- Email: [email protected]
Leukemic reticuloendotheliosis: Presence of a receptor for cytophilic antibody
Leukemic Reticuloendotheliosis:
Presence of a
Receptor for Cytophilic Antibody
ELAINE
S. JAFFE,
ETHAN
M. SHEVACH,
MICHAEL
M. FRANK,
IRA GREEN,
Neoplastic cells from the spleens of two patients with leukemic reticuloendotheliosis were studied in cell suspensions and in frozen sections for the antigen-antibody complement (IgMEAC) receptor of B lymphocytes and for the receptor for cytophilic antibody (IgGEA) of monocytes. A high percentage of the neoplastic cells, both in suspension and in sections, bound the IgGEA reagent but failed to bind the IgMEAC reagent. These findings suggest that these cells belong to the monocyte-histiocyte series.
M.D. M.D. M.D.
M.D.
Bethesda, Maryland
Leukemic reticuloendotheliosis, now generally recognized as a distinct disease entity, is characterized by a usually chronic course,
From
the Hematopathology
Section,
Laboratory
of Pathology, National Cancer Institute; the Laboratory of Immunology, and the Laboratory of Clinical Investigation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland. Requests for reprints should be addressed to Dr. Elaine S. Jaffe, Laboratory of Pathology, National Cancer Institute, Bldg. IO, Room 2A29, National Institutes of Health, Bethesda, Maryland 20014. Manuscript accepted December 20. 1973.
108
July 1974
The American
Journal
marked splenomegaly (often at the time of presentation), and a proliferation of characteristic mononuclear cells in the spleen, bone marrow and lymph nodes. Since Ewald first described this entity in 1923 [ 11, most workers have considered the proliferating cells to be primitive reticulum cells [l-5]. However, morphologically they also resemble lymphocytes, and there has been some functional and ultrastructural evidence to support their lymphocytic origin [6,7]. Other experimental evidence based on similar methods favors their belonging to the histiocytic series [5]. Some workers contend that these cells are primitive reticulum cells capable of undergoing either lymphocytic or histiocytic differentiation [2,3]. Recent evidence indicates that human lymphoreticular cells can be characterized and identified through the demonstration of specific membrane receptors. In most species studied bone marrow derived lymphocytes (B cells), in addition to bearing demonstrable immunoglobulin on their cell surfaces [8,9], often have receptors for the third component of complement (C3) [lo]. Cells having these receptors can be identified by their binding of red cells coated with antibody and complement (EAC). B lymphocytes also bear a receptor for the Fc portion of the immunoglobulin molecule which can be detected with radiolabeled soluble antigen-antibody complexes [ 11,121 or with fluorescein-labeled aggregated human immunoglobulin G (IgG) [ 131. Monocytes, histiocytes and macrophages also bear C3 receptors [ 141 and in addition bear receptors for cytophilic antibody which can be readily detected by IgG coated red blood cells (IgGEA) [ 1%171. Thymus-derived lymphocytes (T cells) bear none of these receptors but under certain conditions can be identified by their ability to form nonimmune rosettes with sheep red blood cells [ 18,191. Studies on human leukemia cells have shown that certain neoplastic cells bear specific membrane receptors, and it is suggested that the presence or absence of
of Medicine
Volume
57
LEUKEMIC RETlCULOENDOTHELlOSlS-JAFFE
these various receptors may indicate the origin of the neoplastic cells in question [20-221. A recent advance has been the adaptation of the technic for demonstrating EAC rosettes to use on frozen tissue sections [23]. We attempted to characterize the cells of leukemic reticuloendotheliosis by examining them for the presence of the IgMEAC and IgGEA receptors, both in cell suspensions and in frozen tissue sections. MATERIALS
AND METHODS
Preparation of Spleens and Lymph Nodes. Control studies were performed on histologically nonneoplastic spleens and lymph nodes obtained at surgery. Immediately after removal, a small portion of the spleen or lymph node was minced finely in RPMI-1640 (Grand Island Biological Co., Grand Island, N.Y.) with 10 per cent fetal calf serum. The minced tissue and supernetant were then filtered through stainless steel mesh. A pellet was obtained, and the red cells were lysed with ammonium chloride. The remaining white cells were then washed three times in serum-free medium and brought to a concentration of 2-3 X 106/ml. This suspension was then used for the rosetteforming assay to be described. For studies of frozen sections, representative tissue blocks were frozen immediately in O.C.T. embedding medium (Ames Co., Div. Miles Laboratories, Inc., Elkhart, Indiana 46514) and maintained at -7O’C until sectioning. The remainder of the tissue was fixed in either formalin or B5 1241. Preparation of IgMEAC, IgGEA and IgMEA. Rabbit IgG and IgM anti-Forssman antibodies to sheep red blood cells (SRBC) were prepared as previously described [25]. They were isolated by gel filtration on Sephadex@ G-200 columns followed by sucrose gradient ultracentrifugation. Sensitized sheep erythrocytes (EA) were prepared with isolated rabbit IgG and IgM antibodies by using the optimal concentration of antibody for maximal complement mediated hemolysis at 37’C. To prepare IgMEAC complexes, normal mouse serum was used as the source of complement (C). It was diluted 1: 10 in veronal buffered saline containing optimal concentrations of calcium and magnesium ions in 0.1 per cent gelatin and then added to an equal volume of IgMEA (5 X iOs/ml in phosphate buffered saline solution [PBS]) and incubated at 37’C for 30 minutes. The cells were then washed twice in PBS and reconstituted to the concentration described. It should be noted that IgGEAC is an unsuitable reagent and is not used because IgGEA sites within the EAC complex would also bind to histiocyte IgGEA receptors. Rosette Assay for the Binding of IgMEAC and IgGEA in Suspensions. Equal volumes of the white cell suspensions (2-3 X 106/ml) and the IgGEA, IgMEAC or IgMEA suspensions (1 X 108/ml) were mixed in plastic tubes and gently rotated at 37’C for 30 minutes. An aliquot of the suspension was then mixed with an equal volume of trypan blue and examined in a hemocytometer chamber. Only viable cells were counted. Any cell binding 3 or more erythrocytes was scored as positive. A minimum of 200 cells was counted, and the percentage of rosette-forming cells was determined. IgMEA, used as a control to detect
ET AL.
any nonspecific binding, was consistently negative under these conditions. For cytologic identification of individual rosetted cells, the rosetted cell suspension was fixed in an equal volume of Perfix@ (Applied Bioscience, Patterson, N.J.), filtered onto a millipore membrane (Millipore Corp., Bedford, Mass. 01730) and stained with hematoxylin and eosin. IgMEAC and IgGEA Rosette Technic for Frozen Sections. Six micron sections were cut with a cryostat and allowed to air dry. The sections were layered with IgGEA, IgMEAC or IgMEA (1 X 1O*/ml) and incubated at room temperature for 30 minutes. The slides were then washed three times in PBS to remove nonadherent red cells. The resultant preparations were fixed for 15 minutes in Perfix, stained with hematoxylin and eosin, and examined by light microscopy. CASE REPORTS The first patient (J.S. CC No. 08-87-04-3) a 33 year old white man, initially noted fatigue and listlessness in the fall of 1969. These symptoms persisted and in the summer of 1970 he was found to have an elevated white blood count with cells in the peripheral blood suggestive of “hairy cell” leukemia. Marked splenomegaly was also noted. The patient was referred to the National Cancer Institute where a diagnosis of leukemic reticuloendotheliosis was confirmed. The white blood cell count on admission was 13,200/mm3 with 37 per cent “lymphoreticular cells.” On Wright-Giemsa smear these cells had characteristic cytoplasmic projections. The phase microscopy preparations showed numerous pseudopods. There was focal marrow involvement. Following chemotherapy the peripheral blood and bone marrow were cleared of abnormal cells, but the spleen remained palpable. The patient was readmitted in December 1972 because of persistent splenomegaly and thrombocytopenia. White blood cell count at this time was 2,500/mm3 with a normal differential. A splenectomy was performed on December 19, 1972. The second patient (H.G. WRH No. 3404001) a 36 year old white man, presented with acute pneumonia in the upper lobe of the left lung. A complete blood count at that time revealed a white blood cell count of 2,000/mm3 with 100 per cent lymphocytes, a hematocrit value of 22 per cent and a platelet count of 45,000/mm3. Reticulocyte count was 0.3 per cent. Physical examination revealed marked splenomegaly without hepatomegaly or lymphadenopathy. Marrow aspiration was achieved with difficulty and revealed 90 per cent “lymphoreticular” cells and 10 per cent plasma cells. The “lymphoreticular” cells demonstrated fine cytoplasmic projections on the Wright-Giemsa stained smear. Bone marrow biopsy revealed a hypercellular marrow largely replaced by “lymphoreticular” cells having abundant clear cytoplasm. A diagnosis of leukemic reticuloendotheliosis was made, and the patient received chemotherapy and splenic radiation, with some subsequent decrease in spleen size. However, because of persistent pancytopenia, splenectomy was performed on May 1, 1973. RESULTS Spleens-Gross and Microscopic Findings. The spleen from patient J.S. weighed 700 g. The cut surJuly 1974
The American Journal of Medicine
Volume 57
109
LEUKEMIC RETiCULOENDOTHELlOSlS-JAFFE ET AL.
Figure 2. Patient H.G. Millipore filter preparation of IgGEA rosettes. Note fine cytoplasmic processes of cell with rosette formation. Cytologic features are similar to those of neoplastic cells in Figure 1. Hematoxylin and eosin stain; original magnification X 3,200.
TABLE II
Patient
Figure 1. Patient J.S. Histology of spleen. Neoplastic cells diffusely infiltrate cords and sinusoids of red pulp. Hematoxylin and eosin stain; original magnification X 400.
TABLE
I
Rosette Formation
B lymphocytes T lymphocytes Monocytes/histiocytes
in Suspension IgMEAC
IgGEA
+ 0
0 0
+
+
J.S. H.G.
face was brick red, homogeneous and firm. The spleen from patient H.G. weighed 1,800 g and contained several small wedge-shaped infarcts in various stages of organization. The remainder of the spleen was brick red and homogeneous. Hematoxylin and eosin stained sections (Figure 1) of both spleens displayed similar histologic features with complete obliteration of normal architecture. Both the red and white pulp regions were diffusely populated by uniform cells with round to oval nuclei and pale cytoplasm. Many of the cells from both spleens were positive for acid phosphatase activity, but this activity was inhibited by tartrate. IgGEA and IgMEAC Rosettes. The reported binding properties of the IgMEAC and the IgGEA reagents to the different cell types are shown in Table I. IgMEAC binds to both B lymphocytes and to monocytes/histiocytes; IgGEA binds only to monocytes/histiocytes. Seventy-one and eight tenths per cent of the cells from patient J.S.‘s splenic suspension and 86.4 per l
l We gratefully acknowledge the assistance of Dr. lsao Katayama who performed the histochemical studies on the biopsy specimens according to his published methods [26].
110
July 1974
The American Journal of Medicine
J.L. R.D. P.P. O.H. H.D. W.S. J.B. R.S. A.M. SM. R.B.
Volume 57
IgMEAC and IgGEA Rosette-Forming Cells in Spleens of Control Subjects and Patients (%) InMEAC Control Subjects 67 33 37 30 50 49 22 21 41 51 52 Patients 4.5 4.0
IaGEA
15 2 2 15 13 9 5 4 10 11 11 71.8 86.4
cent of the cells from patient H.G.‘s splenic suspension formed IgGEA rosettes. On the millipore filter these stained rosetted cells were morphologically identifiable as belonging to the neoplastic population (Figure 2). In contrast, IgGEA rosette-forming cells from 11 histologically normal spleens ranged from 2.0 to 15 per cent with a mean of 9.0 per cent (Table II). All cells from control spleens forming IgGEA rosettes appeared morphologically to be normal histiocytes on the millipore filters. No cells resembling lymphocytes formed IgGEA rosettes. Only 4.5 and 4.0 per cent of the cells from the splenic suspensions of patients J:S. and H.G., respectively, formed IgMEAC rosettes. In contrast, in suspensions from 11 control spleens, cells forming IgMEAC rosettes ranged from 21 to 67 per cent with a mean of 41 per cent (Table II). On the millipore filter preparations almost all these rosetted cells were morphologically identifiable as lymphocytes. Polymorphonuclear leukocytes, which also have C3 receptors, consistently comprised less than 5 per cent of all rosetted cells on the millipore filter preparations. No cells from control spleens with the morphologic
LEUKEMIC RETICULOENDOTHELIOSIS-JAFFE
features of histiocytes IgMEAC rosettes.
were
observed
to
form
Rosette Formation on Frozen Tissue Sections. In histologically normal spleen and lymph node, IgMEAC was seen to adhere to the primary and secondary lymphoid follicles (these areas are known to contain predominantly t3 cells) [27]. The T cell areas (in the spleen, the periarteriolar lymphoid sheath and in the lymph node, the parafollicular zone) were always spared. As expected, the IgGEA reagent adhered to the histiocytes in the subcapsular and medullary sinuses of the lymph node and to the histiocytes of the cords of Billroth in the spleen. The pattern of adherence of the IgMEAC reagent
ET AL.
to the histiocytic areas of lymph node and spleen gave an unexpected result. The IgMEAC reagent adhered to only the sinusoidal histiocytes of lymph node but not to the sinusoidal histiocytes of spleen. It would, therefore, appear that although peripheral blood monocytes and lymph node monocytes have receptors for both C3 and cytophilic antibody, the histiocytes of the spleen have only the receptor for cytophilic antibody. No nonspecific adherence of the IgMEA reagent was ever observed under these conditions. In splenic sections from both patients J.S. and H.G., no adherence of IgMEAC was observed, whereas when the sections were treated with IgGEA,
Figure 3. IgMEAC and IgGEA rosettes on frozen sections. Figures on left are bright field images. Figures on right are identical fields taken with dark field condenser and adherent red cells appear as white dots. a, normal spleen treaded with IgMEAC. IgMEAC adheres to eccentric follicle (6 cells) and spares periarteriolar lymphoid sheath (T cells) and red pulp (histiocytes). b, normal spleen treated with IgGEA. Note adherence to red pulp (histiocytes) and sparing of white pulp (lymphocytes). c, spleen from patient (J.S.) treated with IgGEA. Reagent is diffusely adherent to neoplastic cells in both red and white pulp regions, Ail hematoxylin and eosin stain; original magnification X 63. July 1974
The American Journal of Medicine
Volume 57
111
LEUKEMIC RETICULOENDOTHELIOSIS-JAFFE
ET AL.
intense and diffuse localization of the reagent noted in both red and white pulp areas (Figure IgMEA was again negative.
was 3C).
COMMENTS The technics employed here are known to detect specific membrane receptors on human lymphoretitular cells in suspension [20-221. Dukor [23] showed that the rosette technic for demonstrating C3 receptors can be applied to frozen tissue sections of mouse spleen and lymph nodes. Host red blood cells do not interfere with interpretation since they are lysed by the freezing and thawing process. Our results indicate that this technic works equally well on human tissues. We have further shzwn that the method for demonstrating cells having receptors for cytophilic antibody can also be adapted to use on frozen sections. Frozen sections have the great advantage of enabling the observer to determine the histopathologic localization of cells that formed rosettes and those that did not. In neoplastic conditions, in which a lymphoid organ may be only partially involved by tumor, a study of frozen sections as well as cell suspensions is essential. Furthermore, in conditions such as Hodgkin’s disease, representative cell suspensions often cannot be prepared because of entrapment of the neoplastic cells in a dense connective tissue stroma. Such tissues are readily amenable to study in frozen sections. The patients we studied presented typical clinical and pathologic features of leukemic reticuloendotheliosis. Splenomegaly is often the only abnormality on physical examination [2,4], and the spleen is characteristically the organ most extensively involved pathologically [28]. Biopsied lymph nodes and bone marrow frequently contain increased “reticulum cells” but are rarely infiltrated by neoplastic cells as extensively as the spleen. The importance of the spleen in this disease is further emphasized by the fact that although chemotherapy or radiotherapy is rarely helpful, splenectomy can often produce significant clinical improvement with long-term survival [ 291. These observations all suggest that this neoplasm is a primary splenic tumor. Yam et al. [29,30] described a tartrate-resistant acid phosphatase in a series of cases of leukemic reticuloendotheliosis. That isoenzyme was not present in the cells of the two patients we describe. However, recent observations indicate that the tartrateresistant isoenzyme may not be uniformly present in all cases of leukemic reticuloendotheliosis [3 1,321, and indeed that one cannot distinguish by any conventional clinical or pathologic criteria the tartrateresistant and tartrate-sensitive cases. The origin of the neoplastic cells in this entity has
112
July 1974
The American Journal of Medicine
Volume 57
been controversial, largely because of their apparent hybrid nature. By light and electron microscopy they have many features of lymphocytes [6,7,33]. However, their characteristic cytoplasmic processes and pseudopods as seen on air-dried smears, by phase microscopy and by scanning electron microscopy [34] are more suggestive of reticulum cells or histiocytes. Functionally, although they do not appear to be glass adherent [4,6], they have been reported to adhere to cotton fibers [ 51. Histochemically they also more closely resemble monocytes or histiocytes than lymphocytes [3,5]. Mitus et al. [3] suggested, on the basis of their morphologic and histochemical observations, that they are derived from the primitive reticulum cell and are capable of differentiating towards either a lymphocyte or a histiocyte. Our immunologic studies have shown the cells to have membrane receptors for cytophilic antibody and to lack receptors for C3 as detected with these reagents. In our material these binding properties are characteristic of cells of the histiocytic series, in particular, the splenic histiocytes of the cords of Billroth. That is, although the B cell areas of both control lymph nodes and spleens showed exquisite localization of IgMEAC, histiocytes in the two organs showed different patterns of reactivity with this reagent. IgMEAC localized to histiocytes in subcortical and medullary lymph node sinuses but did not react with histiocytes in the splenic cords of Billroth. Thus splenic histiocytes in frozen sections of normal spleen appear to have only receptors for IgGEA in contrast with sinusoidal histiocytes of normal lymph node, which demonstrate receptors for both IgGEA and C3. One could speculate that these findings are peculiar to the frozen section technic and that perhaps in the spleen the ring fibers of the cords prevent attachment of the sensitized erythrocytes to the histiocyte cell membrane. However, the ring fibers certainly do not prevent attachment of erythrocytes coated with IgG. One might also speculate that the C3 receptor of splenic histiocytes is more temperature dependent, but when the frozen sections are incubated at 37’C, attachment of IgMEAC is still not observed. This finding still could be peculiar to the frozen section technic, but it is our impression, from study of the stained millipore filter-preparations, that normal splenic histiocytes even in suspension do not form IgMEAC rosettes with these reagents. Although diversity among the histiocytic/monocytic population might at first impression seem surprising, such diversity among cells of the immune system is not without precedent. Indeed, there is evidence that not all immunoglobulin-bearing B lymphocytes have the C3 receptor 1221, and by analogy not all histiocytes may bear both the IgG receptor and the C3 receptor. Moreover, one must note that in these studies rabbit
LEUKEMK:RETlCULOENDOTHELlOSlS-JAFFE ET AL.
antibodies and mouse serum as a source of complement were employed. It is possible that membrane receptors on different cells may show different species specificity. Further studies to elucidate the nature of the membrane receptors of histiocytes of different organs are now underway in our laboratory. Although B lymphocytes also bear a receptor for the Fc portion of IgG, we do not think that the receptor being detected on the neoplastic cells is of this type. The Fc receptor of B lymphocytes can most readily be detected with radiolabeled soluble antigenantibody complexes [ 11,121 or with fluorescein-labeled aggregated IgG [ 131. In our present report, frozen sections of both control spleens and lymph nodes treated with the IgGEA reagent showed exquisite localization of the reagent red cells only to histiocytes. When the IgGEA reagent was used with cell suspensions from control spleens and lymph nodes, only a small percentage of the cells formed IgGEA rosettes, and morphologically all appeared to be histiocytes. Therefore, under the conditions used in our study the IgGEA reagent detects only the monocyte IgGEA receptor and not the Fc receptor of the B lymphocyte. Furthermore, we have been unable to detect any binding of this IgGEA reagent to the malignant lymphocytes of five patients with chronic lymphocytic leukemia or to normal human peripheral blood lymphocytes [20]. Other investigators have also been unable to demonstrate binding.of IgGEA to normal mouse B cells [ 161 or to normal or mitogenstimulated human lymphocytes [ 171. However, Brain and Marston [35] recently reported the binding of human red blood cells which had been sensitized with a high titer anti-D serum to normal human B cells. It would, therefore, appear that the ability of different IgGEA reagents to bind to different cell receptors may depend on the source and concentration of the antibody and the nature of the red cells. It is also of interest that in three recent studies [7,36,37] surface immunoglobulin of the IgG class has been demonstrated on the cells from a number of patients with leukemic reticuloendotheliosis. Superficially, this finding might be regarded as evidence for the B cell origin of these malignant cells. How-
ever, it should be noted that cells of the histiocytic series may be stained not because of synthesized surface IgG, but rather because they have a receptor for IgG and therefore they may have been coated by cytophilic IgG in vivo; alternatively, they can bind the fluoresceinated antiimmunoglobulin reagent itself by its Fc moiety. Therefore, any conclusions as to the origin of cells based on their surface staining must be made with caution. It is particularly noteworthy that in two of the three studies [7,36] the surface immunoglobulins detected were polyclonal. Such a result would be expected if the immunoglobulin was present as a consequence of the IgG receptor, rather than of cell synthesis. In only one study [37] did the surface immunoglobulin appear to be monoclonal. If additional cases with monoclonal immunoglobulin are reported, a B cell origin for their cells would seem probable, particularly if such surface immunoglobulin could be removed and regenerated in vitro. In that event it may be that leukemic reticuloendotheliosis as currently defined is complex and includes cases of both B lymphocytic and histiocytic origin in different patients. We have examined the neoplastic cells of two patients with leukemic reticuloendotheliosis for a feature not previously reported, the presence of specific membrane receptors. These receptors can be used as functional markers of possible aid in the identification of cells in a variety of lymphoreticular malignancies and may give a clue as to the state of cellular differentiation. In our two cases of leukemic reticuloendotheliosis, it was shown that the neoplastic cells had a receptor similar to that of normal splenic histiocytes. ACKNOWLEDGMENT We are indebted to Dr. Richard A. Miskoff, Walter Reed Army Medical Center, who provided us with splenic tissue and clinical information on the second patient (H.G.). We are grateful to Mrs. Eileen Sussman for her expert technical assistance and to Mr. Ralph lsenburg for photographic assistance, both of the Laboratory of Pathology of the National Cancer Institute.
REFERENCES 1. Ewald 0: Die leukamische reticuloendotheliose. Deutsches Arch Klin Med 142: 222, 1923. 2. 3.
4.
5.
Bouroncle BA, Wiseman BK, Doan CA: Leukemic reticuloendotheliosis. Blood 13: 609, 1958. Mitus WJ, Mednicoff IB. Wittels B, Dameshek W: Neoplastic lvmohoid reticulum cells in the oerioheral blood: a histochemical study. Blood 17: 206, i97i. Shrek R, Donnelly WJ: “Hairy” cells in blood in lymphoretitular neoplastic disease and “flagellated” cells of normal lymph nodes. Blood 27: 199, 1966. Yam LT. Castoldi GL, Garvey MD, Mitus WJ: Functional cy-
6.
7.
8.
July 1974
togenetic and cytochemical study of the leukemic reticulum cells. Blood 32: 90, 1968. Rubin AD, Douglas SD. Chessin LN, Glade PR, Dameshek W: Chronic reticulolymphocytic leukemia. Am J Med 47: 149, 1969. Burns CP, Maca RD. Hoak JC: Biochemical, morphological, and immunological observations of leukemic reticufoendotheliosis. Cancer Res 33: 1615. 1973. Rabellino E, Colon S. Grey HM, Unanue ER: Immunoglobulins on the surface of lymphocytes. I. Distribution and quantitation. J Exp Med 133: 156. 1971.
The American Journal of Medicine
Volume 57
113
LEUKEMIC RETlCULOENDOTHELlOSlS-JAFFE Ei AL.
9.
10.
11.
12. 13. 14.
15. 16. 17.
18.
19.
20.
21.
22.
114
Unanue El?, Grey HM, Rabellino E, Campbell P, Schmidtke J: lmmunoglobulins on the surface of lymphocytes. II. The bone marrow as the main source of lymphocytes with detectable surface-bound immunoglobulin. J Exp Med 133: 1188,197l. Bianco CR, Patrick R, Nussenzweig V: A population of lymphocytes bearing a membrane receptor for antigen-antibody complement complexes. I. Separation and characterization. J Exp Med 132: 702, 1970. Basten A, Miller JFAP, Sprent J, Pye J: A receptor for antibody on B lymphocytes. I. Method of detection and functional significance. J Exp Med 135: 610, 1972. Basten A, Warner NL, Mandel T: Lymphocytes and the immune response. II. J Exp Med 135: 627, 1972. Dickler HB, Kunkel HG: Interaction of aggregated y-globulin with B lymphocytes. J Exp Med 136: 191, 1972. Huber H, Polley JJ, Linscott WD, Fudenberg HH, MullerEberhard HJ: Human monocytes: distinct receptor sites for the third component of complement and for immunoglobulin G. Science 162: 1281, 1968. Berken B, Benacerraf B: Properties of antibodies cytophilic for macrophages. J Exp Med 123: 119, 1966. Lay WH, Nussenzweig V: Receptors for complement on leukocytes. J Exp Med i28: 991, 1968. Huber H, Douglas SD, Fudenberg HH: The IgG receptor: an immunological marker for the characterization of mononuclear cells. Immunology 17: 7, 1969. Jondal M, Holm G, Wigzell H: Surface markers on human T and B lymphocytes. I. A large population of lymphocytes forming nonimmune rosettes with sheep red blood cells. J Exp Med 136: 207, 1972. Lay WH. Mendes NF. Bianco C, Nussenzweig V: Binding of sheep red blood cells to a large population of human lymphocytes. Nature (Lond) 230: 53 1, 197 1. Shevach EM, Herberman R, Frank MM, Green I: Receptors for complement and immunoglobulin on human leukemic cells and human lymphoblastoid cell lines. J Clin Invest 51: 1933.1972. Pincus S, Bianco C, Nussenzweig V: Increased proportion of complement-receptor lymphocytes in the peripheral blood of patients with chronic lymphocytic leukemia. Blood 40: 303, 1972. Ross GD, Rabellino EM, Polley MJ, Grey HM: Combined studies of complement receptor and surface immunoglobulin bearing cells and sheep erythrocyte rosette-
July 1974
The American Journal of Medicine
Volume 57
23.
24.
25.
26.
27.
28. 29.
30.
31.
32. 33.
34. 35. 36.
37.
forming cells in normal and leukemic human lymphocytes. J Clin Invest 52: 377, 1973. Dukor P, Bianco C, Nussenzweig V: Tissue localization of lymphocytes bearing a membrane receptor for antigenantibody-complement complexes. Proc Natl Acad Sci USA 67: 991, 1970. Bowling MC: Histopathology laboratory procedures of the pathologic anatomy branch of the National Cancer Institute, Washington D.C., U.S. Government Printing Office, 1967. p 2. Frank MM, Gaither T: The effect of temperature on the reactivity of guinea pig complement with y-G and -r-M haemolytic antibodies. Immunology 19: 967, 1970. Katayama I, Li CY, Yam LT: Histochemical study of acid phosphatase isoenzyme in leukemic reticuloendotheliosis. Cancer 29: 157, 1972. Craddock CA, Longmire R, McMillan R: Lymphocytes and the immune response. II. New Engl J Med 285: 378, 1971. lijima S: Leukemic reticuloendotheliosis. Tohoku J Exp Med 89: 35, 1966. Yam LT. Li CY, Finkel H: Leukemic reticuloendotheliosis. The role of tartrate-resistant acid phosphatase in diagnosis and splenectomy in treatment. Arch Intern Med 130: 248, 1972. Yam LT. Li CY, Lam KW: Tartrate-resistant acid phosphatase isoenzyme in the reticulum cells of leukemic reticuloendotheliosis. New Engl J Med 284: 357, 1971. Burke JS, Byrne GE Jr, Rappaport H: Hairy cell leukemia (leukemic reticuloendotheliosis). I. A clinical and pathologic study of 21 patients. Cancer (in press). Lennert K: Personal communication, 1974. Katayama I, Li CY, Yam LT: Ultrastructural characteristics of the hairy cells of leukemic reticuloendotheliosis. Am J Pathol 67: 361. 1972. Trubowitz S, Masek B, Frasca JM: Leukemic reticuloendotheliosis. Blood 38: 288, 197 1. Brain P, Marston RH: Rosette formation by human T and B lymphocytes. Eur J lmmuno13: 6, 1973. Preud’homme JL. Seligmann M: Surface bound immunoglobulins as a cell marker in human lymphoproliferative diseases. Blood 40: 777, 1972. Aisenberg AC, Bloch KJn Long JC: Cell-surface immunoglobulins in chronic lymphocytic leukemia and allied disorders. Am J Med 55: 184, 1973.
Presence of a
Receptor for Cytophilic Antibody
ELAINE
S. JAFFE,
ETHAN
M. SHEVACH,
MICHAEL
M. FRANK,
IRA GREEN,
Neoplastic cells from the spleens of two patients with leukemic reticuloendotheliosis were studied in cell suspensions and in frozen sections for the antigen-antibody complement (IgMEAC) receptor of B lymphocytes and for the receptor for cytophilic antibody (IgGEA) of monocytes. A high percentage of the neoplastic cells, both in suspension and in sections, bound the IgGEA reagent but failed to bind the IgMEAC reagent. These findings suggest that these cells belong to the monocyte-histiocyte series.
M.D. M.D. M.D.
M.D.
Bethesda, Maryland
Leukemic reticuloendotheliosis, now generally recognized as a distinct disease entity, is characterized by a usually chronic course,
From
the Hematopathology
Section,
Laboratory
of Pathology, National Cancer Institute; the Laboratory of Immunology, and the Laboratory of Clinical Investigation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland. Requests for reprints should be addressed to Dr. Elaine S. Jaffe, Laboratory of Pathology, National Cancer Institute, Bldg. IO, Room 2A29, National Institutes of Health, Bethesda, Maryland 20014. Manuscript accepted December 20. 1973.
108
July 1974
The American
Journal
marked splenomegaly (often at the time of presentation), and a proliferation of characteristic mononuclear cells in the spleen, bone marrow and lymph nodes. Since Ewald first described this entity in 1923 [ 11, most workers have considered the proliferating cells to be primitive reticulum cells [l-5]. However, morphologically they also resemble lymphocytes, and there has been some functional and ultrastructural evidence to support their lymphocytic origin [6,7]. Other experimental evidence based on similar methods favors their belonging to the histiocytic series [5]. Some workers contend that these cells are primitive reticulum cells capable of undergoing either lymphocytic or histiocytic differentiation [2,3]. Recent evidence indicates that human lymphoreticular cells can be characterized and identified through the demonstration of specific membrane receptors. In most species studied bone marrow derived lymphocytes (B cells), in addition to bearing demonstrable immunoglobulin on their cell surfaces [8,9], often have receptors for the third component of complement (C3) [lo]. Cells having these receptors can be identified by their binding of red cells coated with antibody and complement (EAC). B lymphocytes also bear a receptor for the Fc portion of the immunoglobulin molecule which can be detected with radiolabeled soluble antigen-antibody complexes [ 11,121 or with fluorescein-labeled aggregated human immunoglobulin G (IgG) [ 131. Monocytes, histiocytes and macrophages also bear C3 receptors [ 141 and in addition bear receptors for cytophilic antibody which can be readily detected by IgG coated red blood cells (IgGEA) [ 1%171. Thymus-derived lymphocytes (T cells) bear none of these receptors but under certain conditions can be identified by their ability to form nonimmune rosettes with sheep red blood cells [ 18,191. Studies on human leukemia cells have shown that certain neoplastic cells bear specific membrane receptors, and it is suggested that the presence or absence of
of Medicine
Volume
57
LEUKEMIC RETlCULOENDOTHELlOSlS-JAFFE
these various receptors may indicate the origin of the neoplastic cells in question [20-221. A recent advance has been the adaptation of the technic for demonstrating EAC rosettes to use on frozen tissue sections [23]. We attempted to characterize the cells of leukemic reticuloendotheliosis by examining them for the presence of the IgMEAC and IgGEA receptors, both in cell suspensions and in frozen tissue sections. MATERIALS
AND METHODS
Preparation of Spleens and Lymph Nodes. Control studies were performed on histologically nonneoplastic spleens and lymph nodes obtained at surgery. Immediately after removal, a small portion of the spleen or lymph node was minced finely in RPMI-1640 (Grand Island Biological Co., Grand Island, N.Y.) with 10 per cent fetal calf serum. The minced tissue and supernetant were then filtered through stainless steel mesh. A pellet was obtained, and the red cells were lysed with ammonium chloride. The remaining white cells were then washed three times in serum-free medium and brought to a concentration of 2-3 X 106/ml. This suspension was then used for the rosetteforming assay to be described. For studies of frozen sections, representative tissue blocks were frozen immediately in O.C.T. embedding medium (Ames Co., Div. Miles Laboratories, Inc., Elkhart, Indiana 46514) and maintained at -7O’C until sectioning. The remainder of the tissue was fixed in either formalin or B5 1241. Preparation of IgMEAC, IgGEA and IgMEA. Rabbit IgG and IgM anti-Forssman antibodies to sheep red blood cells (SRBC) were prepared as previously described [25]. They were isolated by gel filtration on Sephadex@ G-200 columns followed by sucrose gradient ultracentrifugation. Sensitized sheep erythrocytes (EA) were prepared with isolated rabbit IgG and IgM antibodies by using the optimal concentration of antibody for maximal complement mediated hemolysis at 37’C. To prepare IgMEAC complexes, normal mouse serum was used as the source of complement (C). It was diluted 1: 10 in veronal buffered saline containing optimal concentrations of calcium and magnesium ions in 0.1 per cent gelatin and then added to an equal volume of IgMEA (5 X iOs/ml in phosphate buffered saline solution [PBS]) and incubated at 37’C for 30 minutes. The cells were then washed twice in PBS and reconstituted to the concentration described. It should be noted that IgGEAC is an unsuitable reagent and is not used because IgGEA sites within the EAC complex would also bind to histiocyte IgGEA receptors. Rosette Assay for the Binding of IgMEAC and IgGEA in Suspensions. Equal volumes of the white cell suspensions (2-3 X 106/ml) and the IgGEA, IgMEAC or IgMEA suspensions (1 X 108/ml) were mixed in plastic tubes and gently rotated at 37’C for 30 minutes. An aliquot of the suspension was then mixed with an equal volume of trypan blue and examined in a hemocytometer chamber. Only viable cells were counted. Any cell binding 3 or more erythrocytes was scored as positive. A minimum of 200 cells was counted, and the percentage of rosette-forming cells was determined. IgMEA, used as a control to detect
ET AL.
any nonspecific binding, was consistently negative under these conditions. For cytologic identification of individual rosetted cells, the rosetted cell suspension was fixed in an equal volume of Perfix@ (Applied Bioscience, Patterson, N.J.), filtered onto a millipore membrane (Millipore Corp., Bedford, Mass. 01730) and stained with hematoxylin and eosin. IgMEAC and IgGEA Rosette Technic for Frozen Sections. Six micron sections were cut with a cryostat and allowed to air dry. The sections were layered with IgGEA, IgMEAC or IgMEA (1 X 1O*/ml) and incubated at room temperature for 30 minutes. The slides were then washed three times in PBS to remove nonadherent red cells. The resultant preparations were fixed for 15 minutes in Perfix, stained with hematoxylin and eosin, and examined by light microscopy. CASE REPORTS The first patient (J.S. CC No. 08-87-04-3) a 33 year old white man, initially noted fatigue and listlessness in the fall of 1969. These symptoms persisted and in the summer of 1970 he was found to have an elevated white blood count with cells in the peripheral blood suggestive of “hairy cell” leukemia. Marked splenomegaly was also noted. The patient was referred to the National Cancer Institute where a diagnosis of leukemic reticuloendotheliosis was confirmed. The white blood cell count on admission was 13,200/mm3 with 37 per cent “lymphoreticular cells.” On Wright-Giemsa smear these cells had characteristic cytoplasmic projections. The phase microscopy preparations showed numerous pseudopods. There was focal marrow involvement. Following chemotherapy the peripheral blood and bone marrow were cleared of abnormal cells, but the spleen remained palpable. The patient was readmitted in December 1972 because of persistent splenomegaly and thrombocytopenia. White blood cell count at this time was 2,500/mm3 with a normal differential. A splenectomy was performed on December 19, 1972. The second patient (H.G. WRH No. 3404001) a 36 year old white man, presented with acute pneumonia in the upper lobe of the left lung. A complete blood count at that time revealed a white blood cell count of 2,000/mm3 with 100 per cent lymphocytes, a hematocrit value of 22 per cent and a platelet count of 45,000/mm3. Reticulocyte count was 0.3 per cent. Physical examination revealed marked splenomegaly without hepatomegaly or lymphadenopathy. Marrow aspiration was achieved with difficulty and revealed 90 per cent “lymphoreticular” cells and 10 per cent plasma cells. The “lymphoreticular” cells demonstrated fine cytoplasmic projections on the Wright-Giemsa stained smear. Bone marrow biopsy revealed a hypercellular marrow largely replaced by “lymphoreticular” cells having abundant clear cytoplasm. A diagnosis of leukemic reticuloendotheliosis was made, and the patient received chemotherapy and splenic radiation, with some subsequent decrease in spleen size. However, because of persistent pancytopenia, splenectomy was performed on May 1, 1973. RESULTS Spleens-Gross and Microscopic Findings. The spleen from patient J.S. weighed 700 g. The cut surJuly 1974
The American Journal of Medicine
Volume 57
109
LEUKEMIC RETiCULOENDOTHELlOSlS-JAFFE ET AL.
Figure 2. Patient H.G. Millipore filter preparation of IgGEA rosettes. Note fine cytoplasmic processes of cell with rosette formation. Cytologic features are similar to those of neoplastic cells in Figure 1. Hematoxylin and eosin stain; original magnification X 3,200.
TABLE II
Patient
Figure 1. Patient J.S. Histology of spleen. Neoplastic cells diffusely infiltrate cords and sinusoids of red pulp. Hematoxylin and eosin stain; original magnification X 400.
TABLE
I
Rosette Formation
B lymphocytes T lymphocytes Monocytes/histiocytes
in Suspension IgMEAC
IgGEA
+ 0
0 0
+
+
J.S. H.G.
face was brick red, homogeneous and firm. The spleen from patient H.G. weighed 1,800 g and contained several small wedge-shaped infarcts in various stages of organization. The remainder of the spleen was brick red and homogeneous. Hematoxylin and eosin stained sections (Figure 1) of both spleens displayed similar histologic features with complete obliteration of normal architecture. Both the red and white pulp regions were diffusely populated by uniform cells with round to oval nuclei and pale cytoplasm. Many of the cells from both spleens were positive for acid phosphatase activity, but this activity was inhibited by tartrate. IgGEA and IgMEAC Rosettes. The reported binding properties of the IgMEAC and the IgGEA reagents to the different cell types are shown in Table I. IgMEAC binds to both B lymphocytes and to monocytes/histiocytes; IgGEA binds only to monocytes/histiocytes. Seventy-one and eight tenths per cent of the cells from patient J.S.‘s splenic suspension and 86.4 per l
l We gratefully acknowledge the assistance of Dr. lsao Katayama who performed the histochemical studies on the biopsy specimens according to his published methods [26].
110
July 1974
The American Journal of Medicine
J.L. R.D. P.P. O.H. H.D. W.S. J.B. R.S. A.M. SM. R.B.
Volume 57
IgMEAC and IgGEA Rosette-Forming Cells in Spleens of Control Subjects and Patients (%) InMEAC Control Subjects 67 33 37 30 50 49 22 21 41 51 52 Patients 4.5 4.0
IaGEA
15 2 2 15 13 9 5 4 10 11 11 71.8 86.4
cent of the cells from patient H.G.‘s splenic suspension formed IgGEA rosettes. On the millipore filter these stained rosetted cells were morphologically identifiable as belonging to the neoplastic population (Figure 2). In contrast, IgGEA rosette-forming cells from 11 histologically normal spleens ranged from 2.0 to 15 per cent with a mean of 9.0 per cent (Table II). All cells from control spleens forming IgGEA rosettes appeared morphologically to be normal histiocytes on the millipore filters. No cells resembling lymphocytes formed IgGEA rosettes. Only 4.5 and 4.0 per cent of the cells from the splenic suspensions of patients J:S. and H.G., respectively, formed IgMEAC rosettes. In contrast, in suspensions from 11 control spleens, cells forming IgMEAC rosettes ranged from 21 to 67 per cent with a mean of 41 per cent (Table II). On the millipore filter preparations almost all these rosetted cells were morphologically identifiable as lymphocytes. Polymorphonuclear leukocytes, which also have C3 receptors, consistently comprised less than 5 per cent of all rosetted cells on the millipore filter preparations. No cells from control spleens with the morphologic
LEUKEMIC RETICULOENDOTHELIOSIS-JAFFE
features of histiocytes IgMEAC rosettes.
were
observed
to
form
Rosette Formation on Frozen Tissue Sections. In histologically normal spleen and lymph node, IgMEAC was seen to adhere to the primary and secondary lymphoid follicles (these areas are known to contain predominantly t3 cells) [27]. The T cell areas (in the spleen, the periarteriolar lymphoid sheath and in the lymph node, the parafollicular zone) were always spared. As expected, the IgGEA reagent adhered to the histiocytes in the subcapsular and medullary sinuses of the lymph node and to the histiocytes of the cords of Billroth in the spleen. The pattern of adherence of the IgMEAC reagent
ET AL.
to the histiocytic areas of lymph node and spleen gave an unexpected result. The IgMEAC reagent adhered to only the sinusoidal histiocytes of lymph node but not to the sinusoidal histiocytes of spleen. It would, therefore, appear that although peripheral blood monocytes and lymph node monocytes have receptors for both C3 and cytophilic antibody, the histiocytes of the spleen have only the receptor for cytophilic antibody. No nonspecific adherence of the IgMEA reagent was ever observed under these conditions. In splenic sections from both patients J.S. and H.G., no adherence of IgMEAC was observed, whereas when the sections were treated with IgGEA,
Figure 3. IgMEAC and IgGEA rosettes on frozen sections. Figures on left are bright field images. Figures on right are identical fields taken with dark field condenser and adherent red cells appear as white dots. a, normal spleen treaded with IgMEAC. IgMEAC adheres to eccentric follicle (6 cells) and spares periarteriolar lymphoid sheath (T cells) and red pulp (histiocytes). b, normal spleen treated with IgGEA. Note adherence to red pulp (histiocytes) and sparing of white pulp (lymphocytes). c, spleen from patient (J.S.) treated with IgGEA. Reagent is diffusely adherent to neoplastic cells in both red and white pulp regions, Ail hematoxylin and eosin stain; original magnification X 63. July 1974
The American Journal of Medicine
Volume 57
111
LEUKEMIC RETICULOENDOTHELIOSIS-JAFFE
ET AL.
intense and diffuse localization of the reagent noted in both red and white pulp areas (Figure IgMEA was again negative.
was 3C).
COMMENTS The technics employed here are known to detect specific membrane receptors on human lymphoretitular cells in suspension [20-221. Dukor [23] showed that the rosette technic for demonstrating C3 receptors can be applied to frozen tissue sections of mouse spleen and lymph nodes. Host red blood cells do not interfere with interpretation since they are lysed by the freezing and thawing process. Our results indicate that this technic works equally well on human tissues. We have further shzwn that the method for demonstrating cells having receptors for cytophilic antibody can also be adapted to use on frozen sections. Frozen sections have the great advantage of enabling the observer to determine the histopathologic localization of cells that formed rosettes and those that did not. In neoplastic conditions, in which a lymphoid organ may be only partially involved by tumor, a study of frozen sections as well as cell suspensions is essential. Furthermore, in conditions such as Hodgkin’s disease, representative cell suspensions often cannot be prepared because of entrapment of the neoplastic cells in a dense connective tissue stroma. Such tissues are readily amenable to study in frozen sections. The patients we studied presented typical clinical and pathologic features of leukemic reticuloendotheliosis. Splenomegaly is often the only abnormality on physical examination [2,4], and the spleen is characteristically the organ most extensively involved pathologically [28]. Biopsied lymph nodes and bone marrow frequently contain increased “reticulum cells” but are rarely infiltrated by neoplastic cells as extensively as the spleen. The importance of the spleen in this disease is further emphasized by the fact that although chemotherapy or radiotherapy is rarely helpful, splenectomy can often produce significant clinical improvement with long-term survival [ 291. These observations all suggest that this neoplasm is a primary splenic tumor. Yam et al. [29,30] described a tartrate-resistant acid phosphatase in a series of cases of leukemic reticuloendotheliosis. That isoenzyme was not present in the cells of the two patients we describe. However, recent observations indicate that the tartrateresistant isoenzyme may not be uniformly present in all cases of leukemic reticuloendotheliosis [3 1,321, and indeed that one cannot distinguish by any conventional clinical or pathologic criteria the tartrateresistant and tartrate-sensitive cases. The origin of the neoplastic cells in this entity has
112
July 1974
The American Journal of Medicine
Volume 57
been controversial, largely because of their apparent hybrid nature. By light and electron microscopy they have many features of lymphocytes [6,7,33]. However, their characteristic cytoplasmic processes and pseudopods as seen on air-dried smears, by phase microscopy and by scanning electron microscopy [34] are more suggestive of reticulum cells or histiocytes. Functionally, although they do not appear to be glass adherent [4,6], they have been reported to adhere to cotton fibers [ 51. Histochemically they also more closely resemble monocytes or histiocytes than lymphocytes [3,5]. Mitus et al. [3] suggested, on the basis of their morphologic and histochemical observations, that they are derived from the primitive reticulum cell and are capable of differentiating towards either a lymphocyte or a histiocyte. Our immunologic studies have shown the cells to have membrane receptors for cytophilic antibody and to lack receptors for C3 as detected with these reagents. In our material these binding properties are characteristic of cells of the histiocytic series, in particular, the splenic histiocytes of the cords of Billroth. That is, although the B cell areas of both control lymph nodes and spleens showed exquisite localization of IgMEAC, histiocytes in the two organs showed different patterns of reactivity with this reagent. IgMEAC localized to histiocytes in subcortical and medullary lymph node sinuses but did not react with histiocytes in the splenic cords of Billroth. Thus splenic histiocytes in frozen sections of normal spleen appear to have only receptors for IgGEA in contrast with sinusoidal histiocytes of normal lymph node, which demonstrate receptors for both IgGEA and C3. One could speculate that these findings are peculiar to the frozen section technic and that perhaps in the spleen the ring fibers of the cords prevent attachment of the sensitized erythrocytes to the histiocyte cell membrane. However, the ring fibers certainly do not prevent attachment of erythrocytes coated with IgG. One might also speculate that the C3 receptor of splenic histiocytes is more temperature dependent, but when the frozen sections are incubated at 37’C, attachment of IgMEAC is still not observed. This finding still could be peculiar to the frozen section technic, but it is our impression, from study of the stained millipore filter-preparations, that normal splenic histiocytes even in suspension do not form IgMEAC rosettes with these reagents. Although diversity among the histiocytic/monocytic population might at first impression seem surprising, such diversity among cells of the immune system is not without precedent. Indeed, there is evidence that not all immunoglobulin-bearing B lymphocytes have the C3 receptor 1221, and by analogy not all histiocytes may bear both the IgG receptor and the C3 receptor. Moreover, one must note that in these studies rabbit
LEUKEMK:RETlCULOENDOTHELlOSlS-JAFFE ET AL.
antibodies and mouse serum as a source of complement were employed. It is possible that membrane receptors on different cells may show different species specificity. Further studies to elucidate the nature of the membrane receptors of histiocytes of different organs are now underway in our laboratory. Although B lymphocytes also bear a receptor for the Fc portion of IgG, we do not think that the receptor being detected on the neoplastic cells is of this type. The Fc receptor of B lymphocytes can most readily be detected with radiolabeled soluble antigenantibody complexes [ 11,121 or with fluorescein-labeled aggregated IgG [ 131. In our present report, frozen sections of both control spleens and lymph nodes treated with the IgGEA reagent showed exquisite localization of the reagent red cells only to histiocytes. When the IgGEA reagent was used with cell suspensions from control spleens and lymph nodes, only a small percentage of the cells formed IgGEA rosettes, and morphologically all appeared to be histiocytes. Therefore, under the conditions used in our study the IgGEA reagent detects only the monocyte IgGEA receptor and not the Fc receptor of the B lymphocyte. Furthermore, we have been unable to detect any binding of this IgGEA reagent to the malignant lymphocytes of five patients with chronic lymphocytic leukemia or to normal human peripheral blood lymphocytes [20]. Other investigators have also been unable to demonstrate binding.of IgGEA to normal mouse B cells [ 161 or to normal or mitogenstimulated human lymphocytes [ 171. However, Brain and Marston [35] recently reported the binding of human red blood cells which had been sensitized with a high titer anti-D serum to normal human B cells. It would, therefore, appear that the ability of different IgGEA reagents to bind to different cell receptors may depend on the source and concentration of the antibody and the nature of the red cells. It is also of interest that in three recent studies [7,36,37] surface immunoglobulin of the IgG class has been demonstrated on the cells from a number of patients with leukemic reticuloendotheliosis. Superficially, this finding might be regarded as evidence for the B cell origin of these malignant cells. How-
ever, it should be noted that cells of the histiocytic series may be stained not because of synthesized surface IgG, but rather because they have a receptor for IgG and therefore they may have been coated by cytophilic IgG in vivo; alternatively, they can bind the fluoresceinated antiimmunoglobulin reagent itself by its Fc moiety. Therefore, any conclusions as to the origin of cells based on their surface staining must be made with caution. It is particularly noteworthy that in two of the three studies [7,36] the surface immunoglobulins detected were polyclonal. Such a result would be expected if the immunoglobulin was present as a consequence of the IgG receptor, rather than of cell synthesis. In only one study [37] did the surface immunoglobulin appear to be monoclonal. If additional cases with monoclonal immunoglobulin are reported, a B cell origin for their cells would seem probable, particularly if such surface immunoglobulin could be removed and regenerated in vitro. In that event it may be that leukemic reticuloendotheliosis as currently defined is complex and includes cases of both B lymphocytic and histiocytic origin in different patients. We have examined the neoplastic cells of two patients with leukemic reticuloendotheliosis for a feature not previously reported, the presence of specific membrane receptors. These receptors can be used as functional markers of possible aid in the identification of cells in a variety of lymphoreticular malignancies and may give a clue as to the state of cellular differentiation. In our two cases of leukemic reticuloendotheliosis, it was shown that the neoplastic cells had a receptor similar to that of normal splenic histiocytes. ACKNOWLEDGMENT We are indebted to Dr. Richard A. Miskoff, Walter Reed Army Medical Center, who provided us with splenic tissue and clinical information on the second patient (H.G.). We are grateful to Mrs. Eileen Sussman for her expert technical assistance and to Mr. Ralph lsenburg for photographic assistance, both of the Laboratory of Pathology of the National Cancer Institute.
REFERENCES 1. Ewald 0: Die leukamische reticuloendotheliose. Deutsches Arch Klin Med 142: 222, 1923. 2. 3.
4.
5.
Bouroncle BA, Wiseman BK, Doan CA: Leukemic reticuloendotheliosis. Blood 13: 609, 1958. Mitus WJ, Mednicoff IB. Wittels B, Dameshek W: Neoplastic lvmohoid reticulum cells in the oerioheral blood: a histochemical study. Blood 17: 206, i97i. Shrek R, Donnelly WJ: “Hairy” cells in blood in lymphoretitular neoplastic disease and “flagellated” cells of normal lymph nodes. Blood 27: 199, 1966. Yam LT. Castoldi GL, Garvey MD, Mitus WJ: Functional cy-
6.
7.
8.
July 1974
togenetic and cytochemical study of the leukemic reticulum cells. Blood 32: 90, 1968. Rubin AD, Douglas SD. Chessin LN, Glade PR, Dameshek W: Chronic reticulolymphocytic leukemia. Am J Med 47: 149, 1969. Burns CP, Maca RD. Hoak JC: Biochemical, morphological, and immunological observations of leukemic reticufoendotheliosis. Cancer Res 33: 1615. 1973. Rabellino E, Colon S. Grey HM, Unanue ER: Immunoglobulins on the surface of lymphocytes. I. Distribution and quantitation. J Exp Med 133: 156. 1971.
The American Journal of Medicine
Volume 57
113
LEUKEMIC RETlCULOENDOTHELlOSlS-JAFFE Ei AL.
9.
10.
11.
12. 13. 14.
15. 16. 17.
18.
19.
20.
21.
22.
114
Unanue El?, Grey HM, Rabellino E, Campbell P, Schmidtke J: lmmunoglobulins on the surface of lymphocytes. II. The bone marrow as the main source of lymphocytes with detectable surface-bound immunoglobulin. J Exp Med 133: 1188,197l. Bianco CR, Patrick R, Nussenzweig V: A population of lymphocytes bearing a membrane receptor for antigen-antibody complement complexes. I. Separation and characterization. J Exp Med 132: 702, 1970. Basten A, Miller JFAP, Sprent J, Pye J: A receptor for antibody on B lymphocytes. I. Method of detection and functional significance. J Exp Med 135: 610, 1972. Basten A, Warner NL, Mandel T: Lymphocytes and the immune response. II. J Exp Med 135: 627, 1972. Dickler HB, Kunkel HG: Interaction of aggregated y-globulin with B lymphocytes. J Exp Med 136: 191, 1972. Huber H, Polley JJ, Linscott WD, Fudenberg HH, MullerEberhard HJ: Human monocytes: distinct receptor sites for the third component of complement and for immunoglobulin G. Science 162: 1281, 1968. Berken B, Benacerraf B: Properties of antibodies cytophilic for macrophages. J Exp Med 123: 119, 1966. Lay WH, Nussenzweig V: Receptors for complement on leukocytes. J Exp Med i28: 991, 1968. Huber H, Douglas SD, Fudenberg HH: The IgG receptor: an immunological marker for the characterization of mononuclear cells. Immunology 17: 7, 1969. Jondal M, Holm G, Wigzell H: Surface markers on human T and B lymphocytes. I. A large population of lymphocytes forming nonimmune rosettes with sheep red blood cells. J Exp Med 136: 207, 1972. Lay WH. Mendes NF. Bianco C, Nussenzweig V: Binding of sheep red blood cells to a large population of human lymphocytes. Nature (Lond) 230: 53 1, 197 1. Shevach EM, Herberman R, Frank MM, Green I: Receptors for complement and immunoglobulin on human leukemic cells and human lymphoblastoid cell lines. J Clin Invest 51: 1933.1972. Pincus S, Bianco C, Nussenzweig V: Increased proportion of complement-receptor lymphocytes in the peripheral blood of patients with chronic lymphocytic leukemia. Blood 40: 303, 1972. Ross GD, Rabellino EM, Polley MJ, Grey HM: Combined studies of complement receptor and surface immunoglobulin bearing cells and sheep erythrocyte rosette-
July 1974
The American Journal of Medicine
Volume 57
23.
24.
25.
26.
27.
28. 29.
30.
31.
32. 33.
34. 35. 36.
37.
forming cells in normal and leukemic human lymphocytes. J Clin Invest 52: 377, 1973. Dukor P, Bianco C, Nussenzweig V: Tissue localization of lymphocytes bearing a membrane receptor for antigenantibody-complement complexes. Proc Natl Acad Sci USA 67: 991, 1970. Bowling MC: Histopathology laboratory procedures of the pathologic anatomy branch of the National Cancer Institute, Washington D.C., U.S. Government Printing Office, 1967. p 2. Frank MM, Gaither T: The effect of temperature on the reactivity of guinea pig complement with y-G and -r-M haemolytic antibodies. Immunology 19: 967, 1970. Katayama I, Li CY, Yam LT: Histochemical study of acid phosphatase isoenzyme in leukemic reticuloendotheliosis. Cancer 29: 157, 1972. Craddock CA, Longmire R, McMillan R: Lymphocytes and the immune response. II. New Engl J Med 285: 378, 1971. lijima S: Leukemic reticuloendotheliosis. Tohoku J Exp Med 89: 35, 1966. Yam LT. Li CY, Finkel H: Leukemic reticuloendotheliosis. The role of tartrate-resistant acid phosphatase in diagnosis and splenectomy in treatment. Arch Intern Med 130: 248, 1972. Yam LT. Li CY, Lam KW: Tartrate-resistant acid phosphatase isoenzyme in the reticulum cells of leukemic reticuloendotheliosis. New Engl J Med 284: 357, 1971. Burke JS, Byrne GE Jr, Rappaport H: Hairy cell leukemia (leukemic reticuloendotheliosis). I. A clinical and pathologic study of 21 patients. Cancer (in press). Lennert K: Personal communication, 1974. Katayama I, Li CY, Yam LT: Ultrastructural characteristics of the hairy cells of leukemic reticuloendotheliosis. Am J Pathol 67: 361. 1972. Trubowitz S, Masek B, Frasca JM: Leukemic reticuloendotheliosis. Blood 38: 288, 197 1. Brain P, Marston RH: Rosette formation by human T and B lymphocytes. Eur J lmmuno13: 6, 1973. Preud’homme JL. Seligmann M: Surface bound immunoglobulins as a cell marker in human lymphoproliferative diseases. Blood 40: 777, 1972. Aisenberg AC, Bloch KJn Long JC: Cell-surface immunoglobulins in chronic lymphocytic leukemia and allied disorders. Am J Med 55: 184, 1973.