Cell surface antigens on erythroid cells

DEVELOPMENTAL

BIOLOGY

59,

237-240

(1977)

BRIEF Cell Surface

Antigens

A Comparison LORRAINE Division

FLAHERTY,

LINDA

ofLaboratories and Research, Cornell University Medical Received

June

NOTES

of Normal CANTOR,

on Erythroid and Anemic

DEBRA

25,1976;

accepted

(W/W)

ZIMMERMAN,

New York State Department College, 1300 York Avenue, in revised

Cells Mice

AND DOROTHEA

ofHealth, Albany, New York, New form

May

BENNETT’ and

New York 12201, York 10021

4,1977

Cell surface antigens of normal and anemic (W/W) mouse erythroid cells have been examined in cytotoxicity assays with two rat antisera. When tested on fetal liver cells, a rat antierythroblast serum recognized antigen(s) present on erythroid cells early in development, while rat anti-adult red blood cell serum recognized antigen(s) present on mature erythroid cells. Each of these sera had different activity on normal ( +/+ or W/+) as compared to anemic (W/W) erythroid cells. INTRODUCTION

Under the homozygous condition, W mutant alleles produce sterile black-eyed white mice with macrocytic anemia. Transplantation and culture studies reveal that absence of pigment and macrocytic anemia are attributable to inherent defects in the pigment and blood-forming cells themselves (Russell, 1970; Mayer, 1970). Furthermore, McCulloch et al. (1964) have found that there are fewer spleen colonyforming units in W mutant mice and that the resulting colonies are smaller in size. The germ cell defect has only been descriptively analyzed (Mintz and Russell, 1957). In all these affected systems, the migrating stem cells fail or have only limited success in reaching and/or interacting with their host tissues. The study of immunogenetics has contributed new information which suggests that abnormalities in the composition of the cell surface are correlated with abnormalities in differentiation. Bennett et al. (1972) and Yanagisawa et al. (1974) have shown that the T/t locus, a region which ’ Present address: Memorial Cancer Center, 1275 York Avenue, York, 10021.

Sloan-Kettering New York, New

influences cellular interactions during embryonic development, also determines a series of cell surface antigens. We have sought information on the composition of the cell surfaces of developing erythroid cells to determine if the W locus is similarly associated with cell surface abnormalities. In this report, we present data which indicate that W/W erythroid cells differ quantitatively from normal cells in the display of cell surface antigens detected by rat antisera. MATERIALS

AND

METHODS

Antisera. A rat antiserum against normal mouse red blood cells (RBC) was prepared by immunizing rats intraperitoneally against WBBG-+I+ adult red blood cells (dose = 10y cells) at weekly intervals. Serum was collected 7-14 days after the third and subsequent immunizations and was heat-inactivated at 56°C for 30 min. The serum was then assayed with absorbed rabbit serum as a source of complement (Boyse et al., 1970) in cytotoxicity tests on liver cells prepared from 14-dayold fetuses. For this purpose, it was necessary to select rabbit sera which gave low toxicity but high cytotoxic activity on fetal

237 Copyright All rights

0 1977 by Academic Press, Inc. of reproduction in any form reserved.

ISSN

0012-1606

238

DEVELOPMENTAL BIOLOGY

liver cells. Some rabbit sera which provided an active complement for adult cells were not lytic with fetal liver cells. Three doubling dilutions of antisera were picked which, with active complement, gave a range of 30-80% cytotoxicity on fetal liver cells, and aliquots were stored in diluted form for subsequent use. A rat anti-erythroblast serum was prepared by immunizing rats intraperitoneally against the strain 745 tissue culture line of live Friend leukemia-infected mouse cells (dose = 2 x lo7 cells) at weekly intervals. Serum was collected as described above. Absorption of antisera. Preliminary tests of rat anti-adult RBC serum confirmed reports by Cantor et al. (1972) that xenogeneic sera prepared against adult red blood cells have virtually no complement-dependent cytotoxicity for nucleated red cells at high dilutions. Although our rat serum must be assumed to have some anti-mouse antibodies, the titer is apparently far lower than the anti-RBC component. Therefore, we chose to use this serum at high dilutions and without absorption, as a reagent for discriminating between mature and nucleated red cells. Rat anti-erythroblast serum, however, was initially highly cytotoxic for all classes of red cells. After absorption three times at room temperature for 30 min with normal ( +I+) red cells at a ratio of 1 part undiluted serum to 1.5 parts packed red cells, the serum had lost activity for nonnucleated cells but was still actively cytolytic on immature stages. This absorbed serum was stored at three dilutions as described above. Cytotoxicity tests. These two sera were subsequently tested on lCday-old fetal livers obtained from embryos of matings between WBIReJ-WI+ mice. Normal embryos ( + I + and WI + 1 were separated from W/W embryos on the basis of color and size of liver (anemic embryos are pale with smaller livers). Embryos the genotypes of which were in doubt were discarded. The

VOLUME 59, 1977

livers were then removed, and cell suspensions were prepared by mincing with scissors and subsequent washing (15 ml of fluid at 800 rpm for 10 min). Classification of embryos was confirmed by determining the number of yolk sac cells in the cell suspension. (Anemic embryos have a higher ratio of yolk sac to definitive erythroid cells; Russell et al., 1968.) Equal amounts of both normal and anemic liver cells (3.75 x lo5 cells) from the same litter were simultaneously challenged in the presence of complement with rat anti-normal mouse RBC and rat antierythroblast sera. Smears were made using a Cytospin I (Shandon) after challenge to determine the differential count of proerythroblasts, basophilic erythroblasts, polychromatophilic erythroblasts, orthochromic erythroblasts, non-nucleated erythroid cells, primitive blood cells of yolk sac origin (yolk sac cells), and other cell types (lymphoblasts, parenchymal cells, etc.) dimethoxybenzidineusing a Wrights-Giemsa stain (according to Ralph, 1941, except that dimethoxybenzidine from Eastman was used instead of benzidine). Counts of live and dead cells were also made after challenge using trypan blue exclusion (dead cells stained blue). In each test the effects of antiserum and complement were compared to the effects of complement alone. RESULTS

AND

DISCUSSION

Each antiserum alone had no effect on the survival of fetal liver cells. Complement alone had very little effect: Only 510% of the cell population was lysed by this treatment. In contrast, each antiserum in the presence of complement had pronounced effects on specific cell types in the fetal liver cell population (Fig. 1). Immature stages were very sensitive to rat antierythroblast serum; at a l/10 dilution in the cytotoxicity test, it killed cells from normal embryos as follows: approximately 95% of the proerythroblasts, 90% of the basophilic erythroblasts, 80% of the poly-

239

BRIEF NOTES

FIG. 1. Treatment of normal and anemic fetal livers with rat anti-erythroblast serum and rat antiRBC serum. Data represent means of eight separate experiments. Cell types are: Pro, proerythroblasts; Baso, basophilic erythroblasts; Poly, polychromatophilic erythroblasts; Ortho, orthochromic erythroblasts; Non, non-nucleated erythroid cells (reticulocytes and erythrocytes); Other, nonerythroid cells; Yolk, yolk sac cells. Treatments are: open bars, complement alone (control); solid bars, rat antierythroblast serum plus complement (greater destruction of early than of late erythroid precursors); stippled bars, rat anti-RBC serum plus complement (destruction of late precursors and RBC only).

chromatophilic erythroblasts, 45% of the orthochromic erythroblasts, and 65% of the yolk sac cells. Most (65%) of the nonerythroid cells were also killed. It had no cytolytic effect on the non-nucleated cells (reticulocytes and erythrocytes) and possibly even enhanced their survival. On the other hand, treatment with rat anti-adult RBC serum at a l/4000 dilution selectively killed approximately 50% of the non-nucleated cells and 30% of the polychromatophilic erythroblasts, but had no cytolytic effect on proerythroblasts, basophilic erythroblasts, yolk sac cells, or nonerythroid cells. These results suggest that these two rat antisera recognize stage-dependent cell surface antigens. The rat anti-erythroblast serum recognizes an “early” antigen (or antigens) which decreases as erythroid cells mature, while the rat anti-adult RBC serum recognizes a “late” antigen (or antigens) which increases during erythroid cell development. It is more than likely

that these xenogeneic antisera also react against other antigens (mouse-specific antigens, viral antigens, etc.), especially rat anti-erythroblast serum, since it is cytolytic for nonerythroid cells and was not absorbed for this activity. However, in this study we were concerned only with the differential effects of these sera on erythroid cells and their ability to selectively kill different cell populations. Therefore, we compared the effects of these two sera on the survival of mutant as compared to normal cells. Because of the differences observed in the starting populations between W/W and +/+ fetal livers, (W/W mice have lower numbers of orthochromic and non-nucleated red blood cells RAT ANTIERYTHROWJST

RAT ANTI-RBC

I /ANTISERUM MUJTION

2. Antibody treatment of polychromatophilic erythroblasts and orthochromic erythroblasts of normal (solid line) and anemic (dashed line) fetal livers. Data represent means of eight separate experiments. The percentage of original cell population was calculated for each serum dilution, cell type, and experiment by comparing the number of cells after treatment with complement alone with the number of cells after treatment with antiserum plus complement. For each test, the differences between lysis of W/W cells and +I+ cells were computed for each cell type and dilution. A t test was then performed on these paired differences (polychromatophilic erythroblasts: rat anti-erythroblast serum, mean difference d = 2.9, standard error S = 1.9; rat anti-RBC, d = 15.7, B = 3.4; orthochromic erythroblasts: rat antierythroblast, d = 10.9, B = 4.1; rat anti-RBC, d = 13.1, B = 2.4). FIG.

240

DEVELOPMENTAL BIOLOGY

in their fetal livers) (see Fig. 11, at all times, we compared the activity of these sera with the complement control for the same cell type. Although the reactivities of sera against proerythroblasts, basophilic erythroblasts, non-nucleated cells, yolk sac cells, or nonerythroid cells were equivalent (Fig. 11, there were clear-cut differential effects on polychromatophilic orthochromic and erythroblasts. This was more sharply seen in the results of a titration study (Fig. 2). The data shown represent mean values from eight independent experiments in which normal and anemic littermates were tested in the same incubation with three dilutions of each antiserum and were compared with their complement controls. The anti-erythroblast serum selectively killed normal rather than W/W orthochromic erythroblasts (P < 0.05 in the paireddifference t test) and was more active on polychromatophilic erythroblasts, although this latter difference was not statistically significant. The anti-RBC serum killed significantly more maturing stages in W/W, i.e., polychromatophilic and orthochromic erythroblasts (P < 0.005). These differences could not be ascribable to different starting populations of cells nor to differential cell sensitivity to complement, since, in our tests, the survival was compared to the original starting population after treatment with complement alone. The different reactivities of cells from normal and anemic fetuses appear to reflect quantitative differences in the display of antigens associated with differentiation, since absorption of either antiserum with liver cells from either normal or anemic fetuses eliminated the activity. Relative to normal erythroid cells, therefore, W cells appear to be out of phase. Developing cells prematurely lose the “early” antigen(s). recognized by the rat anti-erythroblast serum and gain the “late” antigen(s) recognized by the rat anti-RBC serum. This suggests that the W locus may affect the erythroid cell surface

VOLUME 59, 1977

in such a way as to preclude normal recognition of the mitogenic signals needed for continued proliferation. Obviously, the cell surface defect caused by the W locus may be a primary one, or it could alternatively be a consequence of some abnormal differentiative process of these developing erythroid cells, which then leads to an altered makeup of the cell surface. ACKNOWLEDGMENTS This work was supported in part by GRS Grant No. 5 SO1 RR 05649-09, NIH Grant No. AI 12603 awarded by the National Institute of Allergy and Infectious Diseases, and NIH Grant No. HD 05482 awarded by the National Institute of Child Health and Human Development. REFERENCES BENNETT, D., GOLDBERG, E. H., BOYSE, E. A., and DUNN,L. C. (1972). Serological detection of a cell surface antigen specified by the T (Brachyury) mutant genes in the house mouse. Proc. Nut. Acad. Sci. USA 69, 2076-2080. BOYSE, E. A., HUBBARD, L., STOCKERT, E., and LAMM, M. E. (1970). Improved complementation in the cytotoxic test. TrunspZuntution 10, 446-449. CANTOR, L. N., MORRIS, A. J., MARKS, P. A., and RIFKIND, R. A. (1972). Purification of erythropoietin-responsive cells by immune hemolysis. Proc. Nut. Acad. Sci. USA 69, 1337-1341. MAYER, T. C. (1970). A comparison of pigment cell development in albino, steel, and dominant-spotting mutant mouse embryos. Develop. Biol. 23, 297-309. MCCULLOCH, E. A., SIMINOVITCH, L., and TILL, J. E. (1964). Spleen-colony formation in anemic mice of genotype W/W‘. Science 144, 844-846. MINTZ, B., and RUSSELL, E. S. (1957). Gene-induced embryological modifications of primordial germ cells in the mouse. J. Exp. 2001. 134, 207-237. RALPH, P. H. (1941). The histochemical demonstration of hemoglobin in blood cells and tissue smears. Stain Z’echnol. 16, 105-106. RUSSELL, E. S. (1970). Abnormalities of erythropoiesis associated with mutant genes in mice. Zn Wegulation of Hematopoiesis” (A. S. Gordon, ed.), pp. 649-675. Appleton-Century-Crofts, New York. RUSSELL, E. S., THOMPSON, M. W., and MCFARLAND, E. C. (1968). Analysis of effects of W and F genie substitutions on fetal mouse hematology. Genetics 58, 259-270. YANAGISAWA, K., BENNETT, D., BOYSE, E. A., DUNN, L. C., and DIMEO, A. (1974). Serological identification of sperm antigens specified by lethal t-alleles in the mouse. Immunogenetics 1,5767.