generalized monocyte deficiency in leukaemic reticuloendotheliosis

Scand J Haematol(l982) 29, 153-160

Generalized Monocyte Deficiency in Leukaemic Re t iculoendo t heliosis

ANTHONY J. JANCKILA, JOHN H. WALLACE & LUNG T. YAM

Section of Hematology, Veterans Administration Medical Center, Louisville and

Department of Microbiology and Immunology, University of Louisville, Kentucky, USA

Examination of the blood of 40 patients with leukaemic reticuloendotheliosis (LRE) revealed a drastic reduction in monocyte numbers (mean 74/p1) when compared to a normal group of 33 (mean 442/pI). Repeated blood studies in 8 LRE patients were made over periods of 2 months to 5 years and monocytopenia was observed to be a persistent phenomenon. Similarly, tissues from 29 patients showed an overall decrease in numbers of monocytes/ histiocytes when compared with those of 73 control subjects. Skin window examinations in all 11 patients so studied showed marked reduction or absence of monocyte response to inflammation. 2 exceptional cases to this trend are noted. In one, circulating monocyte levels remained normal over a 14-month period while examinations of blood and marrow in this case showed few hairy cells. In another case, severe and persistent monocyte deficiency was noted during a 2-year period of unsuccessful treatment. When he eventually responded to low dose chlorarnbucil and his Hb rose from 40-140 g/l and WBC became normal, the number of circulating monocytes also became normal.

Key words: leukaemic reticuloendotheliosis - monocyte deficiency

Accepted for publication March 1, 1982

Correspondence to: Dr. Anthony Janckila, Veterans Administration Medical Center, 800 Zorn Avenue, Louisville, Kentucky 40202, USA

ABBREVIATIONS

AcP = acid phosphatase numerous studies have been conducted to characterize these hairy cells, their normal LN = lymphnode

LRE = leukaemic reticuloendotheliosis NSE = non-specific esterase counterpart and cell of origin remain un- NSEF = fluoride-resistant esterase defined ‘(Cawley et a1 1980). Generally TAcP = tartrate-resistant acid phosphatase though hairy cells are considered to be of

either B-lymphocyte or monocyte lineage. Leukaemic reticuloendotheliosis (LRE) is a Major findings in support of a B cell rare form of chronic leukaemia involving origin include lack of monocytic enzymes the proliferation of ‘hairy cells’. While (Stein & Kaiserling 1974, Jansen et a1

0036-553)3/82/070153-08 $02.50/0 0 1982 Munksgaard, Copenhagen

154 JANCKILA, WALLACE & YAM

1979), lack of phagocytic ability (Burns & Cawley 1979, Jansen et a1 1979) and production of one or more classes of immunoglobulin in vitro (Burns et a1 1978, Cohen et a1 1979). Other studies indicate that hairy cells are more similar to monocytes. Thus, they have been reported to possess strong receptors for cytophilic antibody (Jaffe et a1 1974, Scheinberg et a1 1976), have significant phagocytic capacity (Golomb et a1 1978) and staphylocidal activity (King et a1 1975), contain nonspecific esterase (Higgy et a1 1978, Braylan et a1 1978, Golomb et a1 1978) and peroxidase (Reyes et a1 1978) and do not produce or express immunoglobulin (Braylan et a1 1978).

While the experimental results can not be reconciled by either tenet, the clinical and histologic findings are better appreciated in the context of hairy cells representing aber- rant monocytes (Burke 1978, Bouroncle 1979). Seshadri et a1 (1976) noted severe monocytopenia in a series of 6 LRE patients and concluded that in LRE there may be impaired monocyte production and/or maturation. Monocytopenia has since been noted in other studies (Jansen et a1 1979, Makowiak et a1 1980). It has been argued that this lack of circulating monocytes may be due to splenic sequestration or meta- plasia (Braylan et a1 1978). Skin window studies (Seshadri et a1 1976, Yam et a1 1977) disclosed a distinct lack of monocyte response to inflammation and it has been hypothesized (Kjeldsberg 1978) that this may be due to a chemotactic factor in- activator in the blood of LRE patients.

In order to evaluate more critically the status of LRE patients regarding monocytes and macrophages, the present study re- examined blood and tissue of 56 cases of LRE including 8 cases studied repeatedly.

MATERIAL AND METHODS

Patients and controls. 56 patients with LRE were studied. The diagnosis in all cases was confirmed by the detection of hairy cells in blood, marrow or tissue imprints and by the presence of tartrate-resistant acid phosphatase in these cells. 5 patients had undergone splenectomy prior to evaluation, all others being studied before or at the time of splenectomy. None had received any form of chemotherapy. In 1 patient, how- ever, several studies of the blood were done after he was treated with a course of low dose chlorambucil. Peripheral blood was available for study in 40 patients. Marrow or other tissue was evaluated in 29 patients. Blood and tissue were available in 13 patients. The control group consisted of 106 individuals. 33 normal donors provided control material for peripheral blood studies. The control group for tissue studies consisted of 73 patients with a variety of haematologic or on- cologic disorders. Study material is summarized in Table 1.

Evaluation of monocytes in peripheral blood. 200 cell differential leucocyte counts were done on initial blood samples from 40 LRE patients and 33 normal donors and the absolute numbers of monocytes and lymphocytes were determined. In 8 patients, repeated examinations were made over periods of 2 months to 5 years. The nonspecific esterase stain for monocytes (Yam et a1 1971) was done on blood smears from 7 patients and all normals in order to confirm the accuracy of morphologic classification of the mononuclear cells.

Evaluation of monocytes (histiocytes) in tissue. Fresh smears of marrow, and imprints of spleen and lymph node were stained with Wright-Giemsa and for nonspecific esterase. Also, fresh tissues were sectioned and stained for acid phosphatase (AcP), tartrate- resistant acid phosphatase (TAcP), nonspecific esterase (NSE) and fluoride-resistant esterase (NSEF) according to previously described methods (Li et al 1972). These materials were evaluated for the numbers of monocytes or histiocytes on the basis of a semi- quantitative 0 to 4+ scale as follows: an average of 1 to 10 esterase positive cells per representative (40 x) high power field was considered l + ; from 11 to 25 positive cells -2+; from 26 to 40 positive cells - 3 + ; and greater than 40 positive cells -4+. Of necessity, marrow specimens and imprints were evaluated more subjectively taking into account any variation in cellu- larity or uneven distribution of cells.

MONOCYTE DEHCIENCY IN LRE

Group

LRE

Control

Normal

Lymphoproliferative

Myeloproliferative

Miscellaneous**

155

Blood Spleen LN !.larrow

40(37)* 27(23) 4 ( 4 ) 13(13)

33 3

11 10 3(1)

6(2) 1 1

1 4 ( 1 ) 9(1) 18(4)

Evaluation of tissue migration of monocytes. The skin window technique of Rebuck & Crawley (1955) was used to study 11 patients. The results of 6 of these have been published previously (Yam et a1 1977). Skin windows were evaluated at 4, 8, 12, and 24 h for the presence of monocytes at the inflammatory site.

Statistical analyses. The Student's t test was used to compare the mean leucocyte, lymphocyte and monocyte counts of the LRE and normal groups. The paired t test was used to compare monocyte counts obtained by morphologic and cytochemical means. Alpha was set a priori at 0.01 for all statistical analyses.

RESULTS

Leucocytes in blood The numbers of circulating monocytes, lymphocytes and total leucocytes of the LRE and control groups are depicted in Figure 1. While there was no significant difference between the groups in terms of total leucocyte counts, there was a drastic reduction in circulating monocytes in the LRE group. The 99% confidence interval for the difference between the group means is 280.1- 456.9. It may be seen in Figure l c that although a statistical difference was not obtained, many patients with LRE were leucopenic. The lack of significance is prob-

ably due to the extreme skewness in the LRE group. 8 patients were studied on multiple occasions (total of 38 blood samples) and in 7 monocytopenia was found to be persistent over periods of 2 months to 5 years. Only 1 of these 7 had a persistent diminution in lymphocytes (mean 0.745 x lo9/]) but he also had persistent severe leucopenia (mean 1.9 x 109/l). The single patient who maintained adequate levels of monocytes in the blood (mean 0.782 x 109/1) also maintained borderline elevated WBC (mean 11.8 x 109/1) with very few hairy cells (< 10%). A third patient averaged 19% hairy cells and 0.038 x lo9/] monocytes over a 2-year period of unsuccessful treatment. After recently receiving a regimen of low dose chlorambucil, his Hb has risen from 40 to 140 g/l, his WBC and differental have become normal with hairy cells seen only rarely. His absolute monocyte count has risen to 0.672 x lo9/].

The number of blood monocytes were determined on the basis of nonspecific esterase'staining in all 33 normal individuals and in 7 LRE patients. No significant difference (P > 0.05) was noted between this method and morphologic examination for monocyte identification.


24 -

22 -

20 - 18 -

16-

F 14-

L L 12- 0

l0 - 5 -

z - W

2 -

Z 8

6

4

2

0

C Z= 5.935 I LRE 93.7.376 Y-5.826 0 SD= 1.849

K-1.787 I LRE SD=1.301

E.1.780 0 SD=0.185 0 SD=0.648

i.0.074 I LRE SD=0.102 Z.0.442

LRE/NORMAL (distribution overlap)

a = 0.01; Not Significant

LRE/NORMAL (distribution overlap)

a= 0.01; Not Significant

LRE/NORMAL 1 (distribution overlap)

a = 0.01 ; Significant

i n

- 0 0.2 0.4 0.6 0.8 1.0 0 I 2 3 4 5 6 7 0 2 4 6 8 10 12 14 46 48

LYMPHOCYTES ( 109 f ) MONOCYTES ( to9/r 1 WBC(I09/1 1

Figure 1. Sample distributions of absolute monocytes, lymphocytes, and WBC in LRE and normals. (a) Severe monocytopenia in LRE group. (b) Normal numbers of lymphocytes in LRE group. (c) Although statistically non-significant many cases of LRE may be leucopenic.

Monocytes or histiocytes in tissue Results of NSE staining in tissue are summarized in Table 1. Of the 29 cases of LRE examined (44 tissue samples) 4 spleens were classified 3+ for NSE activity; all others being 1+ or 2+ (Figure 2). In contrast, of 73 control individuals (76 tissue samples) 67 were 3+ or 4+ (Figure 2), while 9 were classified as 2+. These 9 tissues considered to contain decreased monocytes/histiocytes consisted of 3 spleens (2 myeloproliferative disorders and 1 reactive hyperplasia), 1 reactive lymph node and 5 marrow specimens ( 3 anaemias, 1 carcinoma and 1 CLL).

Touch impressions of tissues stained for NSE revealed a similar pattern of decreased numbers of monocytes or histiocytes in LRE. In tissue sections of bone marrow, spleen and lymph node stained for tartrate- resistant acid phosphatase, positively stained cells (hairy cells) were always located diffusely throughout the marrow, in the cords and sinuses of splenic red pulp and the pericortical and medullary areas of lymph nodes. Hairy cells were never found in the areas of peripheral lymphoid organs commonly associated with significant numbers of B-lymphocytes (e.g., splenic white

Figure 2. Spleen sections stained for monocytes and histiocytes by the non-specific esterase method. (a) LRE showing overall decrease in positive cells. Note the sinus lining cells (arrow) are also strongly positive. (b) Normal spleen showing typical 4+ esterase activity. ( x 260)

MONOCYTE DEFICIENCY IN LRE 157


Figure 3. Peripheral blood hairy cells showing morphologic heterogeneity. Note gradation from upper left to lower right; lymphocytoid, slightly cleaved or indented, folded and dumbbell shaped respectively. (Wright- Giemsa x 1600)

pulp or lymph node follicles). These latter indicating their inability to migrate under observations, although subjective, are in test conditions. agreement with rigorous studies of others (Katayama & Finkel 1974, Bouroncle 1979).

Monocytes in skin windows All 11 LRE patients showed an absence or marked reduction in monocytes migrating to the skin window over a 24-h period. Neutrophil granulocytes were found throughout the test period but usually were reduced in number. Typically, a normal response yields almost entirely mononuclear cells at 8 h and the response remains predominant- ly mononuclear through 24 h. Hairy cells were extremely rare in the skin windows

DISCUSSION

Results of this study confirm the previous limited observations that monocytopenia is a prominent feature of LRE and is present while adequate numbers of lymphocytes are maintained. In addition, serial studies of peripheral blood revealed that monocytopenia is persistent and is not a transient phenomenon.

Histochemical techniques designed to demonstrate monocytes and histiocytes in

MONOCYTE DEFICIENCY IN LRE 159

tissue by the NSE and NSEF stains con- sistently showed a decrease in overall posi- tivity in tissues from LRE patients. This finding strengthens the probability that the monocyte deficiency in LRE is generalized and not due to storage of these cells in extramedullary tissue compartments. Fur- ther, since none of these patients had received chemotherapy, the monocyte deficiency is not due to therapy-induced myelo- suppression. Skin window studies done by ourselves and others (Seshadri et a1 1976) are consistent with monocyte depletion in LRE since they have shown a marked decrease, in mononuclear cell response to an inflammatory stimulus. The observed decrease in circulating and tissue associated monocytes would argue against a defect in chemotaxis as the sole explanation for the skin window findings. In addition to this defect in innate resistance, there is mount- ing evidence that LRE may be associated with impaired cell mediated immunity (Bouza et a1 1978, Hersh et a1 1980, Macko- wiak 1980).

Monocytopenia in LRE has been shown on occasion but may not have been generally recognized in the past due to the problem of accurate identification of the mononuclear cells. Among the hairy cells, morphologic heterogeneity often exists both within and between patients. We have noted that some hairy cells have indented, folded or ‘dumbbell’ shaped nuclei very closely re- sembling monocytes while others have round, oval or lymphocytoid nuclei (Fig- ure 3). Presently, accurate identification of mononuclear cells is facilitated by the com- bined use of morphologic and cytochemical studies including the TAcP and NSE stains. In addition to the morphologic heterogeneity, hairy cells may exhibit a variety of surface characteristics usually associated

with mutally exclusive cell types. There is also quantitative heterogeneity in the cyto- plasmic enzymes acid phosphatase and nonspecific esterase within and between patients. The various morphologic types (i.e., dumbbell or ‘monocytoid’ versus round or ‘lymphoid’) are also seen to fluctuate in numbers in any particular patient from time to time.

It is apparent that the intrinsic heterogeneity of hairy cells is an impediment to definitively establishing their origin and nature. The laboratory findings of a persistent and systemic decrease in monocytes with normal numbers of lymphocytes in combination with the clinical observations of impaired innate resistance and cell mediated immunity suggests a defect in the production and/or maturation of functional monocytes. While it cannot be ruled out that the monocyte deficiency is somehow an indirect result of the leukaemic process, the simplest interpretation of the available in- formation is that the proliferation of hairy cells is the direct result of ineffective monocyte development. The well defined clinical and histologic features of LRE which allowed it to be recognized as a disease entity are not usually those of other lymphoproliferative disorders (Burke 1978, Bouroncle 1979), and the concept that LRE may represent a myeloproliferative disease should be retained.

ACKNOWLEDGEMENTS

This work‘ was supported in part by an hrnerican Cancer Society Subgrant IN-11IE and by the Research Service of the Veterans Administration. To Mrs. Donna Jones and Ms. Pat Hagan for preparation of the manuscript. To Mr. Don Ferrera and Misses Mary English and Mai Huang for technical assistance.

160 JANCKILA. WALLACE & YAM

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generalized monocyte deficiency in leukaemic reticuloendotheliosis

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