- Email: [email protected]
Rosette-forming cells in the unimmunized mouse: Morphological studies with phase contrast and electron microscopy
CELLULAR
IMMUNOLOGY
2, 182-198 (1971)
Rosette-Forming Morphological
FELIX Institut
Cells in the Unimmunized Studies with Phase Contrast Electron Microscopy REYES 1 AND JEAN-FRANCOIS
de Pathologie
Cellulaire (U 48, INSERM), 94-Le Kremlin-Biche, France Received
December
Mouse: and
BACH
Hdfiital
de Bice^tre,
24,197O
Spontaneous rosettes, obtained with nonimmunized mouse spleen cells and heterologous erythrocytes, have been examined. For electron microscopy, the rosettes are isolated by micromanipulation; embedding is performed by a technique suitable for single-cell study. Furthermore the morphology of these spontaneous rosette-forming cells (RFC sp.) has been investigated in other experiments where spleen cells are incubated with antilymphocyte serum (ALS) and complement, at a concentration which inhibits rosette formation irz vitro. The population of RFC sp. contains two classes of cells: lymphocytes and macrophages. The lymphocytes appear to have a constant morphology, corresponding to the “inactive” monoribosomal small lymphocyte. The macrophages exhibit to a variable degree signs of erythrophagocytosis. These observations are compared with the already known morphology of cells involved in antibody production. The existence of two cell types in the RFC sp. population is discussed in relation to recent data concerning cellular cooperation in the induction of an immunological response to antigens such as heterologous erythrocytes. After incubation with ALS and complement, the RFC sp. show no alterations at the ultrastructural level.
INTRODUCTION
The production of antibodies at the cellular level has been studied by several methods. In one of these, immunocytoadherence, the immunological response to heterologous red cells is studied by incubating lymphoid cells and red cells together in vitro. Heterologous red cells ring themselves around the lymphoid cells forming rosettes. When observed in suspension at low magnification, the rosette-forming cells (RFC) and surrounding erythrocytes assumea “morula form” ( 1) . The term “rosette” is best reserved for the appearance of aggregates in section or between slide and coverslip, where the central lymphoid cell is ringed by erythrocytes. The RFC bears on its membrane specific receptors which have the antigenicity of immunoglobulins, which are synthesized by the RFC, and which are not ad1 Reprint
requests
are to be sent to: Felix Reyes, Mondor, 94- Creteil,
(U 91, INSERM), CHU Hem-i
182
M.D., Unite France.
de Recherche
sur les Anemies
MORPHOLOGY
OF ROSETTE-FORMING
CELLS
183
cells” thus sorbed from circulating antibodies ( 1, 2). The term “antibody-forming appears justified, at least for the great majority of RFC in the spleen or lymph nodes of the immunized animal, although there is in these tissues a low proportion of rosettes formed by cells which have adsorbed “cytophilic” antibodies (2). Morphological studies of RFC after immunization have recently confirmed that they are principally lymphoid cells, which at the ultrastructural level have a variable degree of differentiation, and that some RFC are macrophages (3,4). Rosette-forming cells also exist without immunization. A small number of mouse spleen cells can form a rosette in vitro with sheep red blood cells (SRBC). The number of RFC in the unimmunized mouse is constant for a given strain: for example 0.1% of nucleated splenic cells form rosettes with SRBC in the C,itil,j strain. These “spontaneous” RFC (RFC sp.) are also found in similar small numbers when other heterologous erythrocytes are used. They also occur in lymph nodes, bone marrow, and peritoneal exudate, and in a much smaller number in the thy1ll11s.
The RFC sp. have surface receptors which are probably immunoglobulins specific for the antigen (5, 6). These properties fit with those of the “antigen-sensitive cell” in Burnet’s conception (7j. It has been recently shown that RFC sp. are truly antigen-sensitive cells, since a normal splenic cell population freed of spontaneous rosettes formed with chicken erythrocytes, became specifically nonreactive to that antigen (Sj . This observation confirmed the works of Ada and Byrt (9)) Humphrey and Keller (lo), and Wigzell and Makela (11). It seemed interesting to make a morphological study of these RFC sp. because of their role in immunological response and of their remarkable in vitro and in z&o sensitivity to antilymphocyte serum (ALS) and antimetabolites, suggesting they may be the target of these immunosuppressive agents (12, 13). We observed their morphology and compare it to already known characteristics of cells implicated in immunological response. The very low proportion of rosettes among splenic cells made it necessary to isolate them by micromanipulatiot~ before embedding for electron microscopy. We chose for isolation only the morula form of the clustering of a RFC sp. with surrounding erythrocytes, thus eliminating doubtful or nonspecific agglutination. 1Ve also examined the ultrastructure of RFC sp. treated with ALS in vitro. The findings allow us to conclude that RFC sp. are a population of lymphocytes of constant morphological appearance and macrophages, as suggested by preliminary results (14). We did not observe cytotoxic effects on the RFC sp. with the concentration of ALS and complement used for rosette-formation inhibition. MATERIALS
AND
METHODS
P~~cparationsof rosettes. Stored SRBC and &Bl, mouse spleen cells are used to form rosettes as previously described (6). Rosette formation is obtained by simple centrifugation without incubation. In some instances, fresh chicken erythrocytes are substituted for SRBC. One rosette per 1000 nucleated splenic cells is obtained with SRBC and 4 per 1000 with chicken erythrocytes. Fixation for electron microscopy. After rosettes are formed the whole suspension
184
REYES
AND
BACH
is fixed for 30 min at 4” in 37% glutaraldehyde, washed for 5 min in 0.1 M Seerensen phosphate buffer, and postfixed for 30 min at 4” in 1% osmium tetroxide. After a final washing, cells are prepared for isolation by resuspending in approx. 3 ml of buffer. Isolation (Fig. 1). A 25mm round coverslip is placed on a holder whose position can be adjusted when placed on the mechanical stage of the microscope. The coverslip is divided into two chambers with a strip of silicone paste. In one chamber a 400-p aperture grid, in which rosettes will be placed with a micropipette, is mounted on a drop of 2% agarose. In the other chamber a microdrop of the suspension containing 3000-4000 fixed cells is placed. This chamber is then filled with silicon oil. The coverslip and holder are positioned on the mechanical stage of a Wild inverted microscope and examined at a magnification of 100 diameters. By adjusting the stage, the grid or the cell suspension can be observed. The rosettes, which are easily recognized by their morula form of a mean diameter of 14 p, are aspirated by a micropipette with an internal diameter of 40 p, manipulated by a Fonbrune apparatus. To prevent disruption of the cells, oil is first gently aspirated, then a small quantity of buffer from the microdrop of cell suspension, and finally the chosen rosette. Keeping the rosette at the mouth, the pipette is touched to the surface of the agarose in the other chamber and the rosette is released in the center of the grid. The procedure can be repeated several times, but in practice only one or two rosettes are isolated to facilitate the final sectioning. Dehydratation and embedding. By freeing the agarose from the coverslip, the aperture grid is removed and then covered with a layer of melted agarose. After solidifying, excess agarose is removed with a hot wire and a 2-3-mm agarose cube containing rosettes at the level of the grid is obtained. The block is then dehydrated by the standard method of successive passages in 70, 90, and 100% alcohol and propylene oxide. To permit penetration of agarose, embedding is performed by incubation in Epon overnight at 4” followed by polymerization at +60” for 12 hr. Sectioning. For sectioning, the level at which the rosettes are embedded in Epon is marked by the aperture grid. Sections are cut with a Porter-Blum M T I microtome, mounted on grids covered by a formvar supporting film, and stained with uranyl acetate and lead citrate. Preparations are examined with an Elmiskop 1 electron microscope at 80 kV. Examination with the please contrast microscope. Rosettes are observed in unfixed wet preparations between slide and cover slip with the phase contrast microscope (Zeiss, X 100). For some experiments rosettes are obtained from mice which were injected intravenously several hours before sacrifice with an aqueous colloidal carbon solution (0.2 ml of a solution containing 16 mg carbon/ml). In this manner some rosette-forming cells can be shown to have phagocytized carbon in z&o (“carbon-positive RFC sp.“). Incubation tit,4 ALS. After incubation for 90 min with ALS and complement, normal mouse spleen cells can no longer form rosettes (15). In order to identify and observe the morphology of RFC sp. after a 90-min incubation with ALS, the usual procedure (15) is modified. It has been observed that incubation of splenic cells with ALS for less than 60 min does not prevent rosette formation (12). Therefore splenic cells are incubated for 40 min at 37” with ALS and
MORPHOLOGY
OF ROSETTE-FORMING
185
CELLS
1 Pipette
Oil
Agar
and
FIG. 1. Above: isolation apparatus. and on agar and aperture grid (left).
Objective
Below:
Cellular
morula
form
in cell
suspension
suspension
(arrow,
right)
186
REYES
AND
BACH
complement.’ At the end of this incubation SRBC are added and rosettes are formed by the usual technique. After completing an additional 30-min incubation with ALS and complement, rosettes are examined with the electron microscope and phase contrast microscope as described above. Controls with complement in the absence of ALS are also performed. In total, 40 rosettes, obtained from several mice, were examined by electron microscopy and more than 200 by phase contrast. RESULTS
The RFC sp. which interact with sheep erythrocytes are of two kinds: lymphocytes and macrophages. I-Lymphocytes. These are small round cells with a mean diameter of 5 p. The nucleocytoplasmic ratio is high in most sections. The nucleus is round, with an invagination in the Golgi area ; it has an interphase appearance with dense chromatin-forming blocks at the periphery and the center of the nucleus. The nucleolus, which is small and therefore not always seen in sections, is homogeneousand surrounded by chromatin. The cytoplasm shows few organelles. Besides the perinuclear space, whose external membrane is bordered by ribosomes, only a few short endoplasmic reticulum lamellae are seen. In the cytoplasm the majority of the ribosomesare monosomes.The Golgi apparatus is small and is composed of short lamellae. Mitochondria are small and often concentrated at one pole of the cell. These characteristics are those of the so-called “inactive” small lymphocytes (16, 17) and were found in every lymphocyte examined (Figs. 2 and 4). Lymphocyte-erythrocyte contacts, which are only seen in some sections, vary from a small to a large interface. In other sections the RFC sp. and its surrounding red cells appear separated. This separation may be more apparent than real, resulting perhaps from the angle of section or from shrinkage of cells in preparation. Similar lymphocyte-erythrocyte contacts have been observed in immune rosettes (3). The small lymphocyte exhibits no cytoplasmic processesand there is no pinocytosis. The surrounding erythrocytes are not deformed. Small spherical fragments, apparently from erythrocytes, are seen adhering to the lymphocyte. On sections which are tangential to the surface of the RFC sp., many such spherical fragments are seen. These fragments often appear unconnected to the red cells, but in some sections they form a bridge between a red cell and the RFC sp. (Fig. 6). In spontaneous rosettes formed with chicken erythrocytes lymphocytes have the same electron microscopic appearance, except that the small spherical adhering fragments are not seen (Fig. 7). 2----Macrophagcs. The other cell type in RFC sp. is the macrophage which is easily recognized in most casesby its specialized cytoplasmic organelles and its relation to erythrocytes. In these cells pinocytotic vacuoles are seen; in addition, *A final dilution of 1 :4,000 of a rabbit 1:16,000 and of 1:50 of guinea pig serum.
FIG. 2. Typical small lymphocyte ing to the RFC sp. n = nucleolus.
forming X 17,000.
antithymus
rosette
ALS
with
with
SRBC.
a rosette
Note
small
inhibition
fragments
titer
adher-
of
MORPHOLOGY
FIG. 3. Macrophage between. erythrocytes, x 17,000.
forming rosette and erythrocytic
OF ROSETTE-FORMING
with SRBC. engulfment
Note (arrow).
CELLS
phagosomes, Nucleus
187
cytoplasmic processes not see in this section.
188
REYES
AND
BACH
MORPHOLOGY
OF
ROSETTE-FORMING
189
CELLS
larger vacuoles are present which are variable in size, content, and density of staining. In some cases myelin figures and ferritin deposits are seen. The larger vacuoles appear to be the phagolysosomes of an erythrophagocytic cell (Figs. 3 and 5). Some of the RFC sp. are not easily identifiable as macrophages, since they lack phagolysosomes. In these cases, probable identification is made on the basis of a large Golgi area containing numerous dense granules and an oval, irregularly shaped nucleus with chromatin marginated at the nuclear membrane (Fig. 8). Such an identification is confirmed by the presence of cytoplasmic processes which pass between erythrocytes, deform them, and form a phagocytosis figure (Fig. 5). In the RFC sp. system these characteristics were never seen with lymphocytes. The small spherical fragments, previously described as adhering to the lymphocytes may also be seen outside the macrophage or in pinocytotic vacuoles inside the cell. 3-Phase contrast obsewations. Knowing that the RFC sp. was either a lymphocyte or a macrophage, as shown by electron microscopy, it was possible to identify these two populations of cells with phase contrast microscopy. The distinguishing characteristics were that the lymphocyte was a small round cell (Figs. 9 and 12), while the macrophage is a larger, more irregularly shaped cell, which contains ingested material (Figs. 10 and 13). The presence of carbon particles in some RFC sp. provides additional evidence that they are macrophages (Fig. 11)) and also allows an estimation of the percentage of RFC sp. which are macrophages. In one such study, 30% of RFC sp. were “carbon-positive” (6). In some rosettes with a macrophage as the central cell, a small lymphocyte may adhere in the same manner as do the erythrocytes. This occurred in about 40% of rosettes examined by phase contrast microscopy. 4-Cell moYpho1ogy after incubation with ALS. We have not observed morphological changes in the rosette-forming lymphocytes neither after incubation with ALS and complement nor with complement alone (Fig. 14). At the concentration of ALS used, there is no visible cytotoxicity on any spleen cell in the system. In addition it should be noted that rosette-forming macrophages show more erythrophagocytosis than in unincubated rosette preparations. In numerous sections nearly intact erythrocytes are seen within large phagosomes, reflecting recent engulfment (Fig. 15). Also the ALS incubated rosettes of the macrophage variety may show more lymphocytes adhering to the RFC sp. than in the case of unincubated rosettes. DISCUSSION The primary purpose of this work was to identify the morphology of the spontaneous rosette-forming cells (RFC sp.) in the unimmunized mouse. RFC sp. bear surface receptors which have the antigenicity of immunoglobulins, since inhibition of rosette formation can be accomplished by antiimmunoglobulin
FIG.
4. Detail
of a small
lymphocyte
FIG. 5. Detail of a macrophage cytoplasmic process (c) deforming
forming
rosette
with
SRBC.
forming rosette with SRBC. an erythrocyte. N = nucleus.
g = Golgi
Note pinocytotic X 28,000.
area.
X 38,000.
vacuoles
and
190
REYES
AND
BACH
FIG. 6. Small lymphocyte forming rosette with SRBC. Left: tangential section showing numerous small adhering fragments. X 28,000. Right: another section showing continuity of adhering fragment and neighboring erythrocyte. X 80,000.
sera (6) and anti-F (ab)‘Z sera (2). Recently such an inhibition has been obtained with antilight chain sera, but not with antiheavy chain sera (18). These RFC sp. receptors are specific for the antigen, since no mixed rosettes are found when spleen cells are incubated with a mixture of heterologous erythrocytes from different species (5, 6). This observation also strongly suggeststhat rosette formation is not due in most casesto the adsorption of cytophilic antibodies on the RFC sp. In addition, the inhibition of rosette formation by azathioprine is evidence that the immunoglobulin receptors are synthesized by the RFC sp. (13, 19). Similar receptors have been detected on a few lymphoid cells from nonimmunized animals, using isotope-labeled antigens such as flagellin and hemocyanin (20). In our system, the population of RFC sp. contains two classesof cells : lymphocytes and macrophages. The morphology of the rosette-forming lymphocytes appears to be constant and corresponds to the definition of the monoribosomal “inactive” small lymphocyte (16, 17). This is in contrast with the morphological heterogeneity of the RFC observed in mice immunized with heterologous erythrocytes (3, 4, 21). In the latter situation, RFC appear as lymphocytes or plasma cells, with transitional aspectsand blastlike cells. A similar heterogeneity is found among the antibody-forming cells detected by other techniques (such as hemolysis in gel) (4, 22-24). In these immune systems, antibody-forming cells classified as “lymphocytes” differ from “inactive lymphocytes” since they exhibit a greater development of the organelles related to protein synthesis (polyribosomes and endoplasmic reticulum). These signs of synthetic activity are found also in some small basophilic
MORPHOLOGY
FIG.
7. Small
lymphocyte
FIG. 8. Histiocytic area with numerous x 7,500.
forming
OF
rosette
ROSETTE-FORMING
with
cell forming rosette with granules, and cyto’plasmic
nucleated
191
CELLS
chicken
chicken erythrocytes. process deforming
erythrocytes. Note nuclear an erythrocyte
X 5,400. shape, Golgi (arrow).
192
REYES
AND
FIGS. 9-11. Rosettes with SRBC in phase contrast. 10: macrophage with ingested material. Erythrocyte, carbon-labeled macrophage.
BACH
X 2,500. Fig. 9: small lymphocyte. Fig. probably engulfed (arrow). Fig 11:
hIORPIlOLOG\’
FIG. 12 AND 13. Rosettes with lymphocyte. Fig. 13 : macrophage.
OF ROSETTE-FORMING
chicken erythrocytes Note erythrocyte
in phase deformation.
193.
CELLS
contrast.
X 2,500.
Fig.
12: small
193
REYES
AND
BACH
MORPHOLOGY
OF ROSETTE-FORMING
195
CELLS
mononuclear cells, which could currently be classified as “small lymphocytes” bq’ light microscopy (25). Tl lis emphasizes the importance of electron microscopic examination for the estimation of the functional state of the lymphocytes. Interestingly enough, all rosette-forming lymphocytes appear to be of the monoribosomal type. These cells had long been thought to be incapable of antibody synthesis (26). In fact, their ability to form rosettes strongly suggests that such cells are capable of synthesizing antigen-specific surface receptors, i.e., probably immunoglobulins. In preceding papers (6, 8) data are presented which suggest that RFC sp. are involved in the initiation of the immune response to the corresponding erythrocyte antigen. The rosette-forming monoribosomal small lymphocyte which we have observed is a probable candidate for being the antigen-sensitive cell in this system. Therefore our morphological data would correlate with the conclusion of cell” is a monoriboBosman and Feldman (27) who suggested that the “memory somal small lymphocyte. The small spherical fragments adhering to the RFC sp. may result from the alteration of the SRBC due to the preservation and incubation conditions. They were not seen using chicken erythrocytes from freshly bled anikals. They adhere only to RFC sp. and not to other cells in the preparation. If one assumes that the antigenicity of these fragments is identical to that of the intact erythrocyte membrane. their presence at the surface of lymphocytes may mean an excess of antigen-binding sites available on the RFC sp. membrane. The presence of a large number of antigen-binding sites on these cells would correlate with the results obtained with flagellin- and hemocyanin-binding cells (20). Our observations demonstrate clearly that, in addition to lymphocytes, macrophages are responsible for rosette formation. The macrophages have not been recognized as RFC sp. by other workers (5), probably because these cells escaped microscopic examination, following their adherence to the glass during prolonged incubation. On the other hand, it is highly probable that the few RFC sp. found by Roseman (28) among mouse spleen cells adhering to the glass were macrophages. The RFC sp. which were defined as macrophages possess the characteristics of phagocytic mononuclear cells (29) and specifically those of histiocytic cells involved in erythrophagocytosis (30). However, the degree of erythrophagocytosis which we observed in these rosette-forming macrophages as indicated by the number of erythrocytes engulfed is relatively low. In addition, some of the aspects observed, especially erythrophagosomes at an advanced stage of digestion, probably correspond to previous autoerythrophagocytosis in v&o. However, the reduced phagocytic activity of the rosette-forming macrophages cannot be considered as a peculiarity of these cells, since it is probably explained by the shortness of incubation time (31) in our technique (20 min at room temperature), In fact, a more active erythrophagocytosis can be observed in these cells, following incubation for 90 min at 37” with ALS and complement (see below). FIG. 14. Small lymphocyte forming complement. No cytopathic effect seen. FIG. 15. Macrophage enguIfed erythrocyte
(e).
forming rosette X 18,000.
rosette with X 18,000. with
SRBC.
SRBC; Same
90 min incubation
incubation as in Fig.
with
ALS
14. Note
and
freshly
196
REYES
AND
BACH
Presently it cannot be stated whether or not rosettes formed around macrophages result from an immunological reaction. One may consider that rosette formation is the result of a nonimmune adherence of heterologous erythrocytes on macrophages, as the first stage of phagocytosis. However, only a very small proportion of the spleen macrophages which ingest carbon particles (6) are able to form rosettes. On the other hand, the possibility of the intervention of an immunological reaction in rosette formation by macrophages cannot be excluded. It is known that adherence and engulfment of heterologous erythrocytes by macrophages are favored by opsonizing antibodies (3 1) . Macrophages can acqui,re antigen-reactive sites by adsorbing cytophilic antibodies (32, 33). Rosette-forming macrophages may correspond to the cells which have acquired such cytophilic antibodies at their surface. If cytophilic antibodies are responsible for rosette formation by macrophages, one should expect them to form mixed rosettes with different antigens. In fact, this has been found for macrophages from immunized animals (2). Also, the absence of specificity has been shown in the unimmunized mouse for carbon-labeled RFC sp. (6) ; however, the presence of cytophilic antibodies has not been proven as yet. The present study did not recognize morphological differences between rosette-forming and nonrosette-forming macrophages. However, erythrocyte engulfment was found in RFC sp. and not in other macrophages, suggesting that rosette formation and phagocytosis of the antigen in vitro are closely related phenomena and perhaps two steps of the same process. An understanding of the absence of rosette formation with heterologous erythrocytes by most spleen macrophages from nonimmunized animals awaits further investigation. Mosier (34) and subsequently other authors (35, 36) have shown that the presence of a few glass-adhering cells (presumably macrophages) is required for the induction of a primary immune response in vitro against sheep erythrocytes. Mosier (37) calculated that the minimum number of “adhering cells” necessary for the induction of the response in vitro was 0.1-l/1,000 cells. This figure is remarkably similar to the number of “adherent X-ray resistant cells” found by Roseman to form rosettes with SRBC (l/10,000) (28). It is possible that the rosette-forming adherent cells described in this latter system correspond to the rosette-forming macrophages that we have observed. It is suggested that the rosette-forming macrophages may represent the cells cooperating with lymphocytes in the induction of a primary immune response. The second purpose of the present work was to study at the ultrastructural level the effect of ALS on the RFC sp. an vitro. As previously described (12, 15), inhibition of rosette formation by ALS in vitro can be obtained with a concentration much lower that the concentration of ALS which is cytotoxic in vitro for the whole of lymphoid cells (as measured by the trypan blue test). However, the necessity of complement suggested the possibility that the inhibition phenomenon was due to a cytopathic effect on the RFC sp. The fact that ALS does not destroy preformed rosettes (unpublished observations) does not preclude the possibility of some injury to the RFC sp., since rosettes with a dead central cell are routinely observed in the preparations for phase contrast microscopy. If ALS had some cytotoxic action on RFC sp. responsible for inhibition of rosette formation, some signs would be expected at the ultrastructural level. With the
MORPHOLOGY
OF
ROSETTE-FORMING
197
CELLS
procedure described above, we did not recognize such a cytotoxic effect. Other mechanisms, such as coating of RFC sp. by ALS and some complement components, are suggested by data in another paper ( 12). In these experiments where RFC sp. were treated by ALS i~z vitro, rosetteforming macrophages show increased erythrophagocytosis, and it should be emphasized that several factors acting in concert may be responsible. Erythrophagocytosis increases with incubation time and temperature (31), and might be further facilitated by the low titer (< 1 :16) of anti-SRBC antibodies present in the ALS used [it has been demonstrated that these agglutinins do not interfere with inhibition of rosette formation by ALS (12) 1. Finally, the presence of complement may be an additional factor, since it has been claimed that macrophages bear sites at their surface for the third component of complement which cooperate with sites for the Fc piece of immunoglobulins, in the induction of phagocytosis (33). The clustering of a few lymphocytes around rosette-forming macrophages might be suspected, if one assumes that ,4LS contains cytophilic antibodies (38) responsible for lymphocyte opsonization (39, 40). 0 ur observations do not allow any conclusion on this point, since such a clustering is also seen in some rosette preparations without ALS treatment. The significance of this phenomenon is not known. ACKNOWLEDGMENT The
authors
are indebted
to Pr. Marcel
Bessis
for
constant
support
and interest.
REFERENCES 1. McConnel, I., Munro, A., Gurner, B. W., and Coombs, R. R. A., Int. Arclz. Allergy 35, 209, 1969. 2. Biozzi, G., Stiffel, C., and Mouton, D., In “Immunity, Cancer and Chemotherapy” (E. Mihich, Ed.), pp. 103-139. Academic Press, New York, 1967. 3. Storb, U., Bauer, W., Storb, R., Fliedner, T. M., and Weiser, R. S., J. 1~11mzmol. 102, 1474, 1969. 4. Gudat, F. G., Harris, T. N., Harris, S., and Hummeler, K., J. Exp. Med. 132, 448, 1970. 5. Laskow, R., Snture Lolzdolz 219, 973, 1968. 6. Bach, J. F., Dardenne, M., and Fournier, C., submitted for publication, 1970. 7. Burnet, F. hl., “The Clonal Selection Theory of Acquired Immunity.” Cambridge Univ. Press, London, 1959. 8. Bach, J. F., Muller, J. Y., and Dardenne, M., Nnture London 227, 1251, 1970. 9. Ada, G. L., and Byrt, P., Nature London 222, 1291, 1969. 10. Humphrey, J. H., and Keller, M. V., In “Symposium on Developmental Aspects of Antibody Formation” (J. Sterzl and M. Riha, eds.), Academic Press, New York, 1970. 11. Wigzell, H., and Makela, O., J. Exp. Med. 132, 110, 1970. 12. Bach, J. F., Dardenne, M., and Fournier, C., submitted for publication, 1970. 13. Bach, J. F., and Dardenne, M., submitted for publication, 1970. 14. Reyes, F., and Bach, J. F., C. R. H. Acad. Sci. Skrie D 270, 1702, 1970. 15. Bach, J. F., and Antoine, B., Nature Londolz 217, 658, 1968. 16. Feldman, J. D., and Nordquist, R. E., Lab. Invest. 16, 564, 1967. 17. Tucker-Franklin, D., Se&n. Hew~atol. 6, 4, 1969. 18. Greaves, M. F., and Hoog, J., In “Proceedings of the Third Sigrid Juselius Symposium on Cell Interactions in Immune Responses” (0. Makela and A. Cross Eds.). Academic Press, New York, 1970. 19. Bach, J. F., Dardenne, M., and Fournier, C., Nature London 222, 698, 1969. 20. Byrt, P., and Ada, G. L., Inzmulzologg 17, 503, 1969.
198 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40.
REYES
AND
BACH
Pavlovsky, S., Binet, J. L., Decreusefond, C., Stiffel, C., Mouton, D., Bouthillier, Y.. and Biozzi, G., Ann. Inst. Pasteur Paris 119, 63, 1970. Cunningham, A. J., Smith, J. B., and Mercer, E. H., J. Exp. Med. 124, 701, 1966. Harris, T. N., Hummeler, K., and Harris, S., J. Exp. Med. 123, 161, 1966. Hummeler, K., Harris, T. N., Tomassini, N., Hechtel, M., and Farber, M. B., J. EXD. Med. 124, 255, 1966. Cunningham, A. J., Aust. J. Exp. Biol. Med. Sci. 46, 141, 1968. Granboulan, N., Rev. Hematol. 15, 52, 1960. Bosman, C., and Feldman, J. D., J. Exp. Med. 126, 293, 1968. Roseman, J., Science 165, 1125, 1969. Cohn, Z. A., Hirsch, J. G., and Fedorko, M. E., J. En-p. Med. 123, 747, 1966. Bessis, M., Stand. J. Haematol., Ser. Haematol. 2, 59, 1965. Greendyke, R. hi., Brierty, R. E., and Swisher, S. N., Blood 33, 295, 1963. Berken, A., and Benacerraf, B., J. Exp. Med. 123, 119, 1966. Huber, H., Polley, M. J., Linscott, W. D., Fudenberg, H. H., and Miller-Eberhard, H. J., Science 162, 1281, 1968. Mosier, D. E., Science 156, 1573, 1967. Pierce, C. W., J. Exp. Med. 130, 345, 1969. Shortman, K., Diener, E., Russel, P., and Armstrong, W. D., J. Exp. Med. 131, 461, 1970. Mosier, D. E., and Coppleson, L. W., Proc. Nat. Acad. Sci. U.S.‘4. 61, 542, 1968 Gill, P. G., J. Zmmunol. 102, 1329, 1969. Greaves, M. F., Playfair, J. H. L., Torrigiani, G., Zamir, R., and Roitt, I. M., Lancet 1, 68, 1969. Huber, H., Michlmayr, G., and Fudenberg, H. H., Clin. Exp. Immunol. 5, 607, 1969.
IMMUNOLOGY
2, 182-198 (1971)
Rosette-Forming Morphological
FELIX Institut
Cells in the Unimmunized Studies with Phase Contrast Electron Microscopy REYES 1 AND JEAN-FRANCOIS
de Pathologie
Cellulaire (U 48, INSERM), 94-Le Kremlin-Biche, France Received
December
Mouse: and
BACH
Hdfiital
de Bice^tre,
24,197O
Spontaneous rosettes, obtained with nonimmunized mouse spleen cells and heterologous erythrocytes, have been examined. For electron microscopy, the rosettes are isolated by micromanipulation; embedding is performed by a technique suitable for single-cell study. Furthermore the morphology of these spontaneous rosette-forming cells (RFC sp.) has been investigated in other experiments where spleen cells are incubated with antilymphocyte serum (ALS) and complement, at a concentration which inhibits rosette formation irz vitro. The population of RFC sp. contains two classes of cells: lymphocytes and macrophages. The lymphocytes appear to have a constant morphology, corresponding to the “inactive” monoribosomal small lymphocyte. The macrophages exhibit to a variable degree signs of erythrophagocytosis. These observations are compared with the already known morphology of cells involved in antibody production. The existence of two cell types in the RFC sp. population is discussed in relation to recent data concerning cellular cooperation in the induction of an immunological response to antigens such as heterologous erythrocytes. After incubation with ALS and complement, the RFC sp. show no alterations at the ultrastructural level.
INTRODUCTION
The production of antibodies at the cellular level has been studied by several methods. In one of these, immunocytoadherence, the immunological response to heterologous red cells is studied by incubating lymphoid cells and red cells together in vitro. Heterologous red cells ring themselves around the lymphoid cells forming rosettes. When observed in suspension at low magnification, the rosette-forming cells (RFC) and surrounding erythrocytes assumea “morula form” ( 1) . The term “rosette” is best reserved for the appearance of aggregates in section or between slide and coverslip, where the central lymphoid cell is ringed by erythrocytes. The RFC bears on its membrane specific receptors which have the antigenicity of immunoglobulins, which are synthesized by the RFC, and which are not ad1 Reprint
requests
are to be sent to: Felix Reyes, Mondor, 94- Creteil,
(U 91, INSERM), CHU Hem-i
182
M.D., Unite France.
de Recherche
sur les Anemies
MORPHOLOGY
OF ROSETTE-FORMING
CELLS
183
cells” thus sorbed from circulating antibodies ( 1, 2). The term “antibody-forming appears justified, at least for the great majority of RFC in the spleen or lymph nodes of the immunized animal, although there is in these tissues a low proportion of rosettes formed by cells which have adsorbed “cytophilic” antibodies (2). Morphological studies of RFC after immunization have recently confirmed that they are principally lymphoid cells, which at the ultrastructural level have a variable degree of differentiation, and that some RFC are macrophages (3,4). Rosette-forming cells also exist without immunization. A small number of mouse spleen cells can form a rosette in vitro with sheep red blood cells (SRBC). The number of RFC in the unimmunized mouse is constant for a given strain: for example 0.1% of nucleated splenic cells form rosettes with SRBC in the C,itil,j strain. These “spontaneous” RFC (RFC sp.) are also found in similar small numbers when other heterologous erythrocytes are used. They also occur in lymph nodes, bone marrow, and peritoneal exudate, and in a much smaller number in the thy1ll11s.
The RFC sp. have surface receptors which are probably immunoglobulins specific for the antigen (5, 6). These properties fit with those of the “antigen-sensitive cell” in Burnet’s conception (7j. It has been recently shown that RFC sp. are truly antigen-sensitive cells, since a normal splenic cell population freed of spontaneous rosettes formed with chicken erythrocytes, became specifically nonreactive to that antigen (Sj . This observation confirmed the works of Ada and Byrt (9)) Humphrey and Keller (lo), and Wigzell and Makela (11). It seemed interesting to make a morphological study of these RFC sp. because of their role in immunological response and of their remarkable in vitro and in z&o sensitivity to antilymphocyte serum (ALS) and antimetabolites, suggesting they may be the target of these immunosuppressive agents (12, 13). We observed their morphology and compare it to already known characteristics of cells implicated in immunological response. The very low proportion of rosettes among splenic cells made it necessary to isolate them by micromanipulatiot~ before embedding for electron microscopy. We chose for isolation only the morula form of the clustering of a RFC sp. with surrounding erythrocytes, thus eliminating doubtful or nonspecific agglutination. 1Ve also examined the ultrastructure of RFC sp. treated with ALS in vitro. The findings allow us to conclude that RFC sp. are a population of lymphocytes of constant morphological appearance and macrophages, as suggested by preliminary results (14). We did not observe cytotoxic effects on the RFC sp. with the concentration of ALS and complement used for rosette-formation inhibition. MATERIALS
AND
METHODS
P~~cparationsof rosettes. Stored SRBC and &Bl, mouse spleen cells are used to form rosettes as previously described (6). Rosette formation is obtained by simple centrifugation without incubation. In some instances, fresh chicken erythrocytes are substituted for SRBC. One rosette per 1000 nucleated splenic cells is obtained with SRBC and 4 per 1000 with chicken erythrocytes. Fixation for electron microscopy. After rosettes are formed the whole suspension
184
REYES
AND
BACH
is fixed for 30 min at 4” in 37% glutaraldehyde, washed for 5 min in 0.1 M Seerensen phosphate buffer, and postfixed for 30 min at 4” in 1% osmium tetroxide. After a final washing, cells are prepared for isolation by resuspending in approx. 3 ml of buffer. Isolation (Fig. 1). A 25mm round coverslip is placed on a holder whose position can be adjusted when placed on the mechanical stage of the microscope. The coverslip is divided into two chambers with a strip of silicone paste. In one chamber a 400-p aperture grid, in which rosettes will be placed with a micropipette, is mounted on a drop of 2% agarose. In the other chamber a microdrop of the suspension containing 3000-4000 fixed cells is placed. This chamber is then filled with silicon oil. The coverslip and holder are positioned on the mechanical stage of a Wild inverted microscope and examined at a magnification of 100 diameters. By adjusting the stage, the grid or the cell suspension can be observed. The rosettes, which are easily recognized by their morula form of a mean diameter of 14 p, are aspirated by a micropipette with an internal diameter of 40 p, manipulated by a Fonbrune apparatus. To prevent disruption of the cells, oil is first gently aspirated, then a small quantity of buffer from the microdrop of cell suspension, and finally the chosen rosette. Keeping the rosette at the mouth, the pipette is touched to the surface of the agarose in the other chamber and the rosette is released in the center of the grid. The procedure can be repeated several times, but in practice only one or two rosettes are isolated to facilitate the final sectioning. Dehydratation and embedding. By freeing the agarose from the coverslip, the aperture grid is removed and then covered with a layer of melted agarose. After solidifying, excess agarose is removed with a hot wire and a 2-3-mm agarose cube containing rosettes at the level of the grid is obtained. The block is then dehydrated by the standard method of successive passages in 70, 90, and 100% alcohol and propylene oxide. To permit penetration of agarose, embedding is performed by incubation in Epon overnight at 4” followed by polymerization at +60” for 12 hr. Sectioning. For sectioning, the level at which the rosettes are embedded in Epon is marked by the aperture grid. Sections are cut with a Porter-Blum M T I microtome, mounted on grids covered by a formvar supporting film, and stained with uranyl acetate and lead citrate. Preparations are examined with an Elmiskop 1 electron microscope at 80 kV. Examination with the please contrast microscope. Rosettes are observed in unfixed wet preparations between slide and cover slip with the phase contrast microscope (Zeiss, X 100). For some experiments rosettes are obtained from mice which were injected intravenously several hours before sacrifice with an aqueous colloidal carbon solution (0.2 ml of a solution containing 16 mg carbon/ml). In this manner some rosette-forming cells can be shown to have phagocytized carbon in z&o (“carbon-positive RFC sp.“). Incubation tit,4 ALS. After incubation for 90 min with ALS and complement, normal mouse spleen cells can no longer form rosettes (15). In order to identify and observe the morphology of RFC sp. after a 90-min incubation with ALS, the usual procedure (15) is modified. It has been observed that incubation of splenic cells with ALS for less than 60 min does not prevent rosette formation (12). Therefore splenic cells are incubated for 40 min at 37” with ALS and
MORPHOLOGY
OF ROSETTE-FORMING
185
CELLS
1 Pipette
Oil
Agar
and
FIG. 1. Above: isolation apparatus. and on agar and aperture grid (left).
Objective
Below:
Cellular
morula
form
in cell
suspension
suspension
(arrow,
right)
186
REYES
AND
BACH
complement.’ At the end of this incubation SRBC are added and rosettes are formed by the usual technique. After completing an additional 30-min incubation with ALS and complement, rosettes are examined with the electron microscope and phase contrast microscope as described above. Controls with complement in the absence of ALS are also performed. In total, 40 rosettes, obtained from several mice, were examined by electron microscopy and more than 200 by phase contrast. RESULTS
The RFC sp. which interact with sheep erythrocytes are of two kinds: lymphocytes and macrophages. I-Lymphocytes. These are small round cells with a mean diameter of 5 p. The nucleocytoplasmic ratio is high in most sections. The nucleus is round, with an invagination in the Golgi area ; it has an interphase appearance with dense chromatin-forming blocks at the periphery and the center of the nucleus. The nucleolus, which is small and therefore not always seen in sections, is homogeneousand surrounded by chromatin. The cytoplasm shows few organelles. Besides the perinuclear space, whose external membrane is bordered by ribosomes, only a few short endoplasmic reticulum lamellae are seen. In the cytoplasm the majority of the ribosomesare monosomes.The Golgi apparatus is small and is composed of short lamellae. Mitochondria are small and often concentrated at one pole of the cell. These characteristics are those of the so-called “inactive” small lymphocytes (16, 17) and were found in every lymphocyte examined (Figs. 2 and 4). Lymphocyte-erythrocyte contacts, which are only seen in some sections, vary from a small to a large interface. In other sections the RFC sp. and its surrounding red cells appear separated. This separation may be more apparent than real, resulting perhaps from the angle of section or from shrinkage of cells in preparation. Similar lymphocyte-erythrocyte contacts have been observed in immune rosettes (3). The small lymphocyte exhibits no cytoplasmic processesand there is no pinocytosis. The surrounding erythrocytes are not deformed. Small spherical fragments, apparently from erythrocytes, are seen adhering to the lymphocyte. On sections which are tangential to the surface of the RFC sp., many such spherical fragments are seen. These fragments often appear unconnected to the red cells, but in some sections they form a bridge between a red cell and the RFC sp. (Fig. 6). In spontaneous rosettes formed with chicken erythrocytes lymphocytes have the same electron microscopic appearance, except that the small spherical adhering fragments are not seen (Fig. 7). 2----Macrophagcs. The other cell type in RFC sp. is the macrophage which is easily recognized in most casesby its specialized cytoplasmic organelles and its relation to erythrocytes. In these cells pinocytotic vacuoles are seen; in addition, *A final dilution of 1 :4,000 of a rabbit 1:16,000 and of 1:50 of guinea pig serum.
FIG. 2. Typical small lymphocyte ing to the RFC sp. n = nucleolus.
forming X 17,000.
antithymus
rosette
ALS
with
with
SRBC.
a rosette
Note
small
inhibition
fragments
titer
adher-
of
MORPHOLOGY
FIG. 3. Macrophage between. erythrocytes, x 17,000.
forming rosette and erythrocytic
OF ROSETTE-FORMING
with SRBC. engulfment
Note (arrow).
CELLS
phagosomes, Nucleus
187
cytoplasmic processes not see in this section.
188
REYES
AND
BACH
MORPHOLOGY
OF
ROSETTE-FORMING
189
CELLS
larger vacuoles are present which are variable in size, content, and density of staining. In some cases myelin figures and ferritin deposits are seen. The larger vacuoles appear to be the phagolysosomes of an erythrophagocytic cell (Figs. 3 and 5). Some of the RFC sp. are not easily identifiable as macrophages, since they lack phagolysosomes. In these cases, probable identification is made on the basis of a large Golgi area containing numerous dense granules and an oval, irregularly shaped nucleus with chromatin marginated at the nuclear membrane (Fig. 8). Such an identification is confirmed by the presence of cytoplasmic processes which pass between erythrocytes, deform them, and form a phagocytosis figure (Fig. 5). In the RFC sp. system these characteristics were never seen with lymphocytes. The small spherical fragments, previously described as adhering to the lymphocytes may also be seen outside the macrophage or in pinocytotic vacuoles inside the cell. 3-Phase contrast obsewations. Knowing that the RFC sp. was either a lymphocyte or a macrophage, as shown by electron microscopy, it was possible to identify these two populations of cells with phase contrast microscopy. The distinguishing characteristics were that the lymphocyte was a small round cell (Figs. 9 and 12), while the macrophage is a larger, more irregularly shaped cell, which contains ingested material (Figs. 10 and 13). The presence of carbon particles in some RFC sp. provides additional evidence that they are macrophages (Fig. 11)) and also allows an estimation of the percentage of RFC sp. which are macrophages. In one such study, 30% of RFC sp. were “carbon-positive” (6). In some rosettes with a macrophage as the central cell, a small lymphocyte may adhere in the same manner as do the erythrocytes. This occurred in about 40% of rosettes examined by phase contrast microscopy. 4-Cell moYpho1ogy after incubation with ALS. We have not observed morphological changes in the rosette-forming lymphocytes neither after incubation with ALS and complement nor with complement alone (Fig. 14). At the concentration of ALS used, there is no visible cytotoxicity on any spleen cell in the system. In addition it should be noted that rosette-forming macrophages show more erythrophagocytosis than in unincubated rosette preparations. In numerous sections nearly intact erythrocytes are seen within large phagosomes, reflecting recent engulfment (Fig. 15). Also the ALS incubated rosettes of the macrophage variety may show more lymphocytes adhering to the RFC sp. than in the case of unincubated rosettes. DISCUSSION The primary purpose of this work was to identify the morphology of the spontaneous rosette-forming cells (RFC sp.) in the unimmunized mouse. RFC sp. bear surface receptors which have the antigenicity of immunoglobulins, since inhibition of rosette formation can be accomplished by antiimmunoglobulin
FIG.
4. Detail
of a small
lymphocyte
FIG. 5. Detail of a macrophage cytoplasmic process (c) deforming
forming
rosette
with
SRBC.
forming rosette with SRBC. an erythrocyte. N = nucleus.
g = Golgi
Note pinocytotic X 28,000.
area.
X 38,000.
vacuoles
and
190
REYES
AND
BACH
FIG. 6. Small lymphocyte forming rosette with SRBC. Left: tangential section showing numerous small adhering fragments. X 28,000. Right: another section showing continuity of adhering fragment and neighboring erythrocyte. X 80,000.
sera (6) and anti-F (ab)‘Z sera (2). Recently such an inhibition has been obtained with antilight chain sera, but not with antiheavy chain sera (18). These RFC sp. receptors are specific for the antigen, since no mixed rosettes are found when spleen cells are incubated with a mixture of heterologous erythrocytes from different species (5, 6). This observation also strongly suggeststhat rosette formation is not due in most casesto the adsorption of cytophilic antibodies on the RFC sp. In addition, the inhibition of rosette formation by azathioprine is evidence that the immunoglobulin receptors are synthesized by the RFC sp. (13, 19). Similar receptors have been detected on a few lymphoid cells from nonimmunized animals, using isotope-labeled antigens such as flagellin and hemocyanin (20). In our system, the population of RFC sp. contains two classesof cells : lymphocytes and macrophages. The morphology of the rosette-forming lymphocytes appears to be constant and corresponds to the definition of the monoribosomal “inactive” small lymphocyte (16, 17). This is in contrast with the morphological heterogeneity of the RFC observed in mice immunized with heterologous erythrocytes (3, 4, 21). In the latter situation, RFC appear as lymphocytes or plasma cells, with transitional aspectsand blastlike cells. A similar heterogeneity is found among the antibody-forming cells detected by other techniques (such as hemolysis in gel) (4, 22-24). In these immune systems, antibody-forming cells classified as “lymphocytes” differ from “inactive lymphocytes” since they exhibit a greater development of the organelles related to protein synthesis (polyribosomes and endoplasmic reticulum). These signs of synthetic activity are found also in some small basophilic
MORPHOLOGY
FIG.
7. Small
lymphocyte
FIG. 8. Histiocytic area with numerous x 7,500.
forming
OF
rosette
ROSETTE-FORMING
with
cell forming rosette with granules, and cyto’plasmic
nucleated
191
CELLS
chicken
chicken erythrocytes. process deforming
erythrocytes. Note nuclear an erythrocyte
X 5,400. shape, Golgi (arrow).
192
REYES
AND
FIGS. 9-11. Rosettes with SRBC in phase contrast. 10: macrophage with ingested material. Erythrocyte, carbon-labeled macrophage.
BACH
X 2,500. Fig. 9: small lymphocyte. Fig. probably engulfed (arrow). Fig 11:
hIORPIlOLOG\’
FIG. 12 AND 13. Rosettes with lymphocyte. Fig. 13 : macrophage.
OF ROSETTE-FORMING
chicken erythrocytes Note erythrocyte
in phase deformation.
193.
CELLS
contrast.
X 2,500.
Fig.
12: small
193
REYES
AND
BACH
MORPHOLOGY
OF ROSETTE-FORMING
195
CELLS
mononuclear cells, which could currently be classified as “small lymphocytes” bq’ light microscopy (25). Tl lis emphasizes the importance of electron microscopic examination for the estimation of the functional state of the lymphocytes. Interestingly enough, all rosette-forming lymphocytes appear to be of the monoribosomal type. These cells had long been thought to be incapable of antibody synthesis (26). In fact, their ability to form rosettes strongly suggests that such cells are capable of synthesizing antigen-specific surface receptors, i.e., probably immunoglobulins. In preceding papers (6, 8) data are presented which suggest that RFC sp. are involved in the initiation of the immune response to the corresponding erythrocyte antigen. The rosette-forming monoribosomal small lymphocyte which we have observed is a probable candidate for being the antigen-sensitive cell in this system. Therefore our morphological data would correlate with the conclusion of cell” is a monoriboBosman and Feldman (27) who suggested that the “memory somal small lymphocyte. The small spherical fragments adhering to the RFC sp. may result from the alteration of the SRBC due to the preservation and incubation conditions. They were not seen using chicken erythrocytes from freshly bled anikals. They adhere only to RFC sp. and not to other cells in the preparation. If one assumes that the antigenicity of these fragments is identical to that of the intact erythrocyte membrane. their presence at the surface of lymphocytes may mean an excess of antigen-binding sites available on the RFC sp. membrane. The presence of a large number of antigen-binding sites on these cells would correlate with the results obtained with flagellin- and hemocyanin-binding cells (20). Our observations demonstrate clearly that, in addition to lymphocytes, macrophages are responsible for rosette formation. The macrophages have not been recognized as RFC sp. by other workers (5), probably because these cells escaped microscopic examination, following their adherence to the glass during prolonged incubation. On the other hand, it is highly probable that the few RFC sp. found by Roseman (28) among mouse spleen cells adhering to the glass were macrophages. The RFC sp. which were defined as macrophages possess the characteristics of phagocytic mononuclear cells (29) and specifically those of histiocytic cells involved in erythrophagocytosis (30). However, the degree of erythrophagocytosis which we observed in these rosette-forming macrophages as indicated by the number of erythrocytes engulfed is relatively low. In addition, some of the aspects observed, especially erythrophagosomes at an advanced stage of digestion, probably correspond to previous autoerythrophagocytosis in v&o. However, the reduced phagocytic activity of the rosette-forming macrophages cannot be considered as a peculiarity of these cells, since it is probably explained by the shortness of incubation time (31) in our technique (20 min at room temperature), In fact, a more active erythrophagocytosis can be observed in these cells, following incubation for 90 min at 37” with ALS and complement (see below). FIG. 14. Small lymphocyte forming complement. No cytopathic effect seen. FIG. 15. Macrophage enguIfed erythrocyte
(e).
forming rosette X 18,000.
rosette with X 18,000. with
SRBC.
SRBC; Same
90 min incubation
incubation as in Fig.
with
ALS
14. Note
and
freshly
196
REYES
AND
BACH
Presently it cannot be stated whether or not rosettes formed around macrophages result from an immunological reaction. One may consider that rosette formation is the result of a nonimmune adherence of heterologous erythrocytes on macrophages, as the first stage of phagocytosis. However, only a very small proportion of the spleen macrophages which ingest carbon particles (6) are able to form rosettes. On the other hand, the possibility of the intervention of an immunological reaction in rosette formation by macrophages cannot be excluded. It is known that adherence and engulfment of heterologous erythrocytes by macrophages are favored by opsonizing antibodies (3 1) . Macrophages can acqui,re antigen-reactive sites by adsorbing cytophilic antibodies (32, 33). Rosette-forming macrophages may correspond to the cells which have acquired such cytophilic antibodies at their surface. If cytophilic antibodies are responsible for rosette formation by macrophages, one should expect them to form mixed rosettes with different antigens. In fact, this has been found for macrophages from immunized animals (2). Also, the absence of specificity has been shown in the unimmunized mouse for carbon-labeled RFC sp. (6) ; however, the presence of cytophilic antibodies has not been proven as yet. The present study did not recognize morphological differences between rosette-forming and nonrosette-forming macrophages. However, erythrocyte engulfment was found in RFC sp. and not in other macrophages, suggesting that rosette formation and phagocytosis of the antigen in vitro are closely related phenomena and perhaps two steps of the same process. An understanding of the absence of rosette formation with heterologous erythrocytes by most spleen macrophages from nonimmunized animals awaits further investigation. Mosier (34) and subsequently other authors (35, 36) have shown that the presence of a few glass-adhering cells (presumably macrophages) is required for the induction of a primary immune response in vitro against sheep erythrocytes. Mosier (37) calculated that the minimum number of “adhering cells” necessary for the induction of the response in vitro was 0.1-l/1,000 cells. This figure is remarkably similar to the number of “adherent X-ray resistant cells” found by Roseman to form rosettes with SRBC (l/10,000) (28). It is possible that the rosette-forming adherent cells described in this latter system correspond to the rosette-forming macrophages that we have observed. It is suggested that the rosette-forming macrophages may represent the cells cooperating with lymphocytes in the induction of a primary immune response. The second purpose of the present work was to study at the ultrastructural level the effect of ALS on the RFC sp. an vitro. As previously described (12, 15), inhibition of rosette formation by ALS in vitro can be obtained with a concentration much lower that the concentration of ALS which is cytotoxic in vitro for the whole of lymphoid cells (as measured by the trypan blue test). However, the necessity of complement suggested the possibility that the inhibition phenomenon was due to a cytopathic effect on the RFC sp. The fact that ALS does not destroy preformed rosettes (unpublished observations) does not preclude the possibility of some injury to the RFC sp., since rosettes with a dead central cell are routinely observed in the preparations for phase contrast microscopy. If ALS had some cytotoxic action on RFC sp. responsible for inhibition of rosette formation, some signs would be expected at the ultrastructural level. With the
MORPHOLOGY
OF
ROSETTE-FORMING
197
CELLS
procedure described above, we did not recognize such a cytotoxic effect. Other mechanisms, such as coating of RFC sp. by ALS and some complement components, are suggested by data in another paper ( 12). In these experiments where RFC sp. were treated by ALS i~z vitro, rosetteforming macrophages show increased erythrophagocytosis, and it should be emphasized that several factors acting in concert may be responsible. Erythrophagocytosis increases with incubation time and temperature (31), and might be further facilitated by the low titer (< 1 :16) of anti-SRBC antibodies present in the ALS used [it has been demonstrated that these agglutinins do not interfere with inhibition of rosette formation by ALS (12) 1. Finally, the presence of complement may be an additional factor, since it has been claimed that macrophages bear sites at their surface for the third component of complement which cooperate with sites for the Fc piece of immunoglobulins, in the induction of phagocytosis (33). The clustering of a few lymphocytes around rosette-forming macrophages might be suspected, if one assumes that ,4LS contains cytophilic antibodies (38) responsible for lymphocyte opsonization (39, 40). 0 ur observations do not allow any conclusion on this point, since such a clustering is also seen in some rosette preparations without ALS treatment. The significance of this phenomenon is not known. ACKNOWLEDGMENT The
authors
are indebted
to Pr. Marcel
Bessis
for
constant
support
and interest.
REFERENCES 1. McConnel, I., Munro, A., Gurner, B. W., and Coombs, R. R. A., Int. Arclz. Allergy 35, 209, 1969. 2. Biozzi, G., Stiffel, C., and Mouton, D., In “Immunity, Cancer and Chemotherapy” (E. Mihich, Ed.), pp. 103-139. Academic Press, New York, 1967. 3. Storb, U., Bauer, W., Storb, R., Fliedner, T. M., and Weiser, R. S., J. 1~11mzmol. 102, 1474, 1969. 4. Gudat, F. G., Harris, T. N., Harris, S., and Hummeler, K., J. Exp. Med. 132, 448, 1970. 5. Laskow, R., Snture Lolzdolz 219, 973, 1968. 6. Bach, J. F., Dardenne, M., and Fournier, C., submitted for publication, 1970. 7. Burnet, F. hl., “The Clonal Selection Theory of Acquired Immunity.” Cambridge Univ. Press, London, 1959. 8. Bach, J. F., Muller, J. Y., and Dardenne, M., Nnture London 227, 1251, 1970. 9. Ada, G. L., and Byrt, P., Nature London 222, 1291, 1969. 10. Humphrey, J. H., and Keller, M. V., In “Symposium on Developmental Aspects of Antibody Formation” (J. Sterzl and M. Riha, eds.), Academic Press, New York, 1970. 11. Wigzell, H., and Makela, O., J. Exp. Med. 132, 110, 1970. 12. Bach, J. F., Dardenne, M., and Fournier, C., submitted for publication, 1970. 13. Bach, J. F., and Dardenne, M., submitted for publication, 1970. 14. Reyes, F., and Bach, J. F., C. R. H. Acad. Sci. Skrie D 270, 1702, 1970. 15. Bach, J. F., and Antoine, B., Nature Londolz 217, 658, 1968. 16. Feldman, J. D., and Nordquist, R. E., Lab. Invest. 16, 564, 1967. 17. Tucker-Franklin, D., Se&n. Hew~atol. 6, 4, 1969. 18. Greaves, M. F., and Hoog, J., In “Proceedings of the Third Sigrid Juselius Symposium on Cell Interactions in Immune Responses” (0. Makela and A. Cross Eds.). Academic Press, New York, 1970. 19. Bach, J. F., Dardenne, M., and Fournier, C., Nature London 222, 698, 1969. 20. Byrt, P., and Ada, G. L., Inzmulzologg 17, 503, 1969.
198 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40.
REYES
AND
BACH
Pavlovsky, S., Binet, J. L., Decreusefond, C., Stiffel, C., Mouton, D., Bouthillier, Y.. and Biozzi, G., Ann. Inst. Pasteur Paris 119, 63, 1970. Cunningham, A. J., Smith, J. B., and Mercer, E. H., J. Exp. Med. 124, 701, 1966. Harris, T. N., Hummeler, K., and Harris, S., J. Exp. Med. 123, 161, 1966. Hummeler, K., Harris, T. N., Tomassini, N., Hechtel, M., and Farber, M. B., J. EXD. Med. 124, 255, 1966. Cunningham, A. J., Aust. J. Exp. Biol. Med. Sci. 46, 141, 1968. Granboulan, N., Rev. Hematol. 15, 52, 1960. Bosman, C., and Feldman, J. D., J. Exp. Med. 126, 293, 1968. Roseman, J., Science 165, 1125, 1969. Cohn, Z. A., Hirsch, J. G., and Fedorko, M. E., J. En-p. Med. 123, 747, 1966. Bessis, M., Stand. J. Haematol., Ser. Haematol. 2, 59, 1965. Greendyke, R. hi., Brierty, R. E., and Swisher, S. N., Blood 33, 295, 1963. Berken, A., and Benacerraf, B., J. Exp. Med. 123, 119, 1966. Huber, H., Polley, M. J., Linscott, W. D., Fudenberg, H. H., and Miller-Eberhard, H. J., Science 162, 1281, 1968. Mosier, D. E., Science 156, 1573, 1967. Pierce, C. W., J. Exp. Med. 130, 345, 1969. Shortman, K., Diener, E., Russel, P., and Armstrong, W. D., J. Exp. Med. 131, 461, 1970. Mosier, D. E., and Coppleson, L. W., Proc. Nat. Acad. Sci. U.S.‘4. 61, 542, 1968 Gill, P. G., J. Zmmunol. 102, 1329, 1969. Greaves, M. F., Playfair, J. H. L., Torrigiani, G., Zamir, R., and Roitt, I. M., Lancet 1, 68, 1969. Huber, H., Michlmayr, G., and Fudenberg, H. H., Clin. Exp. Immunol. 5, 607, 1969.