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T-lymphocyte cycling in human cyclic neutropenia: Effects of lithium in vitro and in vivo
CLINICAL
IMMUNOLOGY
AND
IMMUNOPATHOLOGY
23, 586-592 (1982)
T-Lymphocyte Cycling in Human Cyclic Neutropenia: Effects of Lithium in Vitro and in Vivo WILLIAMBORKOWSKY,"
LOUIS SHENKMAN,~ AND AARONRAUSENS
‘Departments of Pediatrics and TMedicine, New York University Medical Center, New York, New York 10016, and SDepartment of Pediatrics. Beth Israel Medical Center, New York. New York 10003 Two patients with cyclic neutropenia demonstrated T-cell lymphopenia during their neutropenic episodes. In addition, they also demonstrated a depressed lymphocyte proliferative response to phytohemagglutinin when neutropenic, which was possibly mediated by a plasma factor. Lithium appeared to normalize T-cell expression and function when used in vitro and in viva. Lithium therapy of one patient resulted in a cessation of T-cell cycling and a transient clinical improvement without affecting neutrophil periodicity.
INTRODUCTION
Cyclic neutropenia is a blood disorder in man and grey collie dogs characterized by episodes of neutropenia at defined regular intervals (usually 21 days in man and 12 days in dogs). During periods of neutropenia, fever, mucosal ulcerations, infections, and lymphadenopathy can occur. Although blood elements other than neutrophils have been observed to cycle (e.g., reticulocyte, platelets, and monocytes) (l), few studies have examined the fate of T lymphocytes, which themselves have documented effects on the maturation of myeloid colonies. Lithium salts have been shown to induce granulocytosis in manic depressive individuals (2) and have recently been used to ameliorate iatrogenic neutropenias in patients treated for malignancy (3). Lithium is believed to function in this capacity by increasing production and/or release of granulocyte colony stimulating factors (4). Treatment of cyclic hematopoiesis with lithium in the grey collie dog has been utilized to eliminate recurrent neutropenia and to normalize levels of other cycling blood cells (5). Lithium has also been observed to enhance various T-lymphocyte functions in vitro (6, 7). We describe two patients with cyclic neutropenia whose T-lymphocyte numbers and function were studied during their cycles. One patient was treated with lithium carbonate. The effects of this treatment on neutropenia and T lymphocytes will be presented. CASE REPORT I
C.R. is a lPyear-old female who was brought to a physician at 9 years of age for a blood count because a male sibling died suddenly of infection and neutropenia. She was found to have neutropenic cycles at 1% to 23-day intervals. Each cycle was 5 to 11 days in duration. The neutropenia was compensated by an absolute monocytosis and eosinophilia. These episodes were accompanied by clinical 586 0090-1229/060586-07$01.00/O Copyright 0 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.
LITHIUM
EFFECTS
ON
T LYMPHOCYTES
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symptoms of fever, adenopathy, and lymphadenitis. Hematologic cycles did not correlate with her menses. Neutropenic intervals were not always associated with any symptoms of illness. CASE REPORT II
B .B. is a 21-year-old male who was diagnosed as having cyclic neutropenia at 4 months of age. His cycles occur every 21 days with each neutropenic interval lasting 5-7 days. During the period his peripheral blood smear shows compensation with eosinophilia while no monocytosis occurs. Clinical evidence of fever, stomatitis, and cellulitis accompany each cycle. Since the diagnosis of his illness he had only once gone through a neutropenic interval without some clinical symptomatology. METHODS Enumeration of lymphocyte subpopulations. T cells were enumerated by the method of Jondal et al. (8), and immunoglobulin-bearing lymphocytes by the method of Winchester and Ross (9) using a goat anti-human immunoglobulin (polyvalent)-fluorescenated antibody (Cappel Ind., Cochronville, Pa.). This method employs a preincubation of peripheral blood mononuclear cells with latex particles (Dow Diagnostics) to verify macrophages. In vitro studies of peripheral blood lymphocyte responses. Ficoll-Hypaquepurified mononuclear cells were suspended in RPM1 1640 supplemented with 20% heat-inactivated pooled AB plasma or autologous plasma and cultured in microtiter plates (Nunc, Denmark) for 72 hr at a concentration of lo5 cells/O.2 ml and with phytohemagglutinin (PHA) at a concentration of 1 &ml (Digro Lab, Detroit, Mich.) and pokeweed mitogen (PWM) at a concentration of 1 pg/ml (Sigma Chemical Co., St. Louis, MO.). [‘*C]Thymidine, 0.03 &i (New England Nuclear, specific activity 50 pCi/mmol) was added to each well for the final 8 hr of culture. Cells were harvested on glass fiber filters and cell-incorporated radioactivity was counted in a liquid scintillation counter. Effects of lithium chloride were determined by adding 2 meq/liter of the salt to parallel cell cultures for the entire culture period. RESULTS T und B Lymphocyte
Evaluation
Both patients were found to have diminished numbers (relative and absolute) of T lymphocyte during periods of neutropenia. Their B-cell number remained in the normal range (5- 15%) throughout their cycle. The T lymphopenia was not due to artifactuai error possibly induced by the relative monocytosis seen in patient C. R. as these cells were excluded from evaluation. Figure 1 represents the cycling of T cells and PMNs in patient C.R. with absolute T-cell numbers ranging from 700 cells/mm3 during neutropenia to 1500 cells/mm3 otherwise. A similar cycling phenomena was present in patient B.B. with absolute T-cell numbers ranging from 1000 cells/mm3 during neutropenic intervals to 2200 cells/mm3 when not neutropenic.
588
BORKOWSKY,
SHENKMAN,
AND RAUSEN
- 60 -w -40 /
,_
I
I 10
I
20
JAN -
FIG. 1. Correlation
I 1 FE8 -
El # -I
I 10
of PMNs and T lymphocytes in patient I.
Effect of in Vitro Preincubation with Li on T-Cell Numbers The preincubation of the patients lymphocytes with LiCl (2 meq/liter) prior to the rosetting procedure increased the number of E-rosettes when T lymphopenia was evident (E-rosettes: 40 + 4% without Li and 61 +- 4% with Li, IZ = 6, P < 0.03) but had no effect when T-lymphocyte numbers were normal (E-rosettes: 55 + 5% without Li and 58 + 6% with Li, n = 6, P < 0.2.) Lymphocyte
Proliferation
to Mitogens
Lymphocytes were stimulated in both autologous or pooled AB plasma. Although there was no difference in response to PHA during cycles if pooled AB plasma was used, a depressed proliferative response to PHA was seen during neutropenia intervals when cultures contained autologous plasma (Fig. 2). The incorporation of therapeutic concentrations of Li in these cultures revealed that Li could reverse the inhibition manifested by neutropenia-associated plasma without having a significant effect on cultures containing either pooled AB plasma or autologous plasma obtained during a nonneutropenia period. Lymphocyte proliferation due to pokeweed mitogen stimulation was normal at all periods of time without significant difference due to either autologous or heterologous plasma. Effect on Li Therapy in Vivo Since Li appeared to augment T-cell expression and function when in vitro incubations were performed, its effects on these parameters as well as neutrophil cycling were examined during in vivo therapy. Li was administered to patient B .B. in doses achieving serum concentrations of 0.50 to 0.75 meq. As seen in Fig. 3, Li therapy appeared to normalize the T-cell cycling after 3 months. Prior to this period, T cycling was still occurring but with an elevated baseline. This effect of Li on T cells was also appreciable by measuring [14C]thymidine incorporation after PHA stimulation in autologous plasma. As seen in Fig. 4, initiation of Li therapy abrogated the abnormally low responses to PHA. This corresponded to the nor-
LITHIUM
EFFECTS
ON T LYMPHOCYTES
IN NEUTROPENIA
589
NON-NEUTROPENIC NEUTROPENIC AUTOLOGOUSPLASMA AUTOLOGOUSPLASMA FIG. 2. Effect of plasma on lymphocyte proliferation to PHA. Differences between values with and without lithium are significant using a paired r test (p < 0.01) only in cultures containing autologous plasma obtained during neutropenic intervals. The mean uptake of [Wlthymidine in cultures containing the same plasma was also significantly lower (P < 0.05 by Student’s t test) than mean values recorded in cultures done using autologous plasma obtained during nonneutropenic intervals or in cultures done using pooled AB plasma. The incorporation of lithium to these cultures rendered these differences nonsignificant. POOLED AB PLASMA
malization in T-cell numbers. Despite these dramatic effects of lithium in lymphocytes, neutrophils continued to cycle. It should be noted, however, that clinical symptomatology during the neutropenic periods was substantially ameliorated. The patient observed that symptoms were less severe and lasted only 1- 3 days as contrasted to the usual 5-7 days he had previously experienced. One neutropenic
--__--_----?
9
50
X
-i n
40 30
1
20 10
-----
DEC. JAN. FEB. MAR. iPR. FIG. 3. Correlation of PMNs and T lymphocytes in patient 2.
BORKOWSKY,
590
SHENKMAN,
AND RAUSEN
------------
t -70 NORMAL RANGE I -60 - 50 1 40 8 I -4 NORMAL RANGE _ x) 0 F L - 20
--_--_------------_---V/////A I
I
I
DEC. FIG.
JAN.
-10
Llrwll# livEAl-YENT y////// I
FEB.
I
MAR
I
APR.
4. Effects of lithium therapy on lymphocyte
indices.
cycle documented by blood count was associated with no evidence of illness. The interval between cycles increased from 21 days to 23-28 days. This also contrasted to previously regular documented cycling. Unfortunately, after 6 months of therapy, this clinical improvement seemed to dissipate. DISCUSSION
Periodicity of neutrophils as well as monocytes, eosinophils, reticulocytes, and lymphocytes has been variously reported to occur in human cyclic neutropenia (1, 10, 11). The possible variability of T lymphocytes and their function was studied in one patient with CN without lymphocyte periodicity. Two measurements done during nonneutropenic periods were significantly above control levels. Stimulation indices of these patients’ lymphocytes were reported to be within the control range. However, a single determination performed on a day when neutropenia was present in a stimulation index more than one standard deviation below normal (12). Grey collie dogs with CN have also had T-cell function studied. Their lymphocyte response to PHA was always reduced when compared to normal dogs. Although no periodicity of lymphocytes was seen in two dogs with CN, lymphocyte responses in one dog with CN appeared to be higher during neutropenic intervals. Depletion of the paracortical (T dependent) thymic lymphocytes was noted in all three dogs (13). The two patients reported here were both found to have cycling of T-cell expression (E-rosette formation) and function (PHA stimulation). Nadirs in T cells and the proliferative response to PHA but not PWM occurred when neutropenia was present. We considered two factitious causes of this phenomenon and discounted both. An increase in monocytes and eosinophils, both of which cosediment with lymphocytes in a Ficoll-Hypaque separation, could cause a false underestimate of E-rosettes and total lymphocytes engaged in a culture. However, a preincubation with latex and the use of a phase microscope was used to differ-
LITHIUM
EFFECTS
ON
T LYMPHOCYTES
IN
NEUTROPENIA
591
entiate lymphocyte from nonlymphocytes. In addition, grey collie dogs with CN have been shown to have increased autorosette formation (14). If our patients exhibited a similar phenomenon then a proportion of their lymphocytes would be lost in the Ficoll-Hypaque separation procedure. However, the ability of an in \?itvo incubation with Li to restore T-cell numbers and PHA hyporesponsiveness to normal would seem to preclude this possibility. Li has been shown to decrease cyclic AMP (CAMP) in a number of tissues. We have observed that Li increases mitogen responses by lymphocytes in normal (6), suppressor cell-enriched, and suppressor cell-depleted cultures (7). Li has also increased low-affinity E-rosette formation (6). We have postulated that these effects are mediated by its antagonism of the CAMP accumulation that is encouraged by agents involved in the pharmacoregulation of immune responses (i.e., prostaglandins, histamine, etc.) (15). Agents that elevate CAMP may also play a role in the maturation of granulocyte progenitor cell. Prostaglandins of the E series are potent in vitro inhibitors of granulocyte colony forming units (16) and may function by suppression of colony stimulating factor(s) (CSF) released by mononuclear phagocytes interacting with stimulated T lymphocytes (17). Our patients may have had a cycling plasma factor which both inhibited lymphocyte proliferation and CSF production. Serum inhibitors of CSF have been described previously (18, 19). Although we never actually measured the latter, levels of CSF in urine and serum of CN dogs cycle inversely with the neutrophil cycle (20). This factor could be mediating its effects by elevating CAMP in the appropriate cells. An early reported “side effect” of lithium therapy of manic depressive individuals was an increase in circulating granulocytes. This effect has recently been used to prevent infections in granulocytopenic patients. The mechanism of lithium-induced granulocytosis is unknown but it has been shown to increase CSF production in mice (21) and in patients with certain neutropenic disorders (22, 23). In fact, lithium therapy was recently successful in normalizing neutrophil counts in grey collie dogs with CN (24). Since Li added in vitro appeared to improve lymphocyte function and since Li therapy of CN grey collie dogs proved successful in eliminating neutrophil cycling we began treating one of our patients with Li carbonate. Within the first 3 months, T-cell cycling had stopped and clinical illness was greatly decreased, although the patient’s neutrophils continued to cycle. A perusal of the course of the grey collie dogs treated with Li revealed that about 10 cycles transpired after initiation of Li therapy prior to the cessation of cycling. The requirement for prolonged therapy with steroids was also recently emphasized in a report documenting the successful treatment of human CN (25). We, therefore, continued therapy for 8 months but never documented an amerlioration of neutrophil periodicity . It was of interest, however, that clinical improvement appeared despite no appreciable changes in the neutropenia. Levamisol, a drug associated with enhancement of T-cell function, has been reported to induce clinical improvement in human CN patients without affecting the actual neutropenia (26, 27). This would suggest that the fever, ulcerations, and adenopathy present during the neutropenia may actually result from T-cell defects. Unfortunately, in the case of our patient, the clinical improvement associated with Li therapy appeared to decrease with time, in spite of the continued normalization of T-cell function. Splenectomy has
592
BORKOWSKY,
SHENKMAN,
AND
RAUSEN
been frequently reported to induce transient clinical benefits to human CN patients (28). It is of interest that Li therapy of mice is associated with a dramatic shrinkage in spleen size (unpublished observations, W. Borkowsky and L. Shenkman) and this effect may account for the similar transient improvement seen in our patient. In summary, we have studied two patients with CN and demonstrated T-cell lymphopenia during neutropenic periods. A plasma factor present during these periods appeared to depress lymphocyte proliferative responses to PHA. The addition of Li, in vitro, to the mononuclear cells resulted in normalization of T-cell expression and function during neutropenic periods. Li therapy of one patient resulted in a cessation of T-cell cycling and a transient clinical improvement without altering significantly neutrophil periodicity. REFERENCES 1. Guerry, D., Dale, D. C., Omine, M., Perry, S., and Wolff, S. M. J. Clin. Invest. 52, 3320, 1973. 2. Shopsin, B., Friedman, R., and Gershon, S., Clin. Pharmacol. Ther. 12, 923, 1971. 3. Stein, R. S., Berman, C., Ali, M. Y., Hansen, R., Jenkins, D. D., and Jumean, H. G., N. Engl. J. Med.
297,
430,
1977.
4. Joyce, R. H., and Chervenick, P. A., Proc. Amer. Sot. Hematol. 18, 126, 1975. 5. Hammond, W. P., and Dale, D. C., Blood 55, 26, 1980. 6. Shenkman, L., Borkowsky, W., Holzman, R. S., and Shopsin, B., Clin. Immunol. Immunopathol. 10, 187, 1978. 7. Shenkman, L., Wadler, S., Borkowsky, W., and Shopsin, B., Immunopharmacology 3, 1, 1981. 8. Jondad, J., Wiggell, H., and Aiut, F., Transplant. Rev. 16, 163, 1973. 9. Winchester, R. J., and Ross, G., In “Manual of Clinical Immunology” (N. R. Rose and H. Freidman, Eds.), p. 64. Amer. Sot. Microbial. Washington, D.C., 1976. 10. Brandt, L., Forssman, O., Mitelman. F., Odeberg, H., Olofsson, T., Olson, I., and Svensson, B.. Stand. J. Haematol. 15, 228, 1975. 11. Greenberg, P. L., Bax, I., Levin, J., and Andrews, T. M., Amer. J. Hematol. 1, 375, 1976. 12. Andrews, R. B., Dunn, C. D. R., Jolly, J., Jones, J. B., and Lange, R. D., Stand. .I. Haemartol. 22, 97, 1979. 13. Angus, K., Wyand, D. S., and Yank, T. S., Clin. Immunol. Immunopathol. 11, 39, 1978. 14. Angus, K., and Yang, T. S., Immunology 35, 1005, 1978. 15. Shenkman, L., Borkowsky, W., and Sbopsin, B., Med. Hypotheses 6, 1, 1980. 16. Motomura, S., and Dexter, T. M., Exp. Hematol. 8, 298, 1980. 17. Cline, M. J., and Golde, D. W., Nature (London) 277, 177, 1979. 18. Cline, M. J., Herman, S. P., and Golde, D. W., Transplant. Prac. 10, 99, 1978. 19. Rudolph, W. G., and Kaneko, J. J., Proc. Sot. Exp. Biol. Med. 163, 421, 1980. 20. Dale, D. C., Brown, C. H., Charbone, P., and Wolff, S. M., Science 173, 152, 1971. 21. Harker, W. G., Rothstein, G., Clarkson, D., Athens, J. W., and McFarlene, J. L., Blood 49, 263, 1977. 22. Robinson, W. A., Entringer, M. A., Haber, J., and Gupta, R., In “Lithium Effects on Granulopoiesis and Immune Function” Vol. 127, “Advances in Experimental Medicine and Biology” (A. H. Rossof and W. A. Robinson, Eds.), p. 281. Plenum, New York, 1980. 23. Chan, H. S. L., Freedman, M. H., and Saunders, E. F., In “Lithium Effects on Granulopoiesis and Immune Function,” Vol. 127, “Advances in Experimental Medicine and Biology” (A. H. Rossof and W. A. Robinson, Eds.), p. 293. Plenum, New York, 1980. 24. Hammond, W. P., and Dale, D. C. Blood 55, 26, 1980. 25. Wright, D. G., Fauci, A. S., Dale, D. C., and Wolff, S. M., N. Engl. J. Med. 298, 295, 1978. 26. Proctor, S. J., Reid, M. M., and Low, W. T., Postgrad. Med. .I. 55, 279, 1979. 27. Senn, J. S., Lai, C. C., and Price, G. B., Brit. J. Cancer 41, 40, 1980. 28. Page, A. R., and Good, R. A., Amer. J. Dis. Child. 94, 623, 1957. Received August 4, 1981; accepted with revisions December 20, 1981.
IMMUNOLOGY
AND
IMMUNOPATHOLOGY
23, 586-592 (1982)
T-Lymphocyte Cycling in Human Cyclic Neutropenia: Effects of Lithium in Vitro and in Vivo WILLIAMBORKOWSKY,"
LOUIS SHENKMAN,~ AND AARONRAUSENS
‘Departments of Pediatrics and TMedicine, New York University Medical Center, New York, New York 10016, and SDepartment of Pediatrics. Beth Israel Medical Center, New York. New York 10003 Two patients with cyclic neutropenia demonstrated T-cell lymphopenia during their neutropenic episodes. In addition, they also demonstrated a depressed lymphocyte proliferative response to phytohemagglutinin when neutropenic, which was possibly mediated by a plasma factor. Lithium appeared to normalize T-cell expression and function when used in vitro and in viva. Lithium therapy of one patient resulted in a cessation of T-cell cycling and a transient clinical improvement without affecting neutrophil periodicity.
INTRODUCTION
Cyclic neutropenia is a blood disorder in man and grey collie dogs characterized by episodes of neutropenia at defined regular intervals (usually 21 days in man and 12 days in dogs). During periods of neutropenia, fever, mucosal ulcerations, infections, and lymphadenopathy can occur. Although blood elements other than neutrophils have been observed to cycle (e.g., reticulocyte, platelets, and monocytes) (l), few studies have examined the fate of T lymphocytes, which themselves have documented effects on the maturation of myeloid colonies. Lithium salts have been shown to induce granulocytosis in manic depressive individuals (2) and have recently been used to ameliorate iatrogenic neutropenias in patients treated for malignancy (3). Lithium is believed to function in this capacity by increasing production and/or release of granulocyte colony stimulating factors (4). Treatment of cyclic hematopoiesis with lithium in the grey collie dog has been utilized to eliminate recurrent neutropenia and to normalize levels of other cycling blood cells (5). Lithium has also been observed to enhance various T-lymphocyte functions in vitro (6, 7). We describe two patients with cyclic neutropenia whose T-lymphocyte numbers and function were studied during their cycles. One patient was treated with lithium carbonate. The effects of this treatment on neutropenia and T lymphocytes will be presented. CASE REPORT I
C.R. is a lPyear-old female who was brought to a physician at 9 years of age for a blood count because a male sibling died suddenly of infection and neutropenia. She was found to have neutropenic cycles at 1% to 23-day intervals. Each cycle was 5 to 11 days in duration. The neutropenia was compensated by an absolute monocytosis and eosinophilia. These episodes were accompanied by clinical 586 0090-1229/060586-07$01.00/O Copyright 0 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.
LITHIUM
EFFECTS
ON
T LYMPHOCYTES
IN
NEUTROPENIA
587
symptoms of fever, adenopathy, and lymphadenitis. Hematologic cycles did not correlate with her menses. Neutropenic intervals were not always associated with any symptoms of illness. CASE REPORT II
B .B. is a 21-year-old male who was diagnosed as having cyclic neutropenia at 4 months of age. His cycles occur every 21 days with each neutropenic interval lasting 5-7 days. During the period his peripheral blood smear shows compensation with eosinophilia while no monocytosis occurs. Clinical evidence of fever, stomatitis, and cellulitis accompany each cycle. Since the diagnosis of his illness he had only once gone through a neutropenic interval without some clinical symptomatology. METHODS Enumeration of lymphocyte subpopulations. T cells were enumerated by the method of Jondal et al. (8), and immunoglobulin-bearing lymphocytes by the method of Winchester and Ross (9) using a goat anti-human immunoglobulin (polyvalent)-fluorescenated antibody (Cappel Ind., Cochronville, Pa.). This method employs a preincubation of peripheral blood mononuclear cells with latex particles (Dow Diagnostics) to verify macrophages. In vitro studies of peripheral blood lymphocyte responses. Ficoll-Hypaquepurified mononuclear cells were suspended in RPM1 1640 supplemented with 20% heat-inactivated pooled AB plasma or autologous plasma and cultured in microtiter plates (Nunc, Denmark) for 72 hr at a concentration of lo5 cells/O.2 ml and with phytohemagglutinin (PHA) at a concentration of 1 &ml (Digro Lab, Detroit, Mich.) and pokeweed mitogen (PWM) at a concentration of 1 pg/ml (Sigma Chemical Co., St. Louis, MO.). [‘*C]Thymidine, 0.03 &i (New England Nuclear, specific activity 50 pCi/mmol) was added to each well for the final 8 hr of culture. Cells were harvested on glass fiber filters and cell-incorporated radioactivity was counted in a liquid scintillation counter. Effects of lithium chloride were determined by adding 2 meq/liter of the salt to parallel cell cultures for the entire culture period. RESULTS T und B Lymphocyte
Evaluation
Both patients were found to have diminished numbers (relative and absolute) of T lymphocyte during periods of neutropenia. Their B-cell number remained in the normal range (5- 15%) throughout their cycle. The T lymphopenia was not due to artifactuai error possibly induced by the relative monocytosis seen in patient C. R. as these cells were excluded from evaluation. Figure 1 represents the cycling of T cells and PMNs in patient C.R. with absolute T-cell numbers ranging from 700 cells/mm3 during neutropenia to 1500 cells/mm3 otherwise. A similar cycling phenomena was present in patient B.B. with absolute T-cell numbers ranging from 1000 cells/mm3 during neutropenic intervals to 2200 cells/mm3 when not neutropenic.
588
BORKOWSKY,
SHENKMAN,
AND RAUSEN
- 60 -w -40 /
,_
I
I 10
I
20
JAN -
FIG. 1. Correlation
I 1 FE8 -
El # -I
I 10
of PMNs and T lymphocytes in patient I.
Effect of in Vitro Preincubation with Li on T-Cell Numbers The preincubation of the patients lymphocytes with LiCl (2 meq/liter) prior to the rosetting procedure increased the number of E-rosettes when T lymphopenia was evident (E-rosettes: 40 + 4% without Li and 61 +- 4% with Li, IZ = 6, P < 0.03) but had no effect when T-lymphocyte numbers were normal (E-rosettes: 55 + 5% without Li and 58 + 6% with Li, n = 6, P < 0.2.) Lymphocyte
Proliferation
to Mitogens
Lymphocytes were stimulated in both autologous or pooled AB plasma. Although there was no difference in response to PHA during cycles if pooled AB plasma was used, a depressed proliferative response to PHA was seen during neutropenia intervals when cultures contained autologous plasma (Fig. 2). The incorporation of therapeutic concentrations of Li in these cultures revealed that Li could reverse the inhibition manifested by neutropenia-associated plasma without having a significant effect on cultures containing either pooled AB plasma or autologous plasma obtained during a nonneutropenia period. Lymphocyte proliferation due to pokeweed mitogen stimulation was normal at all periods of time without significant difference due to either autologous or heterologous plasma. Effect on Li Therapy in Vivo Since Li appeared to augment T-cell expression and function when in vitro incubations were performed, its effects on these parameters as well as neutrophil cycling were examined during in vivo therapy. Li was administered to patient B .B. in doses achieving serum concentrations of 0.50 to 0.75 meq. As seen in Fig. 3, Li therapy appeared to normalize the T-cell cycling after 3 months. Prior to this period, T cycling was still occurring but with an elevated baseline. This effect of Li on T cells was also appreciable by measuring [14C]thymidine incorporation after PHA stimulation in autologous plasma. As seen in Fig. 4, initiation of Li therapy abrogated the abnormally low responses to PHA. This corresponded to the nor-
LITHIUM
EFFECTS
ON T LYMPHOCYTES
IN NEUTROPENIA
589
NON-NEUTROPENIC NEUTROPENIC AUTOLOGOUSPLASMA AUTOLOGOUSPLASMA FIG. 2. Effect of plasma on lymphocyte proliferation to PHA. Differences between values with and without lithium are significant using a paired r test (p < 0.01) only in cultures containing autologous plasma obtained during neutropenic intervals. The mean uptake of [Wlthymidine in cultures containing the same plasma was also significantly lower (P < 0.05 by Student’s t test) than mean values recorded in cultures done using autologous plasma obtained during nonneutropenic intervals or in cultures done using pooled AB plasma. The incorporation of lithium to these cultures rendered these differences nonsignificant. POOLED AB PLASMA
malization in T-cell numbers. Despite these dramatic effects of lithium in lymphocytes, neutrophils continued to cycle. It should be noted, however, that clinical symptomatology during the neutropenic periods was substantially ameliorated. The patient observed that symptoms were less severe and lasted only 1- 3 days as contrasted to the usual 5-7 days he had previously experienced. One neutropenic
--__--_----?
9
50
X
-i n
40 30
1
20 10
-----
DEC. JAN. FEB. MAR. iPR. FIG. 3. Correlation of PMNs and T lymphocytes in patient 2.
BORKOWSKY,
590
SHENKMAN,
AND RAUSEN
------------
t -70 NORMAL RANGE I -60 - 50 1 40 8 I -4 NORMAL RANGE _ x) 0 F L - 20
--_--_------------_---V/////A I
I
I
DEC. FIG.
JAN.
-10
Llrwll# livEAl-YENT y////// I
FEB.
I
MAR
I
APR.
4. Effects of lithium therapy on lymphocyte
indices.
cycle documented by blood count was associated with no evidence of illness. The interval between cycles increased from 21 days to 23-28 days. This also contrasted to previously regular documented cycling. Unfortunately, after 6 months of therapy, this clinical improvement seemed to dissipate. DISCUSSION
Periodicity of neutrophils as well as monocytes, eosinophils, reticulocytes, and lymphocytes has been variously reported to occur in human cyclic neutropenia (1, 10, 11). The possible variability of T lymphocytes and their function was studied in one patient with CN without lymphocyte periodicity. Two measurements done during nonneutropenic periods were significantly above control levels. Stimulation indices of these patients’ lymphocytes were reported to be within the control range. However, a single determination performed on a day when neutropenia was present in a stimulation index more than one standard deviation below normal (12). Grey collie dogs with CN have also had T-cell function studied. Their lymphocyte response to PHA was always reduced when compared to normal dogs. Although no periodicity of lymphocytes was seen in two dogs with CN, lymphocyte responses in one dog with CN appeared to be higher during neutropenic intervals. Depletion of the paracortical (T dependent) thymic lymphocytes was noted in all three dogs (13). The two patients reported here were both found to have cycling of T-cell expression (E-rosette formation) and function (PHA stimulation). Nadirs in T cells and the proliferative response to PHA but not PWM occurred when neutropenia was present. We considered two factitious causes of this phenomenon and discounted both. An increase in monocytes and eosinophils, both of which cosediment with lymphocytes in a Ficoll-Hypaque separation, could cause a false underestimate of E-rosettes and total lymphocytes engaged in a culture. However, a preincubation with latex and the use of a phase microscope was used to differ-
LITHIUM
EFFECTS
ON
T LYMPHOCYTES
IN
NEUTROPENIA
591
entiate lymphocyte from nonlymphocytes. In addition, grey collie dogs with CN have been shown to have increased autorosette formation (14). If our patients exhibited a similar phenomenon then a proportion of their lymphocytes would be lost in the Ficoll-Hypaque separation procedure. However, the ability of an in \?itvo incubation with Li to restore T-cell numbers and PHA hyporesponsiveness to normal would seem to preclude this possibility. Li has been shown to decrease cyclic AMP (CAMP) in a number of tissues. We have observed that Li increases mitogen responses by lymphocytes in normal (6), suppressor cell-enriched, and suppressor cell-depleted cultures (7). Li has also increased low-affinity E-rosette formation (6). We have postulated that these effects are mediated by its antagonism of the CAMP accumulation that is encouraged by agents involved in the pharmacoregulation of immune responses (i.e., prostaglandins, histamine, etc.) (15). Agents that elevate CAMP may also play a role in the maturation of granulocyte progenitor cell. Prostaglandins of the E series are potent in vitro inhibitors of granulocyte colony forming units (16) and may function by suppression of colony stimulating factor(s) (CSF) released by mononuclear phagocytes interacting with stimulated T lymphocytes (17). Our patients may have had a cycling plasma factor which both inhibited lymphocyte proliferation and CSF production. Serum inhibitors of CSF have been described previously (18, 19). Although we never actually measured the latter, levels of CSF in urine and serum of CN dogs cycle inversely with the neutrophil cycle (20). This factor could be mediating its effects by elevating CAMP in the appropriate cells. An early reported “side effect” of lithium therapy of manic depressive individuals was an increase in circulating granulocytes. This effect has recently been used to prevent infections in granulocytopenic patients. The mechanism of lithium-induced granulocytosis is unknown but it has been shown to increase CSF production in mice (21) and in patients with certain neutropenic disorders (22, 23). In fact, lithium therapy was recently successful in normalizing neutrophil counts in grey collie dogs with CN (24). Since Li added in vitro appeared to improve lymphocyte function and since Li therapy of CN grey collie dogs proved successful in eliminating neutrophil cycling we began treating one of our patients with Li carbonate. Within the first 3 months, T-cell cycling had stopped and clinical illness was greatly decreased, although the patient’s neutrophils continued to cycle. A perusal of the course of the grey collie dogs treated with Li revealed that about 10 cycles transpired after initiation of Li therapy prior to the cessation of cycling. The requirement for prolonged therapy with steroids was also recently emphasized in a report documenting the successful treatment of human CN (25). We, therefore, continued therapy for 8 months but never documented an amerlioration of neutrophil periodicity . It was of interest, however, that clinical improvement appeared despite no appreciable changes in the neutropenia. Levamisol, a drug associated with enhancement of T-cell function, has been reported to induce clinical improvement in human CN patients without affecting the actual neutropenia (26, 27). This would suggest that the fever, ulcerations, and adenopathy present during the neutropenia may actually result from T-cell defects. Unfortunately, in the case of our patient, the clinical improvement associated with Li therapy appeared to decrease with time, in spite of the continued normalization of T-cell function. Splenectomy has
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been frequently reported to induce transient clinical benefits to human CN patients (28). It is of interest that Li therapy of mice is associated with a dramatic shrinkage in spleen size (unpublished observations, W. Borkowsky and L. Shenkman) and this effect may account for the similar transient improvement seen in our patient. In summary, we have studied two patients with CN and demonstrated T-cell lymphopenia during neutropenic periods. A plasma factor present during these periods appeared to depress lymphocyte proliferative responses to PHA. The addition of Li, in vitro, to the mononuclear cells resulted in normalization of T-cell expression and function during neutropenic periods. Li therapy of one patient resulted in a cessation of T-cell cycling and a transient clinical improvement without altering significantly neutrophil periodicity. REFERENCES 1. Guerry, D., Dale, D. C., Omine, M., Perry, S., and Wolff, S. M. J. Clin. Invest. 52, 3320, 1973. 2. Shopsin, B., Friedman, R., and Gershon, S., Clin. Pharmacol. Ther. 12, 923, 1971. 3. Stein, R. S., Berman, C., Ali, M. Y., Hansen, R., Jenkins, D. D., and Jumean, H. G., N. Engl. J. Med.
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