sphha mice with chronic hemolytic anemia

CELLULAR

IMMUNOLOGY

138,360-37 1 (1991)

Altered Lymphocyte Populations in sphha/sphha Mice with Chronic Hemolytic Anemia LILLIAN MAGGIO-PRICE,*ANGELIKA GROSSMANN,* DAVID ENGEL,t AND CORNELIUS ROSSE$ Departments of *Comparative Medicine, tPeriodontics, and SBiological Structure, University of Washington, Seattle, Washington 98195 Received May 2, 1991: acceptedJune 25, 1991

Lymphocytekinetics and phenotype were examined in mutant anemic sphh”/sphh” mice that manifest a lifelong lymphocytosis which accompanies their chronic hemolytic anemia. Anemic mice have significant increases in CD4+, CD8+, and sIgM+ lymphocytes in peripheral blood. Pulse and continuous infusion studies with [3H]TdR suggest that this apparent lymphoid expansion is not due to increased production of lymphocytes in bone marrow or thymus but rather to a redistribution of lymphocytes from the spleen to other peripheral lymphoid tissue sites as well as increased proliferation of T and B lymphocytes in lymph nodes. This murine model could be useful to examine lymphocyte perturbations that may accompany chronic hemolytic anemia in humans. 0 1991 Academic Press. Inc. INTRODUCTION Mutant anemic (sphh”/sphha) mice exhibit a congenital, hemolytic anemia attributable to an abnormality in assembly of the erythrocyte membrane protein a-spectrin (l-3). Red blood cells in these mice are spherocytic and fragment easily, resulting in a marked and persistent hemolytic anemia (2). This autosomal recessive defect (4) is characterized by reticulocytosis, marrow erythroid hyperplasia, and extramedullary hematopoiesis in spleen and liver (5). These mice have historically been used in studies of hemoglobin metabolism (6) heme oxygenase inhibitors of hyperbilirubinemia (7, S), and hematopoietic stem cells and marrow transplantation (9- 12). We have noted that anemic mice also exhibit an unexplained peripheral blood lymphocytosis and lymph node hyperplasia. In these studies, we analyzed the kinetics of lymphocyte subpopulations from anemic mice and made comparisons with normal syngeneic animals. MATERIALS

AND

METHODS

Mice Mutant anemic mice (WBB6Fl -sphha/sphh”) were produced by mating heterozygotes from two different inbred strains, WB/Re and C57B1/6J, originally obtained from The Jackson Laboratory, and reared at the University of Washington, School of Medicine. Control mice were WBB6Fl-+/+(+/+). The origin of the mutant has been

0008~8749/91 $3.00 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

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previously described (2). Mice were housed under AAALAC conditions and maintained under specific-pathogen-free (SPF) barrier conditions. A quality assurance program at the University of Washington has ascertained that the colony is serologically negative for major rodent viral pathogens, and culture negative for 10 common bacterial pathogens. Mice are also free of rodent ecto- and endoparasites. Control (WBB6FI-+/+) and anemic (WBB6Fl-sphh”/sphh”) mice were 3 to 5 months old and age- and sexmatched in all experiments.

Reagents and Flow Cytometry Phycoerythrin (PE)-conjugated anti-CD4 mAb, fluorescein isothiocyanate (FITC)conjugated anti-CD8 mAb and Thy 1.2 antibody were obtained from Becton-Dickinson (Mountain View, CA). PE-conjugated goat anti-mouse anti-IgM (Southern Biotechnology, Birmingham, AL) and B220 mAb (14.8) (ATCC; (13)) followed by FITCconjugated goat anti-rat IgG (Kirkegaard and Perry, Gaithersburg, MD) were also used. Cells were stained for 20 min at 22°C with saturating amounts of mAb, washed, and stored in RPM1 with 1% paraformaldehyde in the dark at 4°C before analysis was performed using a Becton-Dickinson FACScan. Forward-angle uv light scatter was used to gate cells from debris and approximately 5 X lo3 cells were analyzed for each sample.

Cell Suspension Preparations Heparinized blood was obtained from the retroorbital sinus of mice and layered over Ficoll-Paque (Pharmacia, Piscataway, NJ), centrifuged at 5OOg,washed in RPM1 with 5% fetal calf serum, and counted using trypan blue supravital stain. Single-cell suspensions of lymph node (axillary, cervical, and inguinal) and thymus were prepared by passing minced, screen-passed tissues through nitex (Tekto, NY), making a singlecell suspension, and counted. The spleen was minced, passed through a metal screen, layered over Lympholyte-M (Cedarlane, Hicksville, NY), washed, passed through nitex, and washed again and the cells were counted. Both femurs were flushed with RPMI, a single-cell suspension was made, and cells were washed and counted.

Cell Kinetics Mice were given [3H]TdR (1 &i/g body weight; 6.7 Ci/mmol; New England Nuclear, Boston, MA) using a single injection or miniosmotic pump. (a) Pulse label. Groups of two to five mice received a single iv injection of [3H]TdR and were sacrificed at 30 min, 24 hr, and 48 hr. (b) Continuous label. Groups of two to five mice were anesthetized with Penthrane (Pitman-Moore, Washington Crossing, PA) and one miniosmotic pump (Alza Corp., Palo Alto, CA) was implanted in the subcutaneous tissue of the back. Each pump was loaded with sufficient [3H]TdR to provide continuous delivery of a total dose of 1 pCi/g body weight per day. Mice were sacrificed after 12, 24, 48, and 72 hr. (c) Radioautography. Single-cell suspension cytospot smears were made of lymph nodes, spleen, thymus, and femoral bone marrow. Cytocentrifuge smears were coated with NTB 2 liquid emulsion (Eastman Kodak) for radioautography, exposed at 4°C in the dark for 6 weeks, developed in Dektol, and fixed with Kodak acid fixer. Smears were then stained with McNeals tetrachrome for determination of cell type.

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(d) Cell counts. Lymphocytes of 9 pm or greater in cell diameter were considered large lymphocytes, and those less than 9 pm were counted as small lymphocytes. Lymphocytes that measured 9 pm or less did not incorporate [3H]TdR in animals injected 30 min before sacrifice and were, therefore, designatedas “small” (postmitotic) lymphocytes. Lymphocytes measuring more than 9 pm in diameter did directly incorporate [3H]TdR, and in this study are designated as “large” (mitotic) lymphocytes. Approximately 1000 consecutive lymphocytes were counted per sample. A cell was counted as labeled with [3H]TdR if it had three or more silver grains over the nucleus. The background mean grain count of 1000 red blood cells was 0.1 per erythrocyte.

RESULTS Bone Marrow Lymphocytes

To gauge whether lymphocyte populations were augmented in the bone marrow, as well as in the periphery of anemic mice, we determined bone marrow cellularity and the incidence of morphologically identifiable lymphocytes along with the incidence of Thy I+, sIg+, and B220+ cells. We confirmed the erythroid hyperplasia we observed in the bone marrow previously (5) and found the total number of cells in the femur to be comparable between anemic and +/+ mice (Table 1). The percentage of total lymphocytes and T and B cell subpopulations is also shown in Table 1. Although the percentage of total lymphocytes and Thy l+ cells was similar in marrows of anemic and +/+ mice, the mean percentage sIgM+ and B220+ cells was significantly lower in marrows of anemic mice (Table 1). Hence, contrary to our expectations, there were notably fewer B lineage lymphocytes in the marrow of mutant anemic mice when compared with marrow of their control +/+ counterparts. To test whether the diminished pool of lymphocytes was due to a slower rate of cell production or an increased rate of cell discharge from the bone marrow, we performed a pulse and chase as well as a continuous labeling study with [3H]TdR. Following a single iv injection of [3H]TdR, labeling of large lymphocytes in +/+ marrow rose from 8.8 + 3.4% at 30 min after the pulse to 15.5 f 5.3% at 48 hr of the chaseperiod. Figure IA indicates that labeling of large lymphocytes in anemic marrow lagged significantly behind that in +/+ marrow as well as throughout the 48-hr chase period. Although small lymphocytes do not actively incorporate [3H]TdR, since they TABLE Bone Marrow Cellularity,”

Cellularitv

+/+ Anemic

5.9 f 1.8 5.9 + 2.4

1

Percentage, and Phenotype of Marrow Lymphocytesb in Anemic and +/+ Mice

%

% Lvmphocvtes

Thy 1.2+

20.9 + 4.1 22.0 f 4.6

2.5 f 1.1 2.0 + 0.7

90 &M+ 32.6 f 4.6’ 20.8 f 6.0’

0 Mean + SD X 10’ per two femurs; derived from 39 +/+ and 37 anemic animals. b Mean + SD derived from five to six animals. ‘P
570 B220+ 53.5 f 6.0“ 22.8 f 9.3d

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B

A 24

WITH

60 50

large lymphocytes

; s

16 12

3 8 g

8 4 20 ::::!-:

small lymphocytes

1

A

0;

0

12

24 36 Time (hrs)

48

0

12

24 36 Time (hrs)

48

FIG. I. Percentages of [‘H]TdR-labeled large (A) and small (B) marrow lymphocytes following a single iv injection of [3H]TdR (pulse and chase). Labeling of both large and small lymphocytes from marrows of anemic (0) mice was initially lower than that of +/+ mice (0) and this persisted throughout the 4%hr chase period. Points represent means +- SD derived from three to four mice.

are the daughter cells of large lymphocytes that do label directly, labeling of small lymphocytes in normal marrow rose from 0 to 49 + 6.5% by 48 hr of the chase period (Fig. IB). Similar to the pattern of large lymphocytes, labeling of small lymphocytes in anemic marrow lagged significantly behind the rate of label accumulation in +/+ marrow. Results of continuous labeling with [3H]TdR were consistent with the findings of the pulse and chase study. Under conditions where every cell that enters S phase acquires label, the rate of accumulation of label in large lymphocytes was retarded in the marrow of anemic mice in comparison with the marrow of +/+ mice (Fig. 2A). In the case of postmitotic small lymphocytes, the rate of labeling under these conditions can be equated with the rate of cell turnover in the population. The data show that the rate of replacement of unlabeled small lymphocytes by newly generated labeled cells was comparable in the marrow of +/+ and anemic mice (Fig. 2B). Thus, the population of morphologically identifiable lymphocytes was unaltered by the erythroid hyperplasia present in the marrow of anemic mice, but the rate of proliferation of large lymphocytes was retarded compared with that in normal +/+ mar-

!;] ;$

;js7 48

Time (hrs)

72

0

; 24

48

72

Time (hrs)

FIG. 2. Percentages of [‘H]TdR-labeled large (A) and small (B) marrow lymphocytes during continuous infusion of [3H]TdR over a 72-hr period. The rate of accumulation of label in large lymphocytes was lower in anemic mice while the rate of replacement of unlabeled small lymphocytes by newly generated labeled cells was comparable in anemic (0) and +/+ (0) mice. Points represent means f SD derived from three to four mice.

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TABLE 2 Thymus Cellularity”

Cellularity +I+ Anemic

5.1 * 2.1 5.8 f 3.4

and Phenotype of Thymocyte&’ in Anemic and +/+ Mice

90

90

90

90 cD4+

CD8+

CD4+ CD8+

CD4- CD8-

9.2 f 1.6 8.2 f 0.8

1.8 f 1.0 1.5 f 0.7

87.0 2 3.1 87.9 f 1.8

1.8 f .08 2.2 f .06

a Mean + SD X 10’ derived from seven to eight animals. b Mean + SD derived from five to six animals.

row. The rate of cell replacement in the postmitotic compartment of small lymphocytes was comparable in anemic and +/+ marrow. The decreasedpopulation of B lineage cells in anemic marrow must be attributed to the increased incidence of cells with lymphoid morphology that do not express Thy 1, sIg, or B220. Thymocytes To determine if there was any alteration in T lymphocyte production, thymic cellularity, phenotype, and kinetics were evaluated. Thymic cellularity as well as the distribution of thymocyte subsetswas not significantly different in +/+ and anemic mice (Table 2). Patterns of cycling cells as determined by pulse (Fig. 3A) or continuous [3H]TdR infusion (Fig. 3B) were also similar in thymocytes from +/+ and anemic animals. Following a single injection of thymidine, labeling was present predominantly in the large thymocytes (approximately 10%) with increasing appearance over the period of the chase in the daughter small thymocytes; this pattern of labeling was noted in thymocytes from both +/+ and anemic animals (Fig. 3A). A similar pattern of labeling was noted when there was continuous availability of thymidine (Fig. 3B). Thus, no significant difference could be detectedin the cellularity, phenotype incidence, rate of proliferation, or cell turnover between thymocytes of anemic and +/+ mice.

A $ ; 0

$j $

50

B 80

40

60

30

small %

40

20

20

10

0

0 0

12

24 36 Time (hrs)

48

b

large

k 0

24

48

72

Time (hrs)

FIG. 3. Percentages of [‘H]TdR-labeled large and small thymocytes during pulse and chase (A) and continuous infusion (B) of [3H]TdR. Large thymocytes in both anemic (0) and +/+ (0) mice showed a lower level of labeling with increasing accumulation in small anemic (0) and +/+ (m) thymocytes. The pattern of cycling cells was similar in both populations from anemic and +/+ mice. Points and bars represent means f SD derived from three to four mice.

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r;

80

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?L’ 60 &

40 20 0

cellularity

FIG. 4. Splenic cellularity and lymphocyte numbers from anemic and +/+ mice. Spleens from anemic mice (a) were significantly (*P < 0.001) more cellular than those of +/+ (u) mice, but absolute numbers of lymphocytes were comparable. Bars represent means + SD derived from 20 to 22 mice.

Splenic Lymphocytes We have confirmed the erythroid hyperplasia in spleens of anemic animals (5, 14) which results in increased total cellularity (Fig. 4) and decreased percentage of lymphocytes in those spleens. Control +/+ spleens consisted of 85 to 90% lymphocytes with the remainder being other myeloid cells, while anemic spleens had approximately 10 to 15% lymphocytes. In terms of absolute numbers, however, the splenic lymphocyte population of anemic animals is comparable to that of +/+ mice (Fig. 4). Although numbers of lymphocytes were similar, the majority of morphologically identifiable lymphocytes in spleens of anemic animals did not express T or B cell surface antigens. There was a significant decrease in the percentage CD4+, CD8+, and sIgM+ cells (Table 3). The percentage B220+ cells was also determined and found to be significantly below that of +/+ animals (Table 3). The pattern of labeling of splenic lymphocytes was somewhat different in anemic and +/+ mice (Fig. 5), and the labeling index was approximately IO-fold higher in lymphocytes from anemic spleens when evaluated by pulse and chase (Figs. 5A and B) or continuous infusion (Figs. 5C and D) of thymidine. Since the majority of splenocytes from anemic animals which are classified as lymphocytes by morphologic criteria did not bear mature T or B cell markers, it is likely that some of these were hematopoietic progenitors like CFLJ-s, BFU-e, and CFU-E (lo), which are increased in anemic spleens. Such heterogeneity may account for the low percentage labeling with lymphocyte surface markers (Table 3).

TABLE 3 Splenic Lymphocyte Lymphocyte numbers +I+ Anemic

9.4 -+ 3.6 10.8 f 3.2

Numbers” and Phenotypeh in Anemic and +/+ Mice

% CD4+

% CDS+

% sIgM+

% B220’

14.9 i 2.4’ 1.8 ?I 0.5’

6.6 f 1.2’ 1.5 f 0.5’

11.9 f 2.6’ 10.4 i 0. IC

51.2 f 11.2’ 10.4 * 3.3’

a Mean f SD X 10’ derived from 20 to 22 animals, ’ Mean + SD derived from three to four animals. ‘P < 0.001; two-tailed t test.

366

MAGGIO-PRICE ET AL. B

A m 0

:: 5 0 g 4 5-u

3o

800

+I+

1 small b

20

anemic

600 400

10

K

200 large

0

0 24

0 C

- --1

“0 120 x

g 80 8

$

+I+

1 0

24

48

24

0

48 D 900

700

72

1

48

anemic

0

T

24

48

72

Time (hrs)

Time (hrs)

FIG. 5. Numbers of [‘H]TdR-labeled large and small splenocytes during pulse and chase (A and B) and continuous infusion (C and D) from anemic (B and D) and +/+ (A and C) mice. The labeling index was higher in lymphocytes from anemic spleens when evaluated by pulse or continuous infusion. Points and bars represent means + SD derived from one to four mice.

Lymph Node Lymphocytes

We pooled cells from groups (axillary, inguinal, cervical) of lymph nodes for each animal studied and determined cell phenotype as well as kinetic behavior. Semiquantitative comparisons of cellularity and lymphocyte subsetswere thus made between +/+ and anemic animals. Lymph nodes from anemic mice were three times more cellular than from +/+ animals. Anemic animals had a higher percentage B cells and consequently lower percentage T cells in their lymph nodes (Table 4). However, since lymph nodes from anemic mice were more cellular, absolute numbers of CD4+ and CDS+ cells, and particularly B cells, as assessedby either sIg or B220 positivity, were increased (Fig. 6) three- to sevenfold. Previous rodent kinetic studies have shown that the large lymphocyte (>9 pm) is in cycle and gives rise to daughter small lymphocytes (15, 16). Thirty minutes after TABLE 4 Lymph Node CellularitP and Lymphocyte Phenotype* in Anemic and +/+ Mice

+I+ Anemic

Cellularity

% T cells

% SIgMf

29.5 + 12.0’ 92.0 + 17.6

64.9 + 6.0’ 42.0 f 2.1’

30.9 + 6.3’ 50.7 + 3.6’

a Mean + SD X lo6 derived from six animals. b Mean + SD derived from six animals. c P < 0.001; two-tailed f test.

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FIG. 6. Lymph node cellularity and numbers of lymphocyte subsets. Lymph nodes from anemic mice (lZ3)were more cellular than those of +/+ (m) mice and contained more CD4+. CDS+, sIgM+, and B220+ (*P < 0.001) cells. Bars represent means f SD derived from six mice.

one pulse dose of thymidine, less than 5% of large lymphocytes were labeled in nodes from both anemic and +/+ mice (data not shown). Since nodes of anemic mice are more cellular, it may be concluded that there is an absolute increase in the number of proliferating lymphocytes in the lymph node compartment of anemic animals compared to controls. Continuous exposure to thymidine revealed similar findings but gave information about the proportion of proliferating cells over a longer period of time. With a continuous 72-hr exposure to thymidine, there was an expected gradual increase in labeled large lymphocytes and daughter small lymphocytes. Nodes from anemic mice had a more rapid increase in labeled cells (Figs. 7A and B), suggesting a higher rate of lymphocyte proliferation in nodes of anemic animals.

Phenotype of PBL. Mutant anemic mice have increased peripheral blood lymphocyte counts (5, 14). Anemic mice evaluated in the present study had absolute lymphocyte counts ranging from 23,850 to 31,970/mm3; lymphocyte counts of +/+ mice were 3920 to 9360/ mm’. Flow cytometric analysis of PBL phenotype showed that the percentage CD4+, CD8+, and sIgM+ cells was similar in anemic and +/+ mice, but there were significant

0

24 Time (h$

72

0

24 Time (h,s48

72

FIG. 7. Numbers of [3H]TdR-labeled large (A) and small (B) lymphocytes from lymph nodes of anemic (0) and +/+ (0) mice during continuous infusion of [3H]TdR. Lymphocytes from anemic mice had a more rapid increase in labeled large and small lymphocytes indicating a higher rate of proliferation compared with +/+ lymphocytes. Points and bars represent means f SD derived from three to four mice.

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increases in the absolute numbers of CD4+, CD8+, and s&M+ lymphocytes in the peripheral blood of anemic mice compared to +/+ animals (Fig. 8). DISCUSSION Anemic mice have increased numbers of circulating B and T lymphocytes, and we wished to determine if that lymphocytosis was due to an increased production of lymphocytes in marrow or thymus, an altered redistribution of lymphocytes from peripheral lymphoid tissue to the blood, or an increased proliferation of lymphocytes in lymph nodes or spleen. Pulse and chase and continuous labeling studies with [3H]TdR found that (a) the size of the lymphocyte populations and rate of lymphocyte production in bone marrow and thymus were comparable in anemic and +/+ animals, (b) the spleen showed increased cellularity with equivalent numbers of lymphocytes, but the majority of lymphocytes in anemic spleensdid not bear mature T and B cell markers, (c) lymph nodes of anemic animals were more cellular, with increased numbers of proliferating cells which were CD4+, CD8+, sIg+, and B220+, and (d) peripheral blood of anemic animals contained increased numbers of CD4+, CD8+, and sIg+ cells. The marrow is the usual site for B lymphopoiesis in the adult mouse (17, 18). Our kinetic studies and phenotypic analysis of marrow lymphocytes confirmed the production of lymphocytes in the marrow, but found no evidence for increased rate of cell production in the marrows of anemic mice. In fact, both the pulse and chase and continuous infusion labeling indicated a lower rate of cell turnover (Fig. 1) and a lower percentage mature and pre-B cells in marrows of anemic animals (Table 1) compared to those of +/+ control mice. The possibility cannot be excluded, however, that there could be more rapid maturation and exit of lymphocytes from anemic marrows to peripheral lymphoid tissue. Kinetic and phenotypic analysis of thymic tissue from anemic animals also did not suggestthat T cell production was increased in the thymus. Thymocytes from anemic animals did not have an increasedpercentage of the early precursor CD4- CDs- population (19), and the surface labeling pattern of thymocytes for the CD4 and CD8 molecule was similar in anemic and +/+ animals. The spleen of the anemic mouse is important for its survival (2) and necessarily becomes an extension of the bone marrow developing extensive extramedullary hematopoiesis (5). Absolute numbers of lymphocytes in spleens of anemic mice were similar to those of +/+ mice. However, the majority of those lymphocytes did not

14000 12000 "E

10000

1E

8000

3

6000

* * 1

l

T

*

T

4000 2000 0 CD4

CD8

slgM

FIG.8. Numbers of lymphocyte subsets in peripheral blood of anemic (I@ and +/+ (m) mice. Lymphocytes from anemic mice had significant increases in numbers of CD4+ (**P < O.OOl), CD8+ (*P < O.Ol), and sIgM+ (**P < 0.001) cells. Bars represent means -I- SD derived from five mice.

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bear mature T or B cell markers (Table 3). Since the anemic mouse must consistently maintain a high level of erythropoiesis, it is likely that some of these cells with lymphocyte morphology are hematopoietic stem cells, committed erythroid progenitors, or natural killer cells. Prior studies in this model ( 10) have shown a marked expansion of CPU-s (Day 8) and CFU-s (Day 12) in spleens of anemic mice. Our kinetic studies showed increased labeling in lymphocytes of anemic spleens consistent with increased turnover of these progenitor cells. It is unlikely that increased T and B cell production is occurring in the spleen since there were low numbers of mature T and B cells and no increase in B220+ cells. B220 is an isoform of the common leukocyte antigen CD45 and is expressed predominantly on “early” B cells (20). An absolute decrease in numbers of splenic T and B lymphocytes would also be consistent with redistribution of these T and B cells to peripheral blood. There were significant changes in the peripheral lymphocyte populations in nodes of anemic animals. Phenotype and continuous infusion labeling studies showed increased numbers of proliferating lymphocytes (Fig. 5) and an expansion of CD4+, CD8+, and particularly B cells in nodes of anemic animals. Some B cell development may also be occurring in nodes since there were increased numbers of B220+ cells. Our labeling index for lymph node lymphocytes in control animals was lower than other published reports in rodents (15, 16, 21). These lower levels of cycling node lymphocytes might be expected in our mice that are specific-pathogen-free (SPF), housed under barrier conditions, and fed autoclaved food and acidified water. Other above-cited studies were not described as being performed in SPF animals, and ubiquitous viruses like mouse hepatitis virus can alter lymphocyte populations (22) and immune function (23). Our findings showed lymphocyte changes in multiple lymphoid compartments in these mutant anemic mice with a chronic hemolytic anemia and the question is raised as to what is driving these lymphocyte population shifts. Increased proliferation of lymph node lymphocytes was the predominant finding, as well as probable redistribution of lymphocytes from the spleen to the blood. It is unlikely that hemolytic anemia alone is producing these lymphocyte changes, since in other murine models of inherited hemolytic anemia, such as a-thalassemia (24,25) and lactate dehydrogenase deficiency (26) these lymphocyte shifts have not been observed. On the other hand, experimental induction of hemolytic anemia by phenylhydrazine has been reported to result in lymphocyte changes in rats (27,28) and mice. Burrows showed a dramatic decrease in pre-B and sIgM+ cells in spleens and marrows of mice receiving shortterm phenylhydrazine treatment (29). Using a phenylhydrazine-induced hemolytic anemia to accelerate Abelson murine leukemia virus-induced lymphomas, Klinken incidentally noted a transient decrease in pre-B, mature B cells, CD4+, and CD8+ cells in spleen and marrow (30). We have also found that chronic administration ofphenylhydrazine to mice results in elevations in all blood lymphocyte subsets, increases in lymph node T and B cells, and other lymphocyte population shifts similar to the anemic mutant mouse (data not shown). Patients with sickle cell anemia have been reported to have increased peripheral blood lymphocytes (3 l-35). Altered cytokine production in response to the anemia and/or to increased uptake of red blood cell degradation products by macrophages could also be playing a role in the lymphoid changes noted in the mutant anemic mouse. In studies by Killar (36) the administration of rIL- 1 to mice resulted in lymphadenosis with lymphocyte population shifts that were remarkably similar to anemic mutant mice. Anemic mice have

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increased numbers of monocytes and macrophages in blood and lymph nodes (5), probably due to the persistent need for handling red cell and hemoglobin degradation products. Increased elaboration of IL- 1 by lymph node macrophagescould be resulting in increased T and B cell proliferation at that site. Indeed, T lymphocytes derived from nodes of anemic mice have enhanced proliferative capacity when activated with anti-CD3 mAb (work in progress), and we are currently determining if macrophages from anemic mice are responsible for increased in vitro proliferation of T cells in anemic animals. The anemic (sphha/sphha)mouse has a persistent anemia as well as persistent hemolysis. Some of the changes noted in this animal are compensatory and necessary for survival while others may be unexpected sequelae to continual hemolysis and chronic demands for hematopoietic stem cells. This mouse model offers an opportunity to study how a defect in the erythroid compartment can result in profound changes in lymphocyte populations and kinetics. ACKNOWLEDGMENTS We are grateful for the expert technical assistance of Miss Faith Shiota in performing these studies. This work was supported in part by NIH Grant RR00032.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26.

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