Morphology of lymphoreticular tissues in mice with reticulosarcoma

Exp. Path ., Bd. Hi, S. 288-295 (1978) Depart ment of General Path ology and Pathologic Anatomy, Medical Facult y, University of Zagreb (Head: Prof. Dr. A. ZUIOLO), and Laborat ory for Exp erimental Therapy, Department of Experimental Biology and ~Iedi cin e " Rudjer Boskovic" Institute (Director: Prof. Dr. )1. BOH.\;';IC), Zagreb, Yugoslavia

Morphology of lymphoreticular tissues in mice with reticulosarcoma By

Z. P. P AVELI C, "M. BORA:'1J(; and K. PAVELIC

With 8 figures (Received March 8, 1978) Address for correspondence: Dr. Z. 1'. PAVELIC (present address): Department of Experim ental Therapeuti cs, Roswell Park Memorial Institute, Buffalo, New York 14263, U.S.A. Key words: lymphoreticular tissue ; reticulosarcoma; tumor immunology; transplantation: antigenicit y ; lymph node; thymus ; spleen; reticulohistiocytes; mouse

Summary The response of murine Iymphoreticular tissues to transplantation of a syngenic reti culosarcoma with mild transplantation antigenicity was studied by recording weight changes and histology. The results were compared with those obtained in normal mice and mice challenged with syngenic spleen cells. The tumor induced vigorous histological response in regional lymph nodes and mild response in nonregionallymph nodes and spleen. Mesenteric lymph nodes and th ymus did not respond to th e tum or. Observed morphological and gravimetrical changes indicate d that both regional and nonregionallymph nodes and spleen take part in host immune response to tumor. A possible contamination of tumor ti ssue by viruses has been excluded. Lymph oreti cular ti ssu es pa rticipate in host response against tumors with st rong transplantation antigenicity (ALEXANDER et al. 1969; BARD et al, 1969 ; EDWARDS et al , 1971 ; FISHER et al. 1973, 1977 ; KRUGER 1967) but inf orm ation about tumors with mild transplant ation antigenicity ar e few in number and cont radicto ry . E DWA RDS et al. (1971) and SYMES (1965) ha ve found that tumors induce vigorous histological resp onse in both regional a nd nonregional lymph nod es. Ot her research workers have onl y noticed st rong response in regional lymph nodes, whil e n o r esp onse (P AVELIe 'et al. 1973) or weak response (F ISHER et al. 1973, 1977) has been ob serve d in n onregionallymph node s. It may be that antigenicity of these tumors was different. Nev ertheless, it seeme d likely that re-inv esti gation of the matter and usage of a tumor model with mild transplantation antigeni city might provide additional inf ormation that would help resolve some of the issues. To this end, we used a murine reticulosarcoma type A described by PAVELIC et al. (1974, 1976, 1977) endowed with mild transplantation antigenicity (PAVELIe 1975). Contamination of t um or tissue by viruses wa s exclu ded by means of elect ro n microscopic examination.

Material and methods An im al s Inbred C57BL mice, 10-14 weeks old (20-24 g) were used in these experiments. They were separated into groups of 6 and supplied with pelleted food and ta p water ad libitum. T um or Reticulosarcoma developed sponta neously in 1971 in an 18-month-old C57BL female breeder and has been maint ained by serial subcuta neous tr ansplant ation. In th ese experiments, t he tumor was in its 18t h generation and was endowed with mild tran splant ation ant igenicity. All animals received subcut aneous inoculations of :2.5 X 1Q6 cells.

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Histolo g y The lymph nodes, spleen, th ymus and tum o: were fi~ed in a mixture of al c~hol (85.%), acet ic acid (5 % ) and formaldeh yde (10 %), embedd ed in paraffin, cut at 511m, and stame~ WIt~ l!ematoxylin and eosin and methyl green-pyronine. Immun~blasts , plasma ~ ells and retIculohI~tlO cytes were count ed in paracortical and medullary area of regional and nonre gional lymph nodes III 10 separate immersion fields. From t his th e mean value was calculated. Immunoblasts a p ~ e are d ~n .greatest intensity in th e paracorti cal area. They measured 15- 2511m and had pale nu clei containing 1 or more pyroninophilic nu cleoli. Their cytoplasm was strongly pyroninophilic. Plasma cells were located predominantly in the medullary area of lymph nodes. They were greater than 811m in diameter. The nucleus contained densely staining chromatin material surrounded by pyroninophilic cytoplasm. Cells designated as reticulohistiocytes measured more than lOl1m in diameter with big, pale, sometimes irregular and often elongated nuclei. The slides were coded so as to eliminate knowledge of th e experimental circumsta nce of the animals from which samples were obtained. Statisti cal analysi s was done by means of the St udent t-test. Electron Mi cros copy T e chnique For electron microscopy, tumor tissue was prepared using th e method previously described by P A VELIC et al. (1977). Blood Co u nts Blood samples for leukocyte counts were taken from the t ail-vein. Groups of mice with tumors were repeatedly bled twice a week. Blood Sm e ars The smears were stained after air dryin g, using the Pappenheim method (May-Grunwald 3 min and 5 % Giemsa st ain, 30 min). Plan of th e Experiment s The animals were divid ed into three groups of 48 mice each. Animals in group I received 2.5 x 1Q6 syngeni c t umor cells inje cted subcuta neously into the left thigh. Animals in group II received 2.5 X lOS syngenic spleen cells in the same mann er. Group III consist ed of 48 normal C57BL mice, separated as controls. At 0, 1, 2, 3, 4, 5, 6 and 7 weeks after tumor implantation, regional (left inguinal and axillary) and nonr egional (right inguinal and axillary) lymph nodes, mesenteric lymph nodes, spleen , t hy mus and t umor were removed from all animals, weighed and then stored for histopathologica.l exa minat ion.

Results The Growth of Tu mor Following subcutaneous inject ion of 2.5 X 106 tumor cells, the implanted reticulosarcoma was removed and weighed at periodic t imes (fig. 1). Th e tumor gr ew in all recipient mice. Weight s o f th e Organ s Regional lymph nod es gained weight more quickly than nonregionallymph nodes (fig. 2). However , the weight curves apparently ran in the same fa shion, peaking b y the end of the 2nd week. Th ereafter they declin ed until the animals di ed. No in crease in the weight of the mesenteric lymph nodes tfig. 1) and thymus (fig. 3) was observed at any time. The weight of the spleen increased rapidly and reached its maxi mal size by the en d of the 4th week (fig. 3). Thereafter it started to decline but remained p ermanently above the normal size. Hematol ogi cal Chan g e s White bl ood cell cou nts remained within normal ranges. No qualitative changes were seen in the p eripheral blood picture. Tumor cells wer e not identified on the smears.

Mi cr o s c o p i c Findin g s Lymph Nodes: No morphologic changes were evident at any time in mesenteri c lymph nodes of the animals in group I (mice injected with tumor cells) and in the lymph nodes of animals in gro ups II (mice injected with syngenic spleen cells) and III (normal mice). After on e week, regional inguinal and axillary lymph nodes of animals injected with tumor cells showed enlarged paracortical areas, densely populated with pyroninophilic cells (or so-called Immun oblasts) and small lymphocytes (fig. 4 and 5). The number of 21*

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Fig. 4. Number of immunoblasts, small lymphocytes, plasma cells and reticulohistiocytes in the nguinal-regional and nonregion al lymph nodes of tumor bearing mice. Bars indicate standard dieviati ons,

immunoblasts appeared ' to increase up to the fourth week when a decrease followed. At that tim e, an increase in th e numb er of reticulohistiocytes (fig. 6) was noticed. It remained high until th e animal s died. The numb er of small lympho cyte s had a tend ency to decrease betw een th e first and th e sevent h weeks. At t he end of th e 7th week th e paracorti cal area was minimal in size, containing mainl y reticulohisti ocyt es and few lymphocyt es. In creased activity of th e medulla, as judged by the numb er of pyronin ophilic plasma cells, was noticed at th e end of t he 2nd week (fig. 7). Th e medulla remain ed crowded with plasma cells until t he 4th week. Afte r that peri od th e numb er of plasma cells pr ogressively decreased until all animal s died. Germinal centers were seen after t wo weeks and remained pr ominent until the end of th e 3rd week. Thereafter occasional germinal cent ers were am ong th e follicles in the cortex. Nonregional inguinal and axillary lymph nodes of tumor bearin g mice showed similar changes as regional lymph nodes. However, t hose cha nges were much less prominent and disapp eared earlier. Th y m u s : No morph ologic changes in th e thymu s of th e tumor bearing anim als and animals inj ected with syngenie spleen cells were observ ed at any time. Spl e en : No morph ologic changes in the spleen of animals in groups II and III were observed at any time. After th e 2nd week, proliferation of immunoblasts and plasma cells occurr ed in the red pulp of th e spleen in tumor bearing mice (f,ig. 8). At the same time the red pulp showed an increa se of peritrabecular myeloblasti c cells an d the megakaryocytes. These cells exhibited pyroninophilia and therefore confused th e histological interpretation and counting of cells in the spleen. The number of immunoblasts reached a peak around th e 4th week, whereafter a decline followed. At the sam e time progessive increase of reticulum cells was noticed up to th e end of the experiment. The increase of the size of white Fig. 1. Changes in weight of tum or ( e - e ) and mesenteri c lymph nodes of tumor bearing mice (0 - 0). Bars indi cate sta ndard deviations. Fig. 2. Changes in weight of inguinal lymph nodes: Nor mal mouse (0-0) ; mouse inje cted with syngenic spleen cells (e -e ); t umor bearin g mouse-regional ( /':" - /':,, ) ; t umor bearing mouse-nonregional ( /':" - /':,, ). Bars indi cate st andard deviati ons. Fig. 3. Changes in weight of spleen (0 - 0 ) and th ymus ( e - e ) of t umor bearin g mice. Bars indicat e standard deviations.

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Fig. 5. Inguinal-regional lymph node of the mouse given injection of syngenic tumor cells two weeks previously. Note densely populated immunoblasts and few lymphocytes in paracortical area. Hematoxylin and eosin, x 670. Fig. 6. Inguinal-regional lymph node of the mouse given injection of syngenic tumor cells four weeks previously. Note increased number of reticulohistiocytes in paracortioal area. Hematoxylin and esoin, X 670.

Fig. 7. Inguinal-regional lymph node of t he mouse given injection of syngenic tumor cells two weeks previously. Note plasma cells in t he medulla. Hemat oxylin and eosin, X 670. Fig. 8. Spleen of the mouse given injection of syngenic tumor cells two weeks previousl y. Note immunoblasts in the red pulp . Hematoxylin and eosin, x 670.

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pulp follicles and their tendency to merge together was observed at the end of the 2nd week. Germinal centers became increasingly prominent. These changes increased until the 4th week, when a decrease was noticed up to the death of animals. Electron Microscopic Findings Contamination of the tumor tissue by viruses, or possible virus etiology of this tumor, was assessed by means of electron microscopy. No virus particles were observed in ultrathin sections of 8 tumor-bearing mice.

Discussion The weight and morphological appearance of regional and nonregional lymph nodes and spleen in tumor hosts suggest that these organs were involved in a response to antigenic stimulation. The appearance of immunoblasts and proliferation of small lymphocytes in the early stages of tumor growth suggest initiation of cell mediated immunity. However, increase in the number of plasma cells and the appearance of active germinal centers in both regional and nonregional lymph nodes and spleen suggest that the tumor also elicited a humoral immune response. In the terminal stages of tumor growth, cells associated with cellular and humoral immunity disappeared and were replaced by reticulohistiocytes. Similar histological changes were observed in draining lymph nodes and spleens of mice given injections of allogenic spleen cells or syngenic tumor cells (BARD et al. 1969; EDWARDS et al. 1971; FISHER et al. 1973; KRUGER 1967), but were not seen in lymph nodes and spleens of animals that received injections of syngenic spleen cells. The stimulated extramedullary myelopoiesis, described by PARSONS and WARREN (1941) in the spleen of mice carrying grafted sarcomas was also seen in our study. The present study has not shown any changes in thymus and mesenteric lymph nodes. This is related to the results of KRUGER (1967). However, Saw et al. (1968) described depletion of thymocytes in response to an allogenic tumor. EDWARDS et al. (1971) showed greater cellular activity in the thymus of tumor bearing animals, implying that the thymus itself was the site of some immunological activity. It seems to us that observed differences in the reaction of lymphoreticular organs in animals with different tumors can be caused by variations in the strength and/or amount of tumor-specific transplantation antigens. It has been shown earlier in this laboratory (PAVELIC 1975) that reticulosarcoma used in this study was endowed with mild transplantation antigenicity. Tumor specific antigens of this reticulosarcoma are probably weaker than tumor specific antigens in neoplasms described by others (ALEXANDER et al. 1969; BARD et al. 1969; EDWARDS et al. 1971; FISHER et al. 1973). It is difficult to be sure that observed antigenicity originated in the tumor itself and was not due to an extraneous agent (such as virus) carried in the tumor tissue. No viruses have been observed by electron microscopic examination of tumor tissues. KRUGER (1967) checked a mouse sarcoma on the presence of 11 viruses and only the lactic dehydrogenase virus was found. Furthermore, transmission of this reticulosarcoma could not be accomplished by injecting cell free extracts of the tumor into newborn mice (PAVELIC et al. 1977). Beside of these facts the possibility of undiscovered viruses of other extraneous agents carried in the tumor tissue cannot be quite surely ruled out. Further studies including virus profile and more extensive electron microscopic examination of tumor tissues should gain more information about factors involved in the antigenicity problem.

Acknowledgements The proficient technical assistance of Ljerka Boskovic, Zlata Jagodic and Jozica Miholjevic is gratefully acknowledged. 294

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