leukemic reticuloendotheliosis: a study of the origin of the malignant cell

Leukemic Reticuloendotheliosis

A Study of the Origin of the Malignant Cell

GERALD W. KING, M.D.

PAUL E. HURTUBISE, Ph.D.

ARTHUR L. SAGONE, Jr., M.D.

ALBERT F. LoBUGLIO, M.D.

EARL N. METZ, M.D.

Columbus, Ohio

From the Departments of Medicine and Patholo- gy. The Ohio State University, Columbus, Ohio. Requests for reprints should be addressed to Dr. Gerald W. King, Division of Hematology and Oncology, Department of Medicine, The Ohio State University Hospitals, 410 West 10th Ave- nue, Columbus, Ohio 43210. Manuscript ac- cepted December 12, 1974.

A highly pure preparation of neoplastic ceils from the spleen of a patient with leukemic reticuloendotheliosis was studied for function, membrane characteristics and glucose metabolism. Glass adherence and phagocytosls of small particles (latex and carbon black) were demonstrated with phase contrast microscopy. Sta- phyiocidai activity was similar to that of normal monocytes. Im- munofiuorescent assays revealed nonspecific uptake of antiserums to immunogiobuiins G (IgG), M (IgM), A (igA) and kappa and lambda light chains. Rosette assays indicated the presence of receptors for IgG on the surface of ail ceils but no receptors for complement (C3) or sheep red blood ceils. Glucose metaboiic studies revealed a pattern that differed from that of normal monocytes or lymphocytes with intermedlate values for giycoiysis, tow hexose monophosphate shunt activity and high Krebs cycle activity. increments in triti@ed (3H)-thymidine uptake and glucose metabolism in response to phytohemaggiutinin stimuiatlon were minimal (5 per cent of normal lymphocyte values) and no response was noted with pokeweed mitogen stimulation. These findings suggest that the leukemic reticuioendotheiiosis ceil most closely resembles ceils of the monocyte-histiocyte series.

The disease entity leukemic reticuloendotheliosis (LRE) or “hairy cell leukemia” has been studied extensively in an attempt to determine the origin of the malignant LRE cell. Various investigators have suggested that this cell is a lymphocyte [l-3], a reticulum cell [4-71 or a “hybrid” of these cells [8]. However, most of these studies have dealt with impure cell preparations and the contribu- tion of contaminating lymphocytes and monocytes could alter the reported results in favor of either a lymphocyte or reticulum cell origin.

In this report, the characteristics of the LRE cell were defined by studying a suspension of cells from the spleen that consisted of greater than 95 per cent LRE cells. Morphologic, functional and metabolic criteria were used to define these cells.

CASE REPORT

This 57 year old physician had a history of excellent health until approxi- mately 6 weeks before his admission to the hospital when he became aware of an unusual degree of fatigability and the appearance of acne lesions over his face and upper part of his chest. He di not seek medical attention until ecchymoses and some hemorrhage developed into the

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acne lesions. Blood counts made at another institution ra vealed pancytopenia and subsequently he was admitted to the Ohio State University Hospitals.

His past medical history and systemic review revealed no important findings other than a history of peptic ulcer disease with two episodes of bleeding, one of which required the transfusion of 2 units of blood. He had not been exposed to any compounds which were potentially myelo- toxic. He had had extensive exposure to diagnostic x-ray in the course of clinical research in cardiac catheteriza- tion. During this period of exposure, the usual safeguards, including the use of a lead apron, had been employed.

The physical examination revealed normal vital signs. The skin was pale, but there was no evidence of jaundice. Small ecchymoses were present over the legs, but no pe- techiae were seen. There were acne lesions on the upper anterior portion of his chest, the side of the face and the scalp. Some of these were hemorrhagic. A small 1.5 by 1.5 cm node was palpable in the left axilla, but otherwise the superficial lymph nodes were not enlarged. The liver was palpable 2 cm below the costal margin, but was not enlarged by percussion. The spleen was easily felt at a point 6 cm below the left costal margin. The remainder of the physical examination was within normal limits.

Initial peripheral blood data were as follows: hemoglobin 8.3 g/l00 ml, hematocrit 24 per cent, reticulocytes 2 per cent, platelets 22,000/mm3, white cell count l,100/mm3. The differential white cell count included 40 neutrophils, 2 eosinophils, 36 lymphocytes, 4 monocytes and 8 cells which appeared to be large lymphocytes with nucleoli. There were 8 nucleated red cells per 100 white cells. Blood urea nitrogen, creatinine, electrolytes, calcium, glucose, serum glutamic oxaloacetic transaminase. serum glutamic pyruvic transaminase, bilirubin, serum protein electrophoresis and quantitative immunoglobulin values and urinalysis were normal. The serum lactic acid dehy- drogenase level was slightly elevated at 344 units. At- tempts at bone marrow aspiration from the sternum and from the posterior superior iliac spine were unsuccessful, but a percutaneous biopsy specimen of the bone marrow was obtained. A touch preparation of this specimen showed a single population of large cells resembling reticulum cells which displayed a large immature nucleus with a nucleolus and an abundant cytoplasm. Supravital examination of the peripheral blood revealed a few cells with fine hairy projections typical of those seen in LRE, and similar findings were noted in peripheral blood which had been cultured for 24 hours. Sections of the fixed tissue from the bone marrow biopsy specimen showed a very cellular marrow also consisting of these cells with small islands of normal marrow and scattered megakaryocytes. Skin tests with streptokinase/streptodornase (SK/SD), mumps, histoplasmin, Candida, tuberculin (PPD) and phytohemagglutinin were positive.

Because hypersplenism was thought to be contributing to the patient’s cytopenia, splenectomy was performed. The spleen weighed 2,500 g and microscopically its usual architecture was obliterated by an infiltration of cells similar to those seen in the bone marrow biopsy specimen. A small accessory spleen also was found, but the abdominal

lymph nodes were not enlarged. A liver biopsy showed foci of extramedullary hematopoiesis.

The patient made an uneventful recovery from the splenectomy and his blood counts improved. The white cell count increased to 3,000 to 4,000/mm3, the platelet count ranged between 100,000 and 155,000/mm3, the hematocrit and hemoglobin remained stable. The patient was dis- charged from the hospital on the 10th postoperative day.

During the 18 months after surgery, the patient kept a regular work schedule but he was hospitalized twice for treatment of infection. Moderate pancytopenia has persist- ed but peripheral blood counts have remained stable. The differential white cell count includes an average of 30 per cent segmented neutrophils and 10 to 20 per cent “hairy cells.” He has not received radiation or chemotherapy.

METHODS

Cell Isolation. Spleen cell suspensions were prepared by mincing fresh splenic tissue with a scalpel and forcing the pieces through a fine wire sieve. Mononuclear cells were isolated by Ficoll-hypaque and sucrose gradient centrifu- gation [9]. The mononuclear cells were then resuspended in Seligman’s balanced salt solution (SBSS) and processed for the remaining studies. Normal lymphocyte suspensions and monocyte monolayers were prepared as previously described [IO-121. Glass Adherence and Phagocytosis. Spleen cell suspensions were incubated in 35 mm plastic dishes (Falcon Plastics, Oxnard, Calif.) with or without glass coverslips in a medium consisting of Hank’s balanced salt solution (HBSS) and 10 per cent pooled AB serum for 2 hours in an atmosphere of 95 per cent air and 5 per cent carbon diox- ide at 37’C. The dishes and/or coverslips were then washed vigorously five times with HBSS. The resulting monolayers were examined microscopically for adherence and then incubated in HBSS plus 10 per cent fresh, pooled human AB serum for 60 minutes with (1) 0.8 p latex particles (Difco Laboratories, Detroit, Mich.) plus carbon black, (2) anti-D coated human red blood cells or (3) viable Cryp- tococcus neoformans organisms. Phagocytosis was determined by microscopic examination of the monolayers. Bactericidal Assay. Bactericidal activity of the LRE cells was determined according to the method of Root [ 131. Briefly, suspensions containing 2.5 X IO6 LRE cells were incubated with 12.5 X lo6 viable Staphylococcus aureus organisms for 60 minutes in HBSS and 10 per cent pooled, fresh AB serum. Aliquots were taken in triplicate at 0 time and after 30 and 60 minute periods of incubation. The cells were lysed with distilled water and the number of remaining viable bacteria was determined by colony counts. The number or organisms killed was determined by subtracting the number of viable bacteria present at 30 or 60 minutes from the number present at 0 time. lmmunofluorescent Assays. Cell suspensions were washed several times in SBSS supplemented with 0.5 per cent gelatin and the final volume was adjusted to contain 10 to 15 X lo6 cells/ml. One-tenth milliliter of LRE cell suspension was mixed with 0.1 ml of each of the monospecific fluorescein isothiocyanate conjugated antiserums (Table I) and reacted at room temperature for 45 minutes.

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TABLE I Membrane Rosette and lmmunofluorescent Characteristics of LRE Cells ______.. -~__ Rosettes (%)* lmmunofluorescence (%)j

Morphology E EA EAC’ IgG IgM IN Kappa Lambda -__ __~

:.95 LRE 3 100 2 100 100 100 100 100

*Per cent cells forming specific rosettes.

t Per cent staining with specific antiserums.

Each antiserum (Meloy Laboratories, Springfield, Va.) was confirmed as monospecific by Ouchterlony double diffu- sion and by hemagglutination with bisdiazotized-benzidine binding of purified myeloma proteins to erythrocytes. Each antiserum was also cleared of aggregated material by passage through a 0.2 w filter. After reacting cells with the appropriate antiserum, they were washed three times with phosphate buffered saline solution, pil 7.8. A drop of the LRE cell suspension was examined using a Leitz Ortholux microscope. Total LRE cells in each field (magnification X 450) were counted first using a visible light source. LRE cells with fluorescence were then counted in the field using an ultraviolet light source. The percentage of cells fluorescing was calculated after examining from 200 to 400 cells for each antiserum used. E, EA and EAC’ Rosette Assays. The E rosette assay for identifying T lymphocytes using sheep red blood cells was performed according to the method of Jondal et al. [ 141. The EAC’ assay for complement (C3) receptors of monocytes and lymphocytes was performed according to the method of Bianco et al. [ 151 and the EA assay of LoBuglio et al. [ 181 was used to determine Fc receptor activity of monocytes or macrophages. Metabolic Studies. Glucose metabolism was determined by incubating the spleen cell suspensions with and without mitogens as previously described [lo]. Briefly, suspensions of 4 X lo6 spleen cells were incubated for three days in metabolic flasks containing 80 per cent minimal essential medium, 20 per cent fetal calf serum, antibiotics and either “C-l-glucose or “C-6-glucose. At the end of this incubation, the medium was acidified, the evolved

14C02 was measured directly with an ionization chamber- electrometer apparatus and the glucose utilization was determined by the glucose oxidase technic. The values for glucose metabolism in normal monocytes and lymphocytes are from studies performed concurrently in our laboratory and reported in part elsewhere [ 10,121. Lymphoblastlc Transformatlon. Spleen cell suspensions were cultured in the presence of phytohemagglutinin-h (PHA-M) or pokeweed mitogen (PWM) for three days and 3H-thymidine uptake was determined as previously described [ lo].

RESULTS

Morphology. Independent evaluation of supravital preparations of the spleen cell suspension by three individual investigators indicated that greater than 95 per cent of the cells were LRE cells. Electron microscopic studies of the cell suspensions confirmed the ultrastructural characteristics ascribed to LRE cells including the ribosome-lamella complex, reported elsewhere as being a characteristic LRE cell organ- elle [ 171 (Figure 1). Glass Adherence and Phagocytosls. When cell suspensions were incubated with glass coverslips or in plastic dishes, monolayers were formed (Figure 2) which were dense and adherent and could not be re- moved by vigorous washing with HBSS. The incubation of iron dextran particles with the LRE cells resulted in adherence of the particles to the LRE cell,

Figure 1. A. LRE cell in spleen cell suspension. B, ribosome lamella complex (RL) in LRE cell from spleen cell suspension.

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LEUKEMIC RETICULOENDOTHELIOSIS-KING ET AL.

Figure 2. LRE cells in glass adherant monolayer.

but definite phagocytosis could not be identified. Cen- trifugation of the cell suspensions and iron dextran resulted in removal of all LRE cells from suspension. When carbon black was added to a suspension of LRE cells and a dilute suspension of latex particles, apparent phagocytosis was noted in the form of phagocytic vacuoles containing latex particles and marginated carbon black, as well as carbon black alone (Figure 3). Antibody coated red cells and Cryp- tococcus neoformans organisms were not phagocy- tized during a P-hour period. Bactericidal Activity. LRE cells killed Staph. aureus organisms at the rate of 5.3 X lo6 in 30 minutes and 7.8 X IO6 in 60 minutes. Studies in our laboratory on 13 normal subjects have shown that normal monocytes kill this organism at the rate of 4.5 f 0.87 X lo6 in 30 minutes and 5.6 f 1.2 X lo6 in 60 minutes. Normal lymphocytes do not exhibit bactericidal activity in this assay system [ 181. lmmunofluorescent and Rosette Assays. Table I presents the results of specific immunofluorescent

Figure 3. L/E cell incubated with 0.8 p latex particles and carbon black. Phagocytic vacuole containing latex particle and marginated carbon black (CB + L) and injest- ed carbon black alone (CB) can be identified.

assays employing monospecific antiserums to the three major immunoglobulin classes as well as to kappa and lambda light chains. LRE cells fluoresced equally with all antiserums. The pattern of this fluorescence was “clumped” and similar to patterns seen with phagocytosis or pinocytosis of immuno- globulins [ 191. Rosette formation by the LRE cells was essentially limited to the EA assay. LRE cells formed EA rosettes which were different from the rosettes formed by normal monocytes in that the LRE- RBC binding was more fragile and was disrupted by agitation. Less than 5 per cent of the cells formed rosettes in the E and EAC’ assays. These data suggest that LRE cells are more closely related to monocytes than to lymphocytes. Glucose Metabolism and Blastogenesis. LRE glucose metabolism differed markedly from that in both normal blood lymphocytes and monocytes (Table II). In the unstimulated state, glucose utilization by LRE cells was intermediate between values for normal lymphocytes and normal monocytes. Krebs cycle activity, as estimated by oxidation of “C-6-glucose (14C-6-O*), was much greater than in normal lymphocytes and similar to that in normal monocytes. Hex- ose monophosphate shunt (HMPS) activity, as estimated by determining the difference in oxidation of 14C-l-glucose (‘4C-1-02) and 14C-6-glucose (14C-6- Oz), was lower in LRE cells than in normal lymphocytes and much less than in normal monocytes. These data suggest that Krebs cycle activity in LRE cells is similar to that of monocytes and is consistent with the abundance of mitochondria noted in LRE cells and monocytes [ 4-61.

With stimulation by the mitogen PHA-M, LRE cells had a minimal increase in HMPS activity compared to that in normal lymphocytes and only a slight increase in glucose utilization and Krebs cycle activity. Blastic transformation as measured by 3H-thymidine uptake was also slight compared to that in normal lymphocytes. When stimulated by PWM, the LRE cells showed no increase in glucose metabolism or 3H-thymidine uptake.

In addition to the blastogenesis studies outlined, the specific antigens SK-SD, PPD and Candida albi- cans produced no blastic transformation of LRE cells in vitro despite the fact that the patient had positive skin tests with all of these antigens.

COMMENTS

In addition to the problem of contamination by normal cells, the identification of the cell of origin of the malignant LRE cell has been plagued by the universal problem of categorizing abnormal cells on the basis of assays usually reserved for identifying normal cells. Whether the LRE cell is an abnormal reticulum

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LEUKEMIC RETICULOENDOTHELIOSIS--KING ET AL.

TABLE ll Glucose Metabolism and DNA Synthesis of LRE Cells ____.._-.

Control+

Normal blood lymphocytes (27)”

‘T-l-0: (nmol/hr/lO’ cells)

‘T-6-0, (nmol/hr/107 cells)

Glucose utilization (nmol/hr/107 cells)

3H-thymidine uptake (cpm/200,000 cells)

Normal blood monocytes (10)” ‘“C-l-O2

‘T-6-0,

Glucose utilization

LRE cells (>95 per cent) ‘T-l-O2

‘T-6-O?

Glucose utilization

‘H-thymidine uptake

6.6 ? 0.5

0.9 i 0.3

72 t 25

900 r 424

45.4 f 11.7

3.19 -i- 1.2

455 i 154

6.2

4.0

146

417

PHA-Mt

20.5 + 4.1

1.4 i 0.5

632 +_ 88

80,625 f 21,616

10.7 6.5

5.4 -

187 147

4,388 492

PWMt

9.6 1 0.8

1.2 1 0.3

306 + 28

49,105 + 36,120

-

-

*Number of normal subjects studied during time of patient evaluation and reported on in part elsewhere [ 10,121. t All data reported as mean ? 1 SD.

cell (monocyte) or an abnormal lymphocyte cannot be clearly answered by this study, but the data suggest that the characteristics of the LRE cell more closely resemble those of the normal monocyte.

Our observations that the LRE cell is capable of phagocytizing small particles and has bactericidal activity against staphylococci is consistent with the observations of Daniel and Flandrin [6] who were able to show LRE cell phagocytosis of latex particles and staphylococcal organisms in vitro using electron microscopy. Others have shown only limited phagocytosis [ 3,4,17,18] or none [2,20,2 11, and microbicidal activity has not been previously demonstrated. Since LRE cells do not phagocytize large particles (red blood cells and cryptococcus organisms), we must assume that their phagocytic mechanism is less effi- cient than that of normal monocytes.

Metabolically, the LRE cell differs from both normal monocytes and lymphocytes. Burns and asso- ciates [22] presented data on glucose utilization by suspensions of LRE cells of greater than 85 per cent purity that closely resemble our results, i.e., intermediate between values for monocytes and lymphocytes, The relatively low resting HMPS activity of the LRE cell when compared to that in both these normal cell types suggests a more total dependence on the Krebs cycle and Embden-Meyerhof pathways for glucose oxidation and presents a unique pattern of glucose metabolism among cells of the hematologic system.

The LRE cell suspension responded minimally to stimulation by the mitogen PHA-M but not PWM. This probably reflects the presence of the few normal lymphocytes contaminating our preparation (less

than 5 per cent) but could be interpreted as indicating the lymphocytic nature of the LRE cells. Abnormal lymphocytes, such as those found in chronic lymphocytic leukemia, respond to PHA but to a lesser degree than normal lymphocytes [23]. Several investigators have reported increased DNA synthesis by LRE cell preparations with PHA stimulation, although it was less than normal [l-3], and others have not been able to demonstrate any response to this mitogen [5,20].

The rosette assays of the LRE cells in our preparation identify this cell type as a member of the monocyte-reticulum cell series. The uniform rosette forma- tions in the EA assay but negative EAC’ assay imply that the LRE cell has no receptor for Cs but does have the Fc receptor required for the EA assay. This is consistent with the observations of Jaffe et al. [7] and correlates well with their observations of normal splenic histiocytes which also have the Fc receptor but not the Cs receptor. The absence of significant rosette formation by the LRE cells in the E and EAC’ assays suggests no similarity between the LRE cells and normal B and T lymphocytes as determined by these markers [ 141.

The immunofluorescent studies suggest nonspecific uptake of all antiserums rather than the specific pattern of identification of surface immunoglobulin. The clumped pattern of the fluorescence on the cells is not characteristic of B lymphocytes in our system, but rather of intracellular phagosomes seen with phagocytic cells. Preud’homme and Seligmann [ 191 have observed similar staining patterns with LRE cells.

In summary, it seems clear that the LRE cell does

September 1975 The American Journal of Medicine Volume 59 415

LEUKEMIC RETlCULOENDOTHELlOSlS-KING ET AL.

not have the functional characteristics of normal lymphocytes or monocytes, although both have been claimed to be the cell of origin. Nevertheless, in our highly pure preparations, LRE cells appeared to share many features with blood monocytes, pat-ticu- larly the ability to phagocytize. to kill microorganisms and to bind antibody coated red blood cells. These observations do not exclude the possibility that the malignant LRE cell does, in fact, represent a primitive reticulum cell or stem cell disorder that in individual

cases may demonstrate characteristics of either lymphocytes or monocytes depending on minimal de- grees of differentiation in the direction of either of these mature cell types.

ACKNOWLEDGMENT

We are indebted to Dr. Kathryn Clausen, Department of Pathology, for providing electron micrographs of the spleen tissue and to Dr. Bertha Bouroncle, De- partment of Medicine, for her assistance and advice.

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leukemic reticuloendotheliosis: a study of the origin of the malignant cell

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