be occurring within the red pulp. These conclusions come from analysis of the data in Figures 3-8 and Tables 1-2 where a decrease in the radioactivity within the lymph nodes and spleen correlated with a decrease in not only number but also intensity of labeled cells present within the deep cortex and white pulp of each organ respectively.

Three questions arise from this data: 1) What is the fate and function of the cells which initially proliferated so rapidly within these areas? Sprent and Miller (1971, 1972) and Sprent (1973) have previously reported in their system that such cells represent the progeny of specifically responding donor cells which rejoin the recirculating pool and are the short-lived effector cells generated against host antigens. While the present study contains no direct information on the fate and function of these cells, it does contain some data which are not entirely compatible with the conclusions from the above study. Foremost is the question of the fate of not only the labeled but also the very prominent disappearance of unlabeled cells from the deep cortex of the lymph nodes and splenic white pulp. Presumably these represented host and unreactive donor small lymphocytes. Certainly not all of these have been mobilized into the recirculating pool. They do not appear to be populating any other lymphoid organ as all appear to show an almost simultaneous drop in activity. Indeed preliminary observations at the light and ultrastructural level indicate a significant degree of cell death occurring within these areas during GVHD (Clancy, unpublished observations). Also, information from other GVH systems indicate a significant degree of specific (Carottini et al., 1970) and non-specific (Singh et al., 1972) cytotoxicity occurring.

2) What is the cause of the decreased level in labeling intensity even among those cells remaining within these areas during the later stages of GVHD? One possibility is a lengthening of the cell cycle generation time amongst these cells. Another possibility is that as a result of extensive cell death within these


areas the environment around these cells would possess a higher concentration of unlabeled pyrimidine nucleosides which would compete with the 3HdT and thus cause a dilution in individual cell labeling. Indeed in vivo reutilization of DNA from dead and dying lymphocytes has previously been reported (Reike, 1962). Also, an effect of tissue edema, reported to occur even within 24 h in GVH reactions (Ford, 1975), on individual cell labeling cannot be ignored. Experiments are in progress assessing the DPM of 3HdT incorporated per microgram of DNA in order to assess the effect of edema. Finally, the very recent evidence of high levels of lymphocyte membrane bound alpha-fetoprotein occurring during murine GVH reactions and possibly functioning as a negative feedback signal (Keller and Tomasi, 1976) could also be operative in the present system.

3) As proliferation ceases within the deep cortex of mesenteric nodes and white pulp of the spleen, what other areas of these organs incorporate 3HdT such that the whole organ may still remain higher than controls? With respect to the lymph nodes and spleen it has been shown in a number of GVHD systems (Elkins, 1964; Lindholm, 1973) that a very significant plasma cell response develops within these organs. Such a plasma cell response of host origin has also been observed in the present system within primarily the medulla and to a lesser extend the red pulp of these organs, respectively (Clancy et al., 1976a). Indeed, assessment of the labeling index and concentration of labeled cells in 3,112 !am 2 microscopic fields of lymph node medullary areas revealed higher values within these subcompartmental areas throughout the intervals examine (Fig. 8). Most of these labeled cells appeared more basophilic than those blast cells in the deep cortex of mesenteric nodes and some labelled cells were distinctly plasma cell precursors. Thus such a proliferation of plasma cell precursors within these areas could account for the level of 3HdT incorporation within the whole organs being maintained even at a background level relative to control animals even with a relatively lower level of 3HdT incorporation than controls occurring within the deep cortex and white pulp. In addition, recent evidence with the present system has also documented the emergence of a new population of host derived Fc receptor lymphocytes within the spleen and lymph nodes during GVHD (Clancy et al., 1976a). Preliminary evidence from cryostat sections indicates that such Fc receptor cells are located primarily within the medulla and red pulp of these organs (Clancy, unpublished observations). It is also noteworthy that the peak intervals for generation of this new population of Fc receptor lymphocytes in neonatal BN GVHD within the spleen and lymph node compartments was days 11-12 (Clancy et al., 1976a). This is the period in which proliferation in the white pulp and deep cortex was less than controls, but the previous paper demonstrated that 3HdT incorporation within the whole spleen and lymph node compartments was still equal to or even slightly higher than controls (Clancy et al., 1976b). Thus the proliferation of two cell populations (plasma cells and Fc receptor lymphocytes), could be re- sponsible for the level of incorporation of 3HdT.

The present study is compatible with the hypothesis that in neonatal rat GVHD a certain percentage of intravenously administered alloreactive donor lymphocytes are recruited from the circulation into the white pulp of the spleen


and deep cortex of lymph nodes. A high proportion (17-22%) of these donor lymphocytes proliferate within 24 h and generate either T cell factors which may stimulate host B cells in other areas of these organs or give rise to direct cytotoxic lymphocytes (Cerottini et al., 1970). Whether the proliferating cells leave these tissue compartments entirely to join the recirculating pool or infiltrate the liver (Clancy, 1973) cannot be said from the present data. Morphologic data indicates that they may exhibit cytotoxic potential before their exit.

Acknowledgements. The author would like to thank Dr. D.G. Scarpelli for his critical review of the manuscript. The secretarial assistance of Miss Joan Rome and Denise Rankin is also gratefully acknowledged.

References

Caffrey, R.W., Everett, N.B., Rieke, W.O.: Radioautographic studies of reticular and blast cells in the hemopoietic tissues of the rat. Anat. Rec. 155, 41 (1966)

Cerottini, J.-C., Nordin, A.A., Brunner, K.T.: In vitro cytotoxic activity of thymus cells sensitized to alloantigens. Nature (Lond.) 227, 72 (1970)

Clancy, J., Jr.: Nonspecific inhibition of adult thoracic duct lymphocyte migration in neonatal graft-versus-host disease. Lab. Invest. 29, 387 (1973)

Clancy, J., Jr., Rieke, W.O.: Unpublished observations Clancy, J., Jr., Tonder, O., Boettcher, C.E.: The effect of neonatal rat graft-versus-host disease

(GVHD) on Fc receptor lymphocytes. J. Immunol. 116, 210 (1976a) Clancy, J., Jr., Chan, P., Schurath, R., Morley, L.: 3H-Deoxythymidine incorporation during

graft-versus-host disease in the neonatal Brown Norway rat. I. Liquid scintillation examination of the whole spleen and lymph nodes. Virchows Arch. Abt. B Cell Path. (1976b)

Elkins, W.L.: Invasion and destruction of homologous kidney by locally inoculated lymphoid cells. J. exp. Med. 120, 329 (1964)

Ford, W.L., Simmonds, S.J., Atkins, R.C.: Early cellular events in a systemic graft-versus-host reaction. II. Autoradiographic estimates of the frequency of donor lymphocytes which respond to each Ag-B-determined antigenic complex. J. exp. Med. 141, 681 (1975)

Gowans, J.L.: Fate of parental strain small lymphocytes in F1 hybrid rats. Ann. N.Y. Acad. Sci. 99, 432 (1962)

Keller, R.H., Tomasi, T.B.: Alpha-fetoprotein synthesis by murine lymphoid cells in allogeneic reactions. J. exp. Med. 143, 1140 (1976)

Lindholm, L., Rydeberg, L., Strannegard, O. : Development of host plasma cells during graft-versus- host reactions in mice. Europ. J. Immunol. 3, 511 (1973)

Porter, K.A., Cooper, E.M. : Transformation of adult allogenic small lymphocytes after transfusion into newborn rats. J. exp. Med. 115, 997 (1962)

Rieke, W.O.: The in vivo reutilization of lymphocyte and sarcoma DNA by cells growing in the peritoneal cavity. J. Cell Biol. 13, 205 (1962)

Singh, J.N., Sabbadini, E., Sehon, A.H.: Cytotoxicity in graft-versus-host reaction. I. Role of donor and host spleen cells. J. exp. Med. 136, 39 (1972)

Sprent, J., Miller, J.F.A.P.: Activation of thymus cells by histocompatibility antigens. Nature (Lond.) 234, 195 (1971)

Sprent, J., Miller, J.F.A.P. : Interaction of thymus lymphocytes with histoincompatible cells. I. Quanti- tation of the proliferative response of thymus cells. Cellular Immunology 3, 361 (1972)

Sprent, J.: The proliferative response of T lymphocytes to alloantigens in irradiated mice: A mixed lymphocyte reaction in vivo. Transpl. Proc. 5, 1725 (1973)

Received March 10, 1976

3h-deoxythymidine incorporation in graft-versus-host disease in the norway rat

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