Distribution and ontogeny of B cells in the garden lizard, Calotes versicolor

DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY, Vol. 9, pp. 3 0 1 - 3 1 0 , 0145-305X/85 $3.00 + .00 Printed in the USA. Copyright (c) 1985 Pergamon Press Ltd. All rights reserved.

DISTRIBUTION

AND

ONTOGENY OF B CELLS CALOTES VERSICOLOR

IN

THE

GARDEN

1985.

LIZARD,

K.Natarajan and VR.Muthukkaruppan Department of Immunology, School of Biological Sciences, Madurai Kamaraj University, Madurai - 625 021, India.

ABSTRACT

Surface immunoglobulin bearing (slg+) cells were identified in the lizard Calotes versicolor by immunofluorescence using a polyvalent antiserum to lizard immunoglobulins and class-specific antibodies to lizard IgM and IgY. 53.3+1.6% of spleen cells, 23.6+0.8% of peripheral blood monoclear cells, 21.5+1.8% of bone marrow mononuclear cells and less than 1% of thymus cells were found to bear immunoglobulin (Ig) on their surface. A similar proportion of cells in each tissue was stained with rabbit antilizard ~ (specific for IgM) whereas only a small proportion of cells were stained with rabbit anti-lizard (q~) (specific for IgY)~ Adult thymectomy significantly increased the proportion of sIgM+ cells in spleen and blood whereas high dose cyclophosphamide (300mg/kg body weight) decreased the proportion of sIgM+ cells thus suggesting that sIg+ cells in the lizard are of B cell lineage as in higher vertebrates. Ontogenetic studies indicate that embryonic liver is an organ enriched for sIgM+ cells at certain stages of development and thus suggest liver to be a site for differentiatin of sIg+ cells in lizard embryos.

INTRODUCTION

Studies on the immune system of phylogenetically key animals could contribute significantly to our understanding of the evolution of the immune response in higher vertebrates. Reptiles being evolutionary precursors of both birds and mammals represent a pivotal phylogenetic group and thus a study of their immune system assumes a special significance. However, relatively few investigations have been made on lymphocyte develpment and heterogeneity in reptiles (1-6). Here, we describe the identification of surface Ig by immunofluorescence on a population of lymphocytes of the garden lizard, Calotes Versicolor and present data consistent with the proposition that s I ~ e l l s in the lizard are B lymphocytes. Further, the ontogeny of B cells has been studied by examining lizard embryonic tissues at various developmental stages. The results show that embryonic liver, at certain stages of development is an organ enriched for sIgM+ cells thus indicating that this is a site for differentiation of sIg+cells. 301

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MATERIALS AND METHODS

Animals: Adult lizards were obtained from the field and maintained in wooden cages and fed with termites and water ad libitum as described earlier (7). The method for obtaining lizard embryos and their staging was as described by Muthukkaruppan et al (7). Purification of lizard serum immunoglobulins and preparation of antisera: Three fractions, designated as lizard Ig,IgM and IgY were isolated from pooled lizard serum by DEAE-cellulose (Sigma,USA) chromatography and gel filtration on Sephadex G-200 or Sepharose 6B (Pharmacia, Sweden). Immunoelectrophoresis against rabbit anti-lizard serum as well as sodium dodecyl sulfate polyacrylamide gel electrophoresis confirmed the purity of the three fractions (8). A polyvalent antiserum to lizard Ig fraction was raised in rabbits. Antisera specific for lizard IgM (anti-~) and IgY (anti-~) were prepared by immunizing rabbits with purified IgM or IgY followed byadsorption of the antisera over immunoadsorbent columns of IgY or IgM respectively bound to cyanogen bromide activated Sepharose 4B (Pharmacia, Sweden). Following this procedure, rabbit anti-lizard ~ and anti-lizard-~ reacted only with lizard IgM and IgY respectively in immunoelectrophoresis(8). Cells: Single cell suspensions from spleen and thymus of adult lizards were prepared as described previously (9). To obtain peripheral blood lymphocytes lizards were bled by cardiac puncture into Alsever's solution, blood was layered over Ficoll-Hypaque (Lymphoprep, density 1.077, Nyegaard, Norway) and centrifuged at 400g for 20 min at 15°C. Cells banding at the interface were isolated and used for immunofluorescence. The preparation was found to contain 3% erythrocytes, 77% mononuclear cells and 20% granulocytes as revealed by May Grunwald-Giemsa staining. Adult bone marrow cells were obtained by aspiration of marrow contents of femurs followed by Ficoll-Hypaque separation as described above. Cells from embryonic liver were obtained by gently teasing the tissue in Alsever's solution with a pair of fine scalpels. Individual livers were dissociated to enumerate total white blood cells per liver. For immunofluorescence studies, the contents of 10-12 livers of a particular stage were pooled. Blood was obtained from lizard embryos by allowing embryos to bleed freely into Alsever's solution in an embryo cup. Cells were then subjected to Ficoll-Hypaque separation as described above. For immunofluorescence studies blood from 10-15 embryos at a particular stage was pooled. Membrane immunofluorescence: Ice-cold L-15 Leibovitz medium (GIBCO, USA) diluted 3:1 with triple distilled water, supplemented with 2% bovine serum albumin (w/v) (Sigma, USA) and containing 20 mM sodium azide (to prevent capping) was used. The whole procedure was carried out at 4°C. All a~tisera were absorbed with lizard erythrocytes before use in immunofluorescence assay. Cells from respective tissues were first exposed to rabbit anti-lizard Ig o~ anti-lizard ~ or anti-lizard ~ for 30 min, washed thrice with medium, exposed to fluorescein isothiocyanate (FITC)-conjugated goat anti-~abbit IgG(Cappel Laboratories,USA) for 30 min, w a s h ~ thrice, mounted in phosphate buffered saline-glycerol and observed in a Zeiss Fluoval Microscope (Zeiss,Jena) fitted with HBN 200 mercury lamp with D224G excitation filter and G247 barrier f~Iter. As controls, the cells were incubated with pre-immune rabbit sera followed by FITC-labelled secondary

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antibody. Cells in each field were examined alternatively under phase contrast and blue-violet excitation. At least 200 mononuclear cells were counted per slide (i.e. excluding erythrocytes and granulocytes)and each sample was counted in duplicate and in most cases after coding the samples. Thymectomy: The method previously described by Pitchappan Muthukkaruppan (I0) was followed to thymectomize adult lizards.

and

Cyclophosphamide: Cyclophosphamide (CY, Endoxan, ASTA, India) was dissolved in sterile distilled water and 300 mg/kg body weight was injected intraperitoneally. This dose was found earlier to deplete B cell functions but not T cell functions in the lizard (11). Histolosy: Smears of embryonic liver from various stages were made, air dried quickly, fixed in methanol and stained with May Grunwald-Giemsa and differential count was performed. Embryonic liver was dissected out, fixed in Bouin's fluid, processed and stained with Harris alum haematoxylin and counter stained with eosin following conventional histological methods. Statistics: The data are expressed as arithmetic mean ~ standard error. P values were determined by a two-tailed students t-test.

RESULTS Distribution of sis+ cells in the adult lizard: Cells from lizardspleen, thymus, blood and bone marrow were exposed to rabbit anti-lizard Ig fraction, anti-lizard ~ or anti-lizard ~ followed by FITC-goat anti-rabbit IgG. The results are presented in Table i. Negligible staining was observed with pre-immune rabbit sera and less than 1% of thymus cells were stained with rabbit anti-lizard Ig. It can be seen from the table that lizard lymphocytes from spleen, blood and bone marrow can be divided into two subpopulations based on the/presence or absence of surface Ig. The major class of surface Ig on the positive cells is IgM. B cell nature of sis+ cells: The absence of slgM+ cells in the lizard thymus suggests that sIgM+ cells in the lizard are independent of the thymus and belong to the B cell lineage. To confirm the B cell nature of sIgM+ cells in the lizard, two approaches werefollowed. It was shown earlier that adult thymectomy results in an abrogation of humoral immunity to sheep red blood cells (SRBC) (ii) and depletion of lymphocytes in periarteriolar region of the lizard spleen (12). It was thus of interest to determine the effect of adult thymectomy on the proportion of slgM+ cells in spleen and blood. It is evident from Table 2, that one month after adult thymectomy there was a highly significant ÷ increase in the proportion of slgM cells in the spleen and peripheral blood. This increase is due to depletion of T cells by thymectomy and suggests that slgM+ cells in the lizard are independentof the thymus.

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TABLE i Distribution of sIg+cells

in lymphoid organs of the adult lizard

% positive cells a stained with

Cells from

Spleen Blood Bone marrow Thymus

Pre immune rabbit sera (control)


anti-lizard Ig fraction

53.3+1.6 23.6+--0.8 21.551.8 <1

anti-lizard ~

anti-lizard I?

52.5+3.2 21.051.3 21.0T0.9
5.4+0.7 6.7T0.9 5.151.2 <1

a positive cells expressed as percentage of mononuclear each value is the mean ! S.E. of eight animals.

Effect

cells;

TABLE 2 of Adult Thymectomy on the Proportion of slgM + Cells Spleen and Blood of the Lizard

Cells from

% positive Sham Controls

Spleen Blood

53.9+1.9 23.551.8

cells a in Thymectomi~ed lizards ~

81.3+2.2 62.5+--2.4

a positive cells expressed as percentage of mononuclear each value is the mean ~S.E. of 8-10 animals. b cells were obtained 30 days after adult thymectomy.

in

p value

<0.001 <0.001

cells;

The second approach used to determine the B cell nature of slgM + cells in the lizard was to study the effect of CY on the proportion of sIgM+cells. Numerous studies with mammals as well as in the lizard indicate that CY at an appropriate dose has a severe effect on the B cell compartment rather than the T cell compartment (11,13-15). As shown in Fig.l, there was a significant decrease in the proportion of sIgM+cells in the spleen four days after CY administration. Normal levels were regained by the 10th day following treatment. Lymphoid cells from blood and bone marrow examined on the 4th day following CY administration also showed a significant reduction of slgM~cells. Thus the dose of CY which abrogates the humoral immune response to SRBC (11) also depletes sIgM+cells significantly. These observations suggest that sIgM + cells in the lizard are B cells similar to those of mammals and birds.

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BONEMARROW

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Fi$.l Effect of CY on the proportion of slgM+ cells. Positive cells are expressed as mean percentage (!S.E.) of mononuclear cells of 8-10 animals. Data for bone marrow were obtained with lymphocytes pooled from 8 animals

0

SPLEEN

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DAYS AFTER CY iNJECTION

Ontoseny of slsM + cells in the lizard: To date, no information is available on the ontogenetic origin of sIg + cells in reptiles. The present investigation suggesting sIgM+cells in adult lizards to be B cells along with the earlier work by Muthukkaruppan et al. (7) describing a series of developmental stages for the lizard has made possible the use of lizard embryos for experimental purposes. To study sIg+ cell ontogeny in the lizard the distribution of sIgM + cells in the liver, thymus, spleen and blood of lizard embryos at various developmental stages was studied. Prior to assaying for slgM + cells, embryonic liver at various stages was examined for the total number of leukocytes per liver. Stained smears were also observed for the proportion of lymphocytes and granulocytes. Liver at stage 33 was predominantly granulocytic. The proportion of granuloeytes was decreased at subsequent stages even though there was an increase in the number of leukocytes per liver (Table 3). The percentage of lymphocytes was only 4 at stage 33. Subsequently, there was an increase in the proportion of lymphocytes reaching a maximum of 65-80% during stages 36 to 42. The interesting point of observation was the tremendous increase in the number of total leukocytes per liver during stages 36 and 38 and this increase was mainly due to the increase in the number of lymphocytes. These observations indicate the occurrence of lymphopoiesis in the liver during stages 36 and 38. At subsequent stages of development, i.e. stages 39 and 42, there was a reduction in the number of total leukocytes per liver. Histological studies of embryonic liver confirm these observations. Lymphocytes were observed only rarely in liver sections at stage 33. However, sections of llver at stage 36 were found to contain lymphocytes in greater frequencies (Fig.2). At stage 42, extensive scanning of liver sections revealed lymphocytes only occasionally.

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TABLE 3 Differentiation of slgM + Developmental

Stage

33 34 36 38 39 41/42

Total leukocytes per liver

0.45xi04 2.90xi04 3.00x104 3.00x104 1.50xlO 4 0.50x104

cells in the liver Stages of the lizard

Differential L

4% 25% 80% 66% 65% 71%

count G

96% 75% 20% 34% 35% 29%

at

Various

Number of lymphocytes per liver

sIgM + cells a

180 7,250 24,000 19,800 9,750 3,550

0 21% 34% 31.2% 8% b

aresults expressed as percentage of mononuclear cells; data are obtained from pooled liver cells. bpredominantly liver parenchyma cells were observed. They were large and non-specifically stained. L - Lymphocytes

G - Granulocytes

Fi$.2 Section of liver from stage 36 embryo of Calotes Versicolor (X 8 5 0 ) - L - Lymphocyte P - Liver parenchyma

Having thus established that lymphocytes are present in significant numbers in embryonic liver, the proportion of sIgM+cells in the liver at various developmental stages was determined (Table 3). sIgM+cells were first seen in the liver at stage 34, increased numbers were evident at stages 36 and 38 and they subsequently decreased at stages 39 and 42. Thus maximal levels of sIgM+cells occurred in the liver during the same stages of development at which large numbers of lymphocytes were present.

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Embryonic blood at various developmental stages was also examined for its content of sIgM+cells. These cells were found at levels of 7.7% at stage 36 and 11-12% from stage 38 till hatching. Adult levels (22%) of sIgM + cells were present in blood two weeks after hatching. In two-day hatchlings, bone marrow contained only 5% lymphocytes as revealed by May Grunwald-Giemsa staining (the remainder being granulocytes), in contrast to 49% lymphocytes in the adult bone marrow. The total number of lymphoid cells in embryonic and hatchling bone marrow was too small to carry out immunofluorescence studies. Thymus cells could be obtained in sufficient numbers for immunofluorescence analysis only at stage 41/42 i.e. the terminal stages of embryonic development. No sIgM+cells were found in the thymus at this stage. Spleen cells could be obtained in sufficient numbers only in two-day old hatchlings at which time 30% of mononuclear cells were sIgM +. Adult levels (52%) of sIgM + cells were observedin the spleen two weeks after hatching. These results indicate that embryonic liver is an organ enriched for sIgM+cells during specific stages of development and thus may be a site for the maturation of B cells in the lizard.

DISCUSSION The present study has identified by immunofluorescence a sIg + population of lymphocytes which is present in lizard spleen, blood and bone marrow but is absent in the thymus. The major Ig class on lizard sIg+lymphocytes is IgM, similar to mammalian and avian B lymphocytes (16,17). The second Ig class found in lizard serum,IgY, is expressed on a relatively small number of lymphocytes as in amphibians and birds (17-19). From the data in Table I it appears that lizard sIgY + cells also express sIgM. Yet no reports are available on the proportion of sIgY+cells in lymphoid organs of other reptiles, though Fiebig and Ambrosius (20) identified IgY immunochemically on the surfaces of tortoise splenocytes. Two evidences indicate that slgM + cells in the lizard are of B cell lineage. Firstly, adult thymectom~ resulted in a highly significant increase in the proportion of sIgM cells in the spleen and blood thus indicating that these cells are independent of thymicinfluence. Secondly, CY administered at a dose which selectively affects the plaque-forming cell response to SRBC (11) also depletes sIgM+cells significantly. The identification of B cells in the lizard on the basis of surface Ig complements our earlier investigations which suggested the existence of B cells in the lizard by functional criteria. A strong indication for the existence of a B cell compartment in the lizard was the observation that adult thymectomy did not affect the response to polyvinyl pyrrolidone, a thymus independent antigen (Jayaramanet al., unpublished), whereas it abrogated the response to SRBC, a thymus dependent antigen (ii). Moreover, plaque-forming cells were not susceptible to the action of a anti-lizard thymocyte serum and complement (I). Taken together, these studies strongly suggest the existence of a B cell compartment in the lizard which is analogous in functional and structural terms to that of higher vertebrates.

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Since there is no information to date on the ontogenetic origin of B cells in reptiles, an attempt was made in the present investigation to study the development of B cells in the lizard. In birds, generation of B cells has been shown to occur in the bursa of Fabricius (21,22). In mammals B cells are generated within the foetal liver and foetal spleen (23-25). Among lower vertebrates studies on B cell ontogeny have been limited to the anuran amphibian Rana pipiens. In this species embryonic urogenital tissues appear to be the earliest sites of B cell generation and B cells were identified in embryonic liver at a later developmental stage (19).

The present investigation in the lizard indicates embryonic liver to be a site for B cell maturation. This is based on the obvervations that i) lymphocytes are detected in embryonic liver as early as stage 33, much earlier to the appearance of lymphocytes in the spleen (26) and the thymus (2) and ii) lymphopoiesis occurs in the liver as indicated by the increasing number of lymphocytes in the liver from stages 34 to 38, coinciding with the time of appearance of a large number of slgM + cells. The site of B cell differentiation thus appears to be similar in both the lizard and the mouse. The occurrence of a large number of granulocytes prior to the appearance of lymphocytes and the transient nature of lymphopoiesis in lizard embryonic liver are also the characteristics of murine foetal liver (23,27). A further point of similarity is the distribution of B cells in adult lizard lymphoid tissues, notably the bone marrow. In the chicken, by contrast, embryonic liver appears to play no role in B cell differentiation (21) and further, slg+cells have been detected in fewer numbers in the adult avian bone marrow (28,29). However, a point of similarity of the lizard and avian immune system is the presence of IgY, distinct from mammalian IgG. Thus the lizard immune system appears to posses characteristics of both avian and mammalian species. Though earlier investigations identified murine foetal liver to be the site of B cell differentiation, it now appears that stem cells may be induced to begin differentiation along B cell lines even prior to their entry into foetal liver. Thus cells capable of B cell differentiation in vitro have been observed in mouse placenta and blood even before the appearance of pre-B cells in the foetal liver (30,31). Pertinent to this is the observation that chick embryo urogenital tissues contain pre-B and B cells from 11-18 days incubation (32). Further studies are thus required to identify other embryonic tissues where early B cell differentiation might occur in the lizard.

ACKNOWLEDGEMENT This work was supported by the Department of Science and Technology No.HCS/DST/512/78) and the Department of Atomic Energy, India.

(Grant

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21. KINCADE,P.W.and COOPER,M.D. Development and distribution of immunoglobulin containing cells [n the chicken. An immunofluorescent analysis using purified antibodies to u, , and light chains. J. Immunol. 106, 371, 1971 22. LYDYARD,P.M., GROSSI,C.E. and COOPER,M.D. Ontogeny of B cells in the chicken. I. Sequential development of clonal diversity in the bursa. J. Exp. Med. 144, 79, 1976 23. OWEN, J.J.T., COOPER, M.D. and RAFF, M.C. In vitro generation of B lymphocytes in mouse foetal liver, a mammalian 'bursa" equivalent. Nature. 249, 361, 1974 24. OWEN,J.J.T., RAFF,M.C. and COOPER,M.D. Studies on the generation of B lymphocytes in the mouse embryos.Eur. J. Immunol. 5, 468, 1975 25. ANDREW,T.A.and OWEN,J.J.T. Studies on the earliest sites of B cells differentiation in the mouse embryo. Dev. Comp. Immunol. 2, 339, 1978 26. KANAKAMBIKA,P. and MUTHUKKARUPPAN,VR. Lymphoid differeniation and organization of the spleen in the lizard, Calotes versicolor. Proc. Ind. Acad. Sci.. LXXV III Sec. B37, 1973 27. OWEN,J.J.T., WRIGHT,D.E., HABU,S., RAFF,M.C. and COOPER, M.D. Studies on the generation of B lymphocytes in foetal liver and bone marrow. J. Immunol. 118, 2067, 1977 28. ALBINI,B and WICK,G. Immunoglobulin determinants on the surface of chicken lymphoid cells Int. Arch. allergy Appl. Immunol. 44, 804, 1973 29. HUDSON,L. and ROITT,I. Immunofluorescent detection of surface antigens specific to B and T lymphocytes in the chicken. Eur. J. Immunol. 3, 63, 1973 30. MELCHERS,F. Murine embryonic B lymphocyte development in the placenta. Nature 277, 219, 1979 31. MELCHERS,F and ABRAMZUK,J. Murine embryonic blood between day 10 and 13 of gestation as a source of immature precursor B cells. Eur. J. Immunol. 10, 763, 1980 32. ZETTERGREN,L.D., CHEN,C.L. and EWART,D.E. Pre-B and B cells in chick embryo urogenital tissues: A newly reported site for cells in early B lineage in endothermic vertebrates. Amer. Zool. 78, 793, 1980 Received : September, 1984 Accepted : October, 1984