InrernarionaI Journalfor Primed
ParasirologyVol. 22, No. I, pp. 4347,
1992
in Great Britain 0
OWO-7519/92 SS.00 + 0.00 Pergamon Press p/c I992 Australian Sociery for Parasitology
A COMPARATIVE STUDY OF THE KINETIC CHANGES OF HEMOPOIETIC STEM CELLS IN MICE INFECTED WITH LETHAL AND NON-LETHAL MALARIA M. ASAMI, Department
of Parasitology,
T. ABE
M. OWHASHI, Miyazaki
and Y.
Medical College, Kiyotake,
NAWA* Miyazaki
889-16, Japan
(Received 12 June 199 1; accepted 23 August 199 1) Abstract-AsAhu M., OWHASHIM., ABE T. and NAWA Y. 1992. A comparative study of the kinetic changes of hemopoietic stem cells in mice infected with lethal and non-lethal malaria. International Journal for Parasitology 22: 4347. The kinetic changes of hemopoietic stem cells in bone marrow and spleen were compared between lethal Plasmodium berghei- and non-lethal P. yoelii 17x-infected mice. P. yoelii 17xinfected mice showed more severe splenomegaly than those infected with P. berghei. P. yoelii 17x-infected mice also showed a greater degree of sustained increase in number of multipotent hemopoietic stem cells (colony-forming units in spleen : CFU-S) and committed stem cells for granulocytes and macrophages (CFU-GM) and for erythrocytes (CFU-E) than P. berghei-infected mice. Such an increase was predominantly seen in the spleen of P. yoelii 17x-infected mice. In P. berghei-infected mice, the number of CFU-S, CFU-GM and also CFU-E only transiently increased and then decreased to a subnormal level at the late stage of infection. The proportion of cycling CFU-S was higher in P. berghei-infected mice than in P. yoelii 17x-infected mice. The IL-3 producing activity per spleen was much higher in P. yoelii 17x-infected than in P. berghei-infected mice at any point in time during the infection. Thus, hemopoietic changes seen after malaria infection seem to be closely related to the pathogenicity of the malaria parasite.
INDEX KEY WORDS: Plasmodium berghei; Plasmodium yoelii 17x; colony-forming units in spleen (CFUS); granulocyte-macrophage colony-forming units (CFU-GM); erythroid colony-forming units (CFU-E); cytosine arabinoside (Ara-C); interleukin-3
(CFU-S) (Silverman, Schooley & Mahlmann, 1987) or the progenitors for erythrocytes measured as erythroid burst-forming units (BFU-E) (Maggio-Price, Brookoff & Wiess, 1985) decreased in bone marrow of mice infected with the lethal malaria. Therefore, the pathogenicity of lethal and non-lethal malaria seems to be related to the hemopoietic changes during infection because altered hemopoiesis should be reflected in the condition of the immune system and of the target cells, namely erythrocytes. In the present study, therefore, we compared the kinetic changes of multipotent hemopoietic stem cells (CFU-S), committed stem cells for granulocytes and macrophages (CFU-GM) and for erythrocytes (CFU-E) in bone marrow and spleen of mice infected with lethal P. berghei and non-lethal P. yoelii 17x.
INTRODUCTION
yoelii 17x is a malaria parasite of rodents which produces a self-limiting infection in mice, whereas another rodent malaria P. berghei is invariably fatal. Such a difference is partly explained on the basis of the difference in host protective immunity induced by lethal and non-lethal Plasmodium infection in mice (Playfair, 1982). Lelchuk, Taverne, Agomo & Playfair (1979) reported that the increase in absolute values for lymphocytes, polymorphs and particularly for monocytes was higher in mice infected with non-lethal P. yoelii than in those infected with lethal P. berghei. Furthermore, blastogenic response of spleen cells to phytohemagglutinin(PHA) (Jayawardena, Targett, Leuchars, Carter, Doenhoff & Davies, 1975) or the capacity to produce interleukins (Lelchuk, Rose & Playfair, 1984) was higher in P. yoelii-infected than in P. berghei-infected mice. In addition to the defective immunogenicity, lethal malaria infection causes hemopoietic suppression. For example, the number of multipotent hemopoietic stem cells measured as colony-forming units in spleen Plasmodium
* To whom all correspondence
MATERIALS
AND METHODS
Mice and infection. Female C57BL/6 mice were raised in the animal center under clean conventional conditions and used at the age of 8-12 weeks. They were infected with P. berghei or P. yoelii I7x by intraperitoneal injection with 1 x lo6 parasitized erythrocytes. Preparation of cells. Mice were killed by an overdose of ether anesthesia. Bone marrow cells were obtained by
should be addressed. 43
44
M. ASAMI,M. OWHASHI,T. ABEand Y. NAWA
flushing the marrow cavity of femurs from infected or normal mice with Hank’s balanced salt solution (HBSS) containing 10% fetal bovine serum (FBS; GIBCO, Grand Island, NY, U.S.A.). The spleen was gently squashed between two frostended slides in alpha-MEM (GIBCO). Erythrocytes were removed by isotonic lysis with a 0.83% ammonium chloride solution. Spleen colony assay. To examine the number of CFU-S, a spleen colony assay (Till & McCulloch, 1961) was performed. Recipient mice (four mice per sample) were exposed to 9.5 Gy from an X-ray source (Hitachi MBR-ISOSR, Japan), and mice were intravenously injected with 5 x 10’ bone marrow or 1 x lo6 spleen cells from donor mice. Recipient mice were killed 7 days after irradiation and cell transfer. The spleen was placed in Carnoy’s fluid overnight and the number of visible colonies (CFU-S) was counted with the aid of a dissecting microscope. All irradiated recipient mice were given chloroquine (200 mg 1-l) in drinking water. The number of CFU-S in total bone marrow was calculated according to Boggs’ finding (1985) that the femurs in the mouse accounted for 5.7% of the total marrow. In vitro erythroid colony assay. The culture procedure was based on the viscous methylcellulose method described by Iscove & Sieber (1975). Briefly, 2 x IO’ bone marrow cells or 2.5 x 10’ spleen cells were plated in a 35 mm plastic dish (Falcon 1008, Becton Dickinson Labware, Oxnard, CA) in 1 ml of a mixture containing alpha-MEM without nucleosides (GIBCO), 0.8% methylcellulose (Nakalai Tesque, Kyoto, Japan), 30% FBS (Flow Laboratories Inc., Mclean, VA, U.S.A.), 10m4 M-2-mercaptoethanol and 2.0 units of recombinant erythropoietin (kindly provided by Chugai Pharmaceutical Co., Japan). The dishes were incubated at 37’C in a humidified atmosphere with 5% CO,-air. Colonies were counted on day 2 for CFU-E. All experiments were performed in quadruplicate and repeated at least twice. In vitro granulocyte-macrophage colony assay. Details of techniques for colony formation in soft agar have been described previously (Owhashi & Nawa, 1985). Briefly, 1 x 10’ bone marrow cells or 1 x lo6 spleen cells were plated in plastic dishes (diameter, 35 mm; Falcon 1008) in 1 ml of a mixture containing McCoy 5A medium (GIBCO), 0.3% agar (Noble agar; Difco Laboratories, Detroit, MI), 10% FBS (Flow Laboratories), and 200 ~1 L929-cell culture supernatant. The dishes were incubated in a humidified atmosphere with 7% CO,air. Colonies were counted on day 7 of culture. Cyfosine arabinoside (Ara-C) treatment. Proliferation activity of CFU-S was assayed as the percentage of CFU-S killed by Ara-C (Cork, Riches & Wright, 1986). Bone marrow or spleen cells were incubated for 1 hat 37°C with or without Ara-C (40 pg ml-‘) in RPM1 1640 (GIBCO), and then washed three times with HBSS. Aliquots of each cell suspension were injected into recipient mice and CFU-S was determined as described above. Assay of IL-3 activity. Spleen cells were obtained on a number of days after infection. They were suspended in RPM1 1640 containing 10% FBS at a cell density of 2 x lo6 cells ml-’ and cultured without stimuli at 37’C for 24 h in 5% CO,. The supernatants were obtained by centrifugation and assayed for IL-3 activity. IL-3 activity was measured by ‘H-
thymidine uptake in FDC-P2 cells (Abe & Nawa, 1987). Briefly, 2 x IO’ FDC-P2 cells suspended in 0.2 ml of the culture medium were incubated in triplicate with 0.02 ml of sample supernatants for 24 h at 37°C in 5% COiair. In the last 6 h of incubation, 18.5 kBq of [‘H]TdR (New England Nuclear, Boston, MA) in 5 ~1 medium was added to the culture. The cells were harvested on glass filter paper and [3H]TdR uptake was measured in a liquid scintillation counter. One unit of IL-3 was defined as the amount of factor that stimulated 50% maximum [3H]TdR uptake (Ihle, Keller, Henderson, Klein & Palaszynski, 1982). RESULTS After infection with lethal P. berghei, C57BL/6 mice showed a logarithmic increase of parasitemia up to 80% and all mice died at around day 21. However, non-lethal P. yoelii 17x-infection was self-limiting
with a maximum parasitemia of 4&50% on day 24, and then parasitized red cells were rapidly cleared from the blood by day 32 (Fig. 1A). Splenomegaly became noticeable on day 4 in both infections. The total spleen cell number of P. berghei-infected mice reached a peak on day 8 (8.9-fold increase from uninfected level) whereas that of P. yoelii 17x-infected mice reached a peak (19. l-fold) on day 20 (Fig. 1B).
B 30 =cn 8 5 20 2 B 6 10 P
t
IL!zzrL
OO
10
Days after
20
30
infection
FIG. 1. Kinetic changes of parasitemia (A) and number of spleen cells (B) in mice infected with P. berghei (0) or P. yoelii 17.x(0). Each point represents the mean f SD. of five mice.
To examine the difference of hemopoietic changes between lethal P.berghei and non-lethal P. yoelii 17x infection in mice, the numbers of CFU-S, CFU-GM and CFU-E in bone marrow and spleen were examined at various days after infection.
Hemopoietic stem cell changes in murine malaria
45
TABLE I-EFFECT OF Au-C TREATMENTON THE VIABILITYOF CFU-S FROM BONE MARROWOR SPLEENCELLSOFNORMAL,P. berghei- ORP. yoeili 17x-INFECTEDMICE
Ara-C treatment* Cell source
Mouse
Bone marrow
cant P.b Id7 P.Y IdI cant P.b 16d P.y 16d cant P.b Id P.y Id cant P.b 16d P.y 16d
Spleen
+
59 57 14 68 59 62 59 55 62 62 31 69
f f f f f f f f f f f f
1.3 2.3 2.7 1.8 0.9 3.0 2.0 1.2 3.6 4.0 3.0 9.0
52 f 42 f 60 f 6Of 18 f 35 f 52 f 43 f 45 f 55 f 12 f 37 f
Reduction(%) 1.3 2.0 5.1 1.9 1.4 5.4 1.6 1.8 6.0 1.2 2.2 6.4
12 21 18 12 69 44 13 34 29 12 62 41
* Proliferation activity of CFU-S was assayed as the percentage CFU-S killed by Ara-C (40 pg ml-‘). Mean f S.D.of four mice. t Seven days after P. berghei infection. $ Seven days after P. yoelii 17x infection.
In bone marrow of P. berghei-infected mice, the number of CFU-S increased only 1.6-fold over the normal level on day 6 and then rapidly decreased to l/ 10 of the normal level on day 20 (Fig. 2A). In bone marrow of P. yoelii 17x-infected mice, the number of CFU-S gradually increased and reached a peak on day 20 (2.8-fold) and then decreased to the normal level. In spleen of P. berghei-infected mice, the number of CFU-S increased up to 14.3-fold on day 10, whereas that in spleen of P. yoelii 17X-infected mice increased up to 47.2-fold on day 20 (Fig. 2B). In uninfected mice, the proportion of CFU-S in spleen and in bone marrow was about 1:4 (Fig. 2A, B). Thus, the increase in number of CFU-S in both malarial infections mainly occurred in spleen, especially in the case of P. yoelii 17x infection. r^ 0
30
B
A
5 u! ;
B
5
s 1
20
A
7
o5
8
$
s
z ii ; f :
4
10
B
2
li?!
0L!!!X& 0
I----
0 I?!55 0 10
To determine the proportion of cycling CFU-S, bone marrow and spleen cells of P. berghei- or P. yoelii 17x-infected mice were treated with Ara-C and were assayed for the number of remaining CFU-S (Table 1). The proportion of cycling CFU-S in bone marrow cells of P. berghei-infected mice was about 69% whereas that of P. yoelii 17x-infected mice was about 44% on day 16. In normal mice, only 12% of CFU-S in bone marrow cells were Ara-C sensitive. The proportions of Ara-C sensitive CFU-S in the spleen were essentially similar to those in bone marrow regardless of whether before or after infection.
_I
102030
0
10
20
30
Days after infection
FIG. 3. Changes in number of CFU-GM in total bone marrow (A) or total spleen(B) during P.berghei(O) or P. yoelii 17x (0) infection. Each point represents the mean f S.D.of five dishes.
20
30
0
10
20
30
Days after infection
FIG. 2. Changes in number of CFU-S in total bone marrow (A) or total spleen (B) during P. berg& (0) or P. yoelii 17x (0) infection. Each point represents the mean f S.D.of four mice.
Figure 3 shows the kinetics of CFU-GM in total bone marrow or the spleen of mice after infection with lethal and non-lethal malaria. In P. berghei infection, the number of CFU-GM in bone marrow increased to 15fold on day 6 and then decreased to a subnormal level. In P. yoelii 17x-infected mice, the number of
ASAMI,
M.
46
M.
OWHASHI, T.
CFU-GM in bone marrow increased up to 5.4 fold on day 21 and then decreased with time. In normal adult mice, only about 1% of the total number of CFU-GM resided in the spleen. In P. ~e~g~e~-infested mice, the number of CFU-GM in the spleen slightly increased. However, the number of CFU-GM in the spleen of P. yoelii 17x-infected mice markedly increased by up to 580-fold of the normal level on day 20 and exceeded the number of CFU-GM in total bone marrow. B i
1.0;”
W ’
100
iz ij
50
t
E 2
II
Oo
102030
10
20 30
Days after infection
FIG.4. Changes in number of CFU-E in total bone marrow (A) or total spleen (B) during P. berghei (a) or P. yoelii 17x (0) infection. Each point represents the mean f S.D.of four dishes.
In normal adult mice, the spleen is an important erythropoietic organ and contained about 40% of bone marrow CFU-E (Fig. 4). In both malarial infections, the increase in number of CFU-E was seen mainly in the spleen, though those in bone marrow also slightly increased. The number of splenic CFU-E of P. berghei-infected mice reached a peak on day 10 (57-fold), whereas that of P. yoelii 17x-infected mice reached a peak on day 14 (430-fold).
Oo 1
10
20
30
’
01 0
10
20
30
1
Days after infection
FIG. 5. Time course of IL-3 production during malaria infection. Spleen cells of P. berghei (O)- or P. yoelii 17x(O)infected mice were examined for IL-3 producing activity. One unit of IL-3 activity is defined as the amount of IL-3 required per milliliter to induce 50% of ma~mum proliferation of the FDC-P2 cell line. Results were expressed as the mean f S.D. of total IL-3 units produced by IO6spkn cells (A, x 10’ units) and by the total spleen (B, x IO”units).
ABE and Y. NAWA
IL-3 producing
activities of the spleen cells from
P. berghei- or P. yoelii 17x-infected mice were ex-
amined. The IL-3 producing activity per determined number (1 06) of spleen cells from P. berghei-infected mice reached a peak on day 3 and then returned to the normal level (Fig. SA). Based on the unit number of cells, the IL-3 producing activity of the spleen cells from non-lethal P. yoelii 17x-infected mice was similar to that of the spleen cells from P. berghei-infected mice until day 18 and slightly exceeded the normal level on day 30 (Fig. SA). Since the degree of splenomegaly was markedly different between lethal and non-lethal infections, these results were converted into IL-3 producing activity per spleen (Fig. 5B). Obviously the IL-3 producing activity per spleen was much higher in the P. yoelii 17x- than in the P. berghei-infected mice at any point in time during infection. DISCUSSION The results reported here show that the virulence of murine malaria was closely related to the proliferation and differentiation of CFU-S and also of committed stem cells (CFU-GM and CFU-E) in bone marrow and, especially, in the spleen of the host. Splenomegaly with macrophage hyperplasia is a prominent feature of malaria (Taliaferro & Mullligan, 1937). The spleen is known to play an important role in the immunological defense of the host against malaria infection (Wyler, 1983). The present results show that the spleen is important as the hemopoietic organ responding to the acute demand for hemopoiesis during malarial infection. Non-lethal P. yoelii 17x-infected mice showed more severe splenomegaly than lethal P. berghciinfected mice. In P. yoefii 17x-infected mice, splenomegaly was reduced in accordance with the decline of hemopoiesis (Figs. lB, 2-4). In our recent study (submitted for publication), CFU-S-deficient W/W” mice did not elicit splenomegaly 7 days after P. berghei infection. Thus, splenomegaly is closely related to the increase of hemopoietic stem cells. In normal uninfected mice, the proportion of Ara-C sensitive CFU-S was only about 1213% of the total number of CFU-S in bone marrow and also in the spleen. After infection with P. berghei, however, those in the spleen and bone marrow increased up to 6269%. In the case of P. yoelii 17.x infection, the proportion of cycling CFU-S in bone marrow and in the spleen was 4447% (Table 1). Such an increase in the proportion of cycling CFU-S was seen in mice after irradiation (Lajtha, Gilbert & Guzman, 1971), and especially when mice were irradiated with the lethal dose, the proportion of cycling CFU-S reached over 60% (Becker, McCulloch, Siminovitch & Till, 1965). Thus, the extremely high proportion of cycling CFU-S may indicate that the increased demand for bIood cells
Hemopoietic stem cell changes in murine malaria
exceeds the capacity of blood cell production. IL-3, the multi-lineage hemopoietic cell growth factor, is a lymphokine having an effect on the proliferation and the differentiation of multipotent hemopoietic stem cells and also committed progenitor cells in vivo (Metcalf, Begley, Johnson, Nicola, Lopez & Williamson, 1986) and in vitro (Spivak, Smith & Ihle, 1985). We could detect only transient increase in IL-3 producing activity of the spleen cells in the early stage of infection in two strains of murine malaria when it was measured and calculated on the unit number of cells (Fig. 5A). However, when the IL-3 producing activity was converted to per spleen, it was much higher in P. yoelii 17x- than in P. bergheiinfected mice (Fig. 5B). Obviously such a difference is directly related to the absolute number and function of T cells in the spleen. Related to this, Gross, Geva & Frankenburg (1988) reported that splenic T cetls of P. berghei-infected mice increased in number but decreased in percentage. Our results indicate that one of the important causes of the difference in hemopoiesis in lethal and non-lethal murine malarial infection is the difference in hemopoietic factor production in both infections. In conclusion, the difference in the hemopoietic proliferative responses of the host after lethal and nonlethal murine malaria infection is one of the critical factors determining the virulence of malarial parasites. The actual mechanism of the disturbance of the accumulation and differentiation of CFU-S in lethal P. berghei infection should be clarified in future investigations. thank MS E. Ohno and A. Tanaka for their excellent technical assistance
Acknowledgements-We
REFERENCES ABE T. & NAWAY. 1987. Localization of mucosal mast cells in W/W mice after reconstitution with bone marrow cells or cultured mast cells, and its relation to the protective capacity to Strongyloides ratti infection. Parasite Immunology 9:477-48X
BECKERA.J., MCCULL~CHE.A., SIMINOVIXH L. & TILLJ.E. 1965. The effect of differing demands for blood cell production on DNA synthesis by hemopoietic colonyforming cells of mice. Blood Z&296-308. BOGGSD.R. 1985. Hematopoiesis and aging: IV. Mass and distribution of erythroid marrow in aged mice. Experimental Hemaiology 13: 10441047. CORK M.J., RICHESA.C. & WRIGHTE.G. 1986. A stimulator of murine haemopoietie stem cell proliferation produced by human fetal liver cells. British Journal of~aemarology 63:775-783.
47
Gross A., GEVAS. & FRANKENBURG S. 1988. Pl~mod~um berghei: lymphocyte and macrophage dynamics in the
spleen of Balb/c mice in the course of infection and after rechallenge of cured mice. Experimental Parasitology 6!WO-60.
KELLERJ., HENDERSONL., KLEINF. & PALASZYNZKI E. 1982. Procedures for the purification of interleukin 3 to homogeneity. Journal of Immunology 129243 l-2436. ISCOVEN.N. & S~EBERF. 1975. Erythroid progenitors in mouse bone marrow detected by macroscopic colony formation in culture. Experimental Hematology Z32-43. JAYAWARDENA A.N., TARGETS G.A.T., LEUCHAR~E., CARTER R.L., DOENHOFF M.J. & DAVIES A.J.S. 1975. T-cell activation in murine malaria. Nature (London) 258: 149-I 5 1. LAJTHA L.G., GILBERT C.W. & GUZMAN E. 1971. Kinetics of haemopoietic colony growth. British Journal of HaematoIHLE J.N.,
iogy 2&343-354. LELCHUK R., TAVERNE J., AG~MO P.U. & PLAYFAIR J.H.L. 1979. ~velopment and suppression of a ~p~ation of late-adhering macrophages in mouse malaria Parasite Immunology 1: 61-78. LELCHIJK R., ROSE G. & PLAYFAIRJ.H.L. 1984. Changes in the capacity of macrophages and T cells to produce interleukins during murine malaria infection. Cellular Immunology 84:253-263. MAGGIO-PRICE L., BROOKOFFD. &WEISS L. 1985. Changes in hematopoietic stem cells in bone marrow of mice with Plasmodium berghei malaria. Blood 66: 1080-1085. METCALF D., BEG~EY C.G., JOHNSON G.R., NICOLA N.A., LOPEZ A.F. & WILLIAMSOND.J. 1986. Effects of purified
bacterially synthesized murine multi-CSF (IL-3) on hematopoiesis in normal adult mice. Blood 68:4657. OWHASHI M. & NAWA Y. 1985. Granulocyte-macrophage colony-stimulating factor in the sera of Schistosoma japonicum-infected mice. Infection and Immunity 49:.533537. PLAYFAIRJ.H.L. 1982. Immunity to malaria. British Medical
Bullei~n~l53-159. SILVERMANP.H., SCHOOLEYJ.C. & MAHLMANN L.J. 1987. Murine malaria decreases hemopoietic stem cells. Blood 69:408413. SPIVAKJ.L., SMITH R.R.L. & IHLE J.N. 1985. Interleukin 3 promotes the in vitro proliferation of murine pluripotent hematopoietic stem cells. Journal of Clinical Investigation 76:1613-1621. TALIAFERRO W.H. & MULLIGAN H.W. 1937. The histopathology of malaria with special reference to the function and origin of the macrophages in defence. Indian Medical Research Memoirs 29: l-138. TILL J.E. & MCCLL~CH E.A. 1961. A direct measurement of the radiation sensitivity of normal mouse bone marrow cells. Radiation Research 14~213-222. WYLER D.J. 1983. The spleen in malaria. In: Malaria and the Red Cell (Ciba Foundation Symposium 94) (Edited by EVERKI D. & WHELAN J.), pp. 98-l 16. Pitman, London.
ParasirologyVol. 22, No. I, pp. 4347,
1992
in Great Britain 0
OWO-7519/92 SS.00 + 0.00 Pergamon Press p/c I992 Australian Sociery for Parasitology
A COMPARATIVE STUDY OF THE KINETIC CHANGES OF HEMOPOIETIC STEM CELLS IN MICE INFECTED WITH LETHAL AND NON-LETHAL MALARIA M. ASAMI, Department
of Parasitology,
T. ABE
M. OWHASHI, Miyazaki
and Y.
Medical College, Kiyotake,
NAWA* Miyazaki
889-16, Japan
(Received 12 June 199 1; accepted 23 August 199 1) Abstract-AsAhu M., OWHASHIM., ABE T. and NAWA Y. 1992. A comparative study of the kinetic changes of hemopoietic stem cells in mice infected with lethal and non-lethal malaria. International Journal for Parasitology 22: 4347. The kinetic changes of hemopoietic stem cells in bone marrow and spleen were compared between lethal Plasmodium berghei- and non-lethal P. yoelii 17x-infected mice. P. yoelii 17xinfected mice showed more severe splenomegaly than those infected with P. berghei. P. yoelii 17x-infected mice also showed a greater degree of sustained increase in number of multipotent hemopoietic stem cells (colony-forming units in spleen : CFU-S) and committed stem cells for granulocytes and macrophages (CFU-GM) and for erythrocytes (CFU-E) than P. berghei-infected mice. Such an increase was predominantly seen in the spleen of P. yoelii 17x-infected mice. In P. berghei-infected mice, the number of CFU-S, CFU-GM and also CFU-E only transiently increased and then decreased to a subnormal level at the late stage of infection. The proportion of cycling CFU-S was higher in P. berghei-infected mice than in P. yoelii 17x-infected mice. The IL-3 producing activity per spleen was much higher in P. yoelii 17x-infected than in P. berghei-infected mice at any point in time during the infection. Thus, hemopoietic changes seen after malaria infection seem to be closely related to the pathogenicity of the malaria parasite.
INDEX KEY WORDS: Plasmodium berghei; Plasmodium yoelii 17x; colony-forming units in spleen (CFUS); granulocyte-macrophage colony-forming units (CFU-GM); erythroid colony-forming units (CFU-E); cytosine arabinoside (Ara-C); interleukin-3
(CFU-S) (Silverman, Schooley & Mahlmann, 1987) or the progenitors for erythrocytes measured as erythroid burst-forming units (BFU-E) (Maggio-Price, Brookoff & Wiess, 1985) decreased in bone marrow of mice infected with the lethal malaria. Therefore, the pathogenicity of lethal and non-lethal malaria seems to be related to the hemopoietic changes during infection because altered hemopoiesis should be reflected in the condition of the immune system and of the target cells, namely erythrocytes. In the present study, therefore, we compared the kinetic changes of multipotent hemopoietic stem cells (CFU-S), committed stem cells for granulocytes and macrophages (CFU-GM) and for erythrocytes (CFU-E) in bone marrow and spleen of mice infected with lethal P. berghei and non-lethal P. yoelii 17x.
INTRODUCTION
yoelii 17x is a malaria parasite of rodents which produces a self-limiting infection in mice, whereas another rodent malaria P. berghei is invariably fatal. Such a difference is partly explained on the basis of the difference in host protective immunity induced by lethal and non-lethal Plasmodium infection in mice (Playfair, 1982). Lelchuk, Taverne, Agomo & Playfair (1979) reported that the increase in absolute values for lymphocytes, polymorphs and particularly for monocytes was higher in mice infected with non-lethal P. yoelii than in those infected with lethal P. berghei. Furthermore, blastogenic response of spleen cells to phytohemagglutinin(PHA) (Jayawardena, Targett, Leuchars, Carter, Doenhoff & Davies, 1975) or the capacity to produce interleukins (Lelchuk, Rose & Playfair, 1984) was higher in P. yoelii-infected than in P. berghei-infected mice. In addition to the defective immunogenicity, lethal malaria infection causes hemopoietic suppression. For example, the number of multipotent hemopoietic stem cells measured as colony-forming units in spleen Plasmodium
* To whom all correspondence
MATERIALS
AND METHODS
Mice and infection. Female C57BL/6 mice were raised in the animal center under clean conventional conditions and used at the age of 8-12 weeks. They were infected with P. berghei or P. yoelii I7x by intraperitoneal injection with 1 x lo6 parasitized erythrocytes. Preparation of cells. Mice were killed by an overdose of ether anesthesia. Bone marrow cells were obtained by
should be addressed. 43
44
M. ASAMI,M. OWHASHI,T. ABEand Y. NAWA
flushing the marrow cavity of femurs from infected or normal mice with Hank’s balanced salt solution (HBSS) containing 10% fetal bovine serum (FBS; GIBCO, Grand Island, NY, U.S.A.). The spleen was gently squashed between two frostended slides in alpha-MEM (GIBCO). Erythrocytes were removed by isotonic lysis with a 0.83% ammonium chloride solution. Spleen colony assay. To examine the number of CFU-S, a spleen colony assay (Till & McCulloch, 1961) was performed. Recipient mice (four mice per sample) were exposed to 9.5 Gy from an X-ray source (Hitachi MBR-ISOSR, Japan), and mice were intravenously injected with 5 x 10’ bone marrow or 1 x lo6 spleen cells from donor mice. Recipient mice were killed 7 days after irradiation and cell transfer. The spleen was placed in Carnoy’s fluid overnight and the number of visible colonies (CFU-S) was counted with the aid of a dissecting microscope. All irradiated recipient mice were given chloroquine (200 mg 1-l) in drinking water. The number of CFU-S in total bone marrow was calculated according to Boggs’ finding (1985) that the femurs in the mouse accounted for 5.7% of the total marrow. In vitro erythroid colony assay. The culture procedure was based on the viscous methylcellulose method described by Iscove & Sieber (1975). Briefly, 2 x IO’ bone marrow cells or 2.5 x 10’ spleen cells were plated in a 35 mm plastic dish (Falcon 1008, Becton Dickinson Labware, Oxnard, CA) in 1 ml of a mixture containing alpha-MEM without nucleosides (GIBCO), 0.8% methylcellulose (Nakalai Tesque, Kyoto, Japan), 30% FBS (Flow Laboratories Inc., Mclean, VA, U.S.A.), 10m4 M-2-mercaptoethanol and 2.0 units of recombinant erythropoietin (kindly provided by Chugai Pharmaceutical Co., Japan). The dishes were incubated at 37’C in a humidified atmosphere with 5% CO,-air. Colonies were counted on day 2 for CFU-E. All experiments were performed in quadruplicate and repeated at least twice. In vitro granulocyte-macrophage colony assay. Details of techniques for colony formation in soft agar have been described previously (Owhashi & Nawa, 1985). Briefly, 1 x 10’ bone marrow cells or 1 x lo6 spleen cells were plated in plastic dishes (diameter, 35 mm; Falcon 1008) in 1 ml of a mixture containing McCoy 5A medium (GIBCO), 0.3% agar (Noble agar; Difco Laboratories, Detroit, MI), 10% FBS (Flow Laboratories), and 200 ~1 L929-cell culture supernatant. The dishes were incubated in a humidified atmosphere with 7% CO,air. Colonies were counted on day 7 of culture. Cyfosine arabinoside (Ara-C) treatment. Proliferation activity of CFU-S was assayed as the percentage of CFU-S killed by Ara-C (Cork, Riches & Wright, 1986). Bone marrow or spleen cells were incubated for 1 hat 37°C with or without Ara-C (40 pg ml-‘) in RPM1 1640 (GIBCO), and then washed three times with HBSS. Aliquots of each cell suspension were injected into recipient mice and CFU-S was determined as described above. Assay of IL-3 activity. Spleen cells were obtained on a number of days after infection. They were suspended in RPM1 1640 containing 10% FBS at a cell density of 2 x lo6 cells ml-’ and cultured without stimuli at 37’C for 24 h in 5% CO,. The supernatants were obtained by centrifugation and assayed for IL-3 activity. IL-3 activity was measured by ‘H-
thymidine uptake in FDC-P2 cells (Abe & Nawa, 1987). Briefly, 2 x IO’ FDC-P2 cells suspended in 0.2 ml of the culture medium were incubated in triplicate with 0.02 ml of sample supernatants for 24 h at 37°C in 5% COiair. In the last 6 h of incubation, 18.5 kBq of [‘H]TdR (New England Nuclear, Boston, MA) in 5 ~1 medium was added to the culture. The cells were harvested on glass filter paper and [3H]TdR uptake was measured in a liquid scintillation counter. One unit of IL-3 was defined as the amount of factor that stimulated 50% maximum [3H]TdR uptake (Ihle, Keller, Henderson, Klein & Palaszynski, 1982). RESULTS After infection with lethal P. berghei, C57BL/6 mice showed a logarithmic increase of parasitemia up to 80% and all mice died at around day 21. However, non-lethal P. yoelii 17x-infection was self-limiting
with a maximum parasitemia of 4&50% on day 24, and then parasitized red cells were rapidly cleared from the blood by day 32 (Fig. 1A). Splenomegaly became noticeable on day 4 in both infections. The total spleen cell number of P. berghei-infected mice reached a peak on day 8 (8.9-fold increase from uninfected level) whereas that of P. yoelii 17x-infected mice reached a peak (19. l-fold) on day 20 (Fig. 1B).
B 30 =cn 8 5 20 2 B 6 10 P
t
IL!zzrL
OO
10
Days after
20
30
infection
FIG. 1. Kinetic changes of parasitemia (A) and number of spleen cells (B) in mice infected with P. berghei (0) or P. yoelii 17.x(0). Each point represents the mean f SD. of five mice.
To examine the difference of hemopoietic changes between lethal P.berghei and non-lethal P. yoelii 17x infection in mice, the numbers of CFU-S, CFU-GM and CFU-E in bone marrow and spleen were examined at various days after infection.
Hemopoietic stem cell changes in murine malaria
45
TABLE I-EFFECT OF Au-C TREATMENTON THE VIABILITYOF CFU-S FROM BONE MARROWOR SPLEENCELLSOFNORMAL,P. berghei- ORP. yoeili 17x-INFECTEDMICE
Ara-C treatment* Cell source
Mouse
Bone marrow
cant P.b Id7 P.Y IdI cant P.b 16d P.y 16d cant P.b Id P.y Id cant P.b 16d P.y 16d
Spleen
+
59 57 14 68 59 62 59 55 62 62 31 69
f f f f f f f f f f f f
1.3 2.3 2.7 1.8 0.9 3.0 2.0 1.2 3.6 4.0 3.0 9.0
52 f 42 f 60 f 6Of 18 f 35 f 52 f 43 f 45 f 55 f 12 f 37 f
Reduction(%) 1.3 2.0 5.1 1.9 1.4 5.4 1.6 1.8 6.0 1.2 2.2 6.4
12 21 18 12 69 44 13 34 29 12 62 41
* Proliferation activity of CFU-S was assayed as the percentage CFU-S killed by Ara-C (40 pg ml-‘). Mean f S.D.of four mice. t Seven days after P. berghei infection. $ Seven days after P. yoelii 17x infection.
In bone marrow of P. berghei-infected mice, the number of CFU-S increased only 1.6-fold over the normal level on day 6 and then rapidly decreased to l/ 10 of the normal level on day 20 (Fig. 2A). In bone marrow of P. yoelii 17x-infected mice, the number of CFU-S gradually increased and reached a peak on day 20 (2.8-fold) and then decreased to the normal level. In spleen of P. berghei-infected mice, the number of CFU-S increased up to 14.3-fold on day 10, whereas that in spleen of P. yoelii 17X-infected mice increased up to 47.2-fold on day 20 (Fig. 2B). In uninfected mice, the proportion of CFU-S in spleen and in bone marrow was about 1:4 (Fig. 2A, B). Thus, the increase in number of CFU-S in both malarial infections mainly occurred in spleen, especially in the case of P. yoelii 17x infection. r^ 0
30
B
A
5 u! ;
B
5
s 1
20
A
7
o5
8
$
s
z ii ; f :
4
10
B
2
li?!
0L!!!X& 0
I----
0 I?!55 0 10
To determine the proportion of cycling CFU-S, bone marrow and spleen cells of P. berghei- or P. yoelii 17x-infected mice were treated with Ara-C and were assayed for the number of remaining CFU-S (Table 1). The proportion of cycling CFU-S in bone marrow cells of P. berghei-infected mice was about 69% whereas that of P. yoelii 17x-infected mice was about 44% on day 16. In normal mice, only 12% of CFU-S in bone marrow cells were Ara-C sensitive. The proportions of Ara-C sensitive CFU-S in the spleen were essentially similar to those in bone marrow regardless of whether before or after infection.
_I
102030
0
10
20
30
Days after infection
FIG. 3. Changes in number of CFU-GM in total bone marrow (A) or total spleen(B) during P.berghei(O) or P. yoelii 17x (0) infection. Each point represents the mean f S.D.of five dishes.
20
30
0
10
20
30
Days after infection
FIG. 2. Changes in number of CFU-S in total bone marrow (A) or total spleen (B) during P. berg& (0) or P. yoelii 17x (0) infection. Each point represents the mean f S.D.of four mice.
Figure 3 shows the kinetics of CFU-GM in total bone marrow or the spleen of mice after infection with lethal and non-lethal malaria. In P. berghei infection, the number of CFU-GM in bone marrow increased to 15fold on day 6 and then decreased to a subnormal level. In P. yoelii 17x-infected mice, the number of
ASAMI,
M.
46
M.
OWHASHI, T.
CFU-GM in bone marrow increased up to 5.4 fold on day 21 and then decreased with time. In normal adult mice, only about 1% of the total number of CFU-GM resided in the spleen. In P. ~e~g~e~-infested mice, the number of CFU-GM in the spleen slightly increased. However, the number of CFU-GM in the spleen of P. yoelii 17x-infected mice markedly increased by up to 580-fold of the normal level on day 20 and exceeded the number of CFU-GM in total bone marrow. B i
1.0;”
W ’
100
iz ij
50
t
E 2
II
Oo
102030
10
20 30
Days after infection
FIG.4. Changes in number of CFU-E in total bone marrow (A) or total spleen (B) during P. berghei (a) or P. yoelii 17x (0) infection. Each point represents the mean f S.D.of four dishes.
In normal adult mice, the spleen is an important erythropoietic organ and contained about 40% of bone marrow CFU-E (Fig. 4). In both malarial infections, the increase in number of CFU-E was seen mainly in the spleen, though those in bone marrow also slightly increased. The number of splenic CFU-E of P. berghei-infected mice reached a peak on day 10 (57-fold), whereas that of P. yoelii 17x-infected mice reached a peak on day 14 (430-fold).
Oo 1
10
20
30
’
01 0
10
20
30
1
Days after infection
FIG. 5. Time course of IL-3 production during malaria infection. Spleen cells of P. berghei (O)- or P. yoelii 17x(O)infected mice were examined for IL-3 producing activity. One unit of IL-3 activity is defined as the amount of IL-3 required per milliliter to induce 50% of ma~mum proliferation of the FDC-P2 cell line. Results were expressed as the mean f S.D. of total IL-3 units produced by IO6spkn cells (A, x 10’ units) and by the total spleen (B, x IO”units).
ABE and Y. NAWA
IL-3 producing
activities of the spleen cells from
P. berghei- or P. yoelii 17x-infected mice were ex-
amined. The IL-3 producing activity per determined number (1 06) of spleen cells from P. berghei-infected mice reached a peak on day 3 and then returned to the normal level (Fig. SA). Based on the unit number of cells, the IL-3 producing activity of the spleen cells from non-lethal P. yoelii 17x-infected mice was similar to that of the spleen cells from P. berghei-infected mice until day 18 and slightly exceeded the normal level on day 30 (Fig. SA). Since the degree of splenomegaly was markedly different between lethal and non-lethal infections, these results were converted into IL-3 producing activity per spleen (Fig. 5B). Obviously the IL-3 producing activity per spleen was much higher in the P. yoelii 17x- than in the P. berghei-infected mice at any point in time during infection. DISCUSSION The results reported here show that the virulence of murine malaria was closely related to the proliferation and differentiation of CFU-S and also of committed stem cells (CFU-GM and CFU-E) in bone marrow and, especially, in the spleen of the host. Splenomegaly with macrophage hyperplasia is a prominent feature of malaria (Taliaferro & Mullligan, 1937). The spleen is known to play an important role in the immunological defense of the host against malaria infection (Wyler, 1983). The present results show that the spleen is important as the hemopoietic organ responding to the acute demand for hemopoiesis during malarial infection. Non-lethal P. yoelii 17x-infected mice showed more severe splenomegaly than lethal P. berghciinfected mice. In P. yoefii 17x-infected mice, splenomegaly was reduced in accordance with the decline of hemopoiesis (Figs. lB, 2-4). In our recent study (submitted for publication), CFU-S-deficient W/W” mice did not elicit splenomegaly 7 days after P. berghei infection. Thus, splenomegaly is closely related to the increase of hemopoietic stem cells. In normal uninfected mice, the proportion of Ara-C sensitive CFU-S was only about 1213% of the total number of CFU-S in bone marrow and also in the spleen. After infection with P. berghei, however, those in the spleen and bone marrow increased up to 6269%. In the case of P. yoelii 17.x infection, the proportion of cycling CFU-S in bone marrow and in the spleen was 4447% (Table 1). Such an increase in the proportion of cycling CFU-S was seen in mice after irradiation (Lajtha, Gilbert & Guzman, 1971), and especially when mice were irradiated with the lethal dose, the proportion of cycling CFU-S reached over 60% (Becker, McCulloch, Siminovitch & Till, 1965). Thus, the extremely high proportion of cycling CFU-S may indicate that the increased demand for bIood cells
Hemopoietic stem cell changes in murine malaria
exceeds the capacity of blood cell production. IL-3, the multi-lineage hemopoietic cell growth factor, is a lymphokine having an effect on the proliferation and the differentiation of multipotent hemopoietic stem cells and also committed progenitor cells in vivo (Metcalf, Begley, Johnson, Nicola, Lopez & Williamson, 1986) and in vitro (Spivak, Smith & Ihle, 1985). We could detect only transient increase in IL-3 producing activity of the spleen cells in the early stage of infection in two strains of murine malaria when it was measured and calculated on the unit number of cells (Fig. 5A). However, when the IL-3 producing activity was converted to per spleen, it was much higher in P. yoelii 17x- than in P. bergheiinfected mice (Fig. 5B). Obviously such a difference is directly related to the absolute number and function of T cells in the spleen. Related to this, Gross, Geva & Frankenburg (1988) reported that splenic T cetls of P. berghei-infected mice increased in number but decreased in percentage. Our results indicate that one of the important causes of the difference in hemopoiesis in lethal and non-lethal murine malarial infection is the difference in hemopoietic factor production in both infections. In conclusion, the difference in the hemopoietic proliferative responses of the host after lethal and nonlethal murine malaria infection is one of the critical factors determining the virulence of malarial parasites. The actual mechanism of the disturbance of the accumulation and differentiation of CFU-S in lethal P. berghei infection should be clarified in future investigations. thank MS E. Ohno and A. Tanaka for their excellent technical assistance
Acknowledgements-We
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