Clin. exp. Immunol. (1976) 23, 471-476.
Peripheral lymphocyte subpopulations in human fakiparum malaria D. J. WYLER Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases National Institutes of Health, Bethesda, Maryland, U.S.A.
(Received 8 August 1975)
SUMMARY
The concentration of circulating T, B, and 'null' lymphocytes was determined in thirty children and three adults with Plasmodium falciparum infections in West Africa. During infection, both percentage as well as concentration of T cells were decreased as compared to levels following treatment. The percentage but not concentration of B cells was increased. Both percentage and concentration of 'null' cells were increased in malaria. Patients with splenomegaly had the most severe alterations in T-cell number; no other historic or clinical parameter correlated with the degree or pattern of change in circulating lymphocyte subpopulations. These alterations were rapidly reversible after antimalarial treatment and presumably represent the sequestration of T cells in the spleen or other organs.
INTRODUCTION Two subpopulations of lymphocytes have been recognized which have separate but sometimes interacting immunological functions: immunoglobulin-forming cells and their direct precursors (B cells) and thymus-derived lymphocytes (T cells) which are responsible for mediating cellular immune responses and for modulating B-cell function. By light microscopy these subpopulations are morphologically indistinguishable, but they can be identified on the basis oftheir specialized membrane receptors. Human T cells form rosettes (E rosettes) with sheep erythrocytes (SRBC). B cells have surface immunoglobulin and receptors for the third component of complement (C'3). These characteristics of B cells are recognized with fluorescein-conjugated anti-immunoglobulin or SRBC complexed with C'3 which form rosettes (EAC rosettes) around the lymphocytes. Lymphocytes lacking demonstrable receptors are referred to as 'null' cells. Disturbance in the distribution of these subpopulations in peripheral blood has been demonstrated in several diseases (Wybran & Fudenberg, 1973; Williams et al., 1973; Niklasson & Williams, 1974) and may reflect important aspects of the immune response. Although much has been learned about immune mechanisms in rodent and simian malaria which suggests important roles for both T and B cells in the development of protective immunity, less is understood of the immune responses in human infection with Plasmodium falciparum. The production of antibody in malaria and its probable protective role in falciparum infections (Cohen, McGregor & Carrington, 1961) indicates that B-cell activity is important. Peripheral blood T and B lymphocytes from patients with previous falciparum malaria infections proliferate in vitro in response to malaria antigen (Wyler & Oppenheim, 1974; Wyler & Smith, unpublished results), although the protective role of these cells in infection is unknown. In an effort to learn more about these lymphocytes in human malaria, the concentraCorrespondence: Dr D. J. Wyler, Laboratory of Parasitic Diseases, National Institute of Allergy and Diseases, National Institutes of Health, Bethesda, Maryland 20014, U.S.A.
Infectious
471
472
D. J. Wyler
tion of T, B, and 'null' cells in peripheral blood was measured in Africans with P. falciparum infections. It was observed that a marked but rapidly reversible decrease in circulating T cells (occasionally associated with alteration in B cells) and increase in 'null' cells occurred during infection. These alterations may represent non-specific disturbances or could represent redistribution of circulating lymphocytes to lymphoid organs as part of the normal immune response to malaria. MATERIALS AND METHODS Patients. Thirty children (nineteen males, eleven females) age 6 months to 9 years (mean 2J years) and three adults with falciparum malaria were selected at random from the outpatient department of the Medical Research Council Laboratories, Fajara, The Gambia, West Africa, between November 1974 and February 1975 (dry season). Medical histories were obtained and complete physical examinations were performed. Malaria patients with concomitant malnutrition, bacterial or viral infections, or chronic diseases or who had received antimalarial medication or tribal potions were not studied. Packed cell volume (PCV), white blood count (WBC), and differential leucocyte counts were determined. Thick and thin blood smears were stained with Giemsa and parasite density was quantitated by counting the number of parasitized erythrocytes per fifty leucocytes in blood smears and relating this to the WBC (i.e. parasites/mm3 = parasites/50 leucocytes x WBC/50). Patients were treated with oral pyrimethamine (Daraprim, Burroughs Wellcome; 12-5 or 25 mg daily for 1-3 days depending upon age). Four patients did not require treatment but were followed with repeat blood smears. Two patients received intramuscular chloroquine hydrochloride (Aralen, Winthrop; 5 mg base/kg). Nineteen patients returned for follow-up studies 1-43 days (mean: 15-6 days) after treatment. Interim histories, physical examinations and blood tests and smears were again performed, and results suggested that these patients did not represent a clinically selected group. Identification of lymphocyte subpopulations. Heparinized whole blood obtained from jugular or femoral vein in children or antecubital vein in adults was fractionated on Ficoll-Hypaque density gradients according to the method of Boyum (1968). The mononuclear cells were washed three times in Earle's balanced salt solution (BSS), centrifuging each time at 250 g for 10 min at 20'C. Concentration of cells was adjusted to 4 x 106 mononuclear cells/ml in BSS and found to be > 90% viable by Trypan Blue dye exclusion. Rosettes were prepared according to the methods of Jondal, Holm & Wigzell (1972). E rosettes were prepared by incubating at 370C for 5 min 0-25 ml of the mononuclear cell suspension with 0-25 ml of a 0.5% suspension in BSS of thrice-washed SRBC. Cells were centrifuged at 100 g for 5 min at 40C and the supernatant gently replaced by 0 5 ml of SRBC-absorbed, heat-inactivated foetal calf serum (Grand Island Biological Company, Grand Island, New York). Cells were again centrifuged for 5 min at 100 g and placed at 40C overnight. EAC rosettes were prepared from SRBC sensitized with 19S rabbit anti-SRBC antibody using fresh mouse serum as a source of complement. A 0-25-ml aliquot of a 0.5% suspension of the EAC reagent was incubated with 0-25 ml of the mononuclear cell suspension at 37°C for 30 min. All rosettes were gently resuspended and fixed in 1% buffered glutraldehyde (pH 7 0), puddled on glass slides, stained with Giemsa and partially destained with absolute methanol to improve tinctural quality. Slides were coded and read without knowledge of the source of lymphocytes. Four hundred mononuclear cells were counted in oil immersion fields and the percentage of rosette-forming cells determined. The concentration of rosetteforming cells was calculated by multiplying percentage rosette times total concentration of lymphocytes.
RESULTS Parasite densities in thirty patients ranged from 1100 to 334,000 trophozoites/mm3 (geometric mean 24,694). Temperatures (rectal) in these patients ranged from 35-6 to 41O0°C (mean 38 70C) when first seen in the clinic. Ten of the patients were found to have significantly enlarged spleens by examination, with spleen edge palpable between 2 and 8 cm (mean 3 cm) below the left costal margin in the mid-clavicular line. Patients with splenomegaly also tended to have hepatomegaly. In none of the patients were lymphadenopathy, petechiae, rash, or herpes labialis observed. Table 1 summarizes the results of blood tests and rosette assays performed on these patients. Since patients were studied sequentially in relation to their infection, the alterations in values for these parameters during infection is felt to reflect the effects of acute malaria. The proportional distribution of lymphocyte subpopulations in the patients after treatment
e|\o_+',DEi
Peripheral lymphocytes in malaria
"
A
X
|
~~~~~~~00 m t
8 :C C27,
Nt
e < E
+1
.
£
6z
CI
V
~+1
~o
N
.0 ,_
t=oYn
'D: CO:
A
sV
EnX
\
;~~~~~~~~~~~~~C
EE
473
474
D. J. Wyler
was found to be no different from nine randomly studied healthy African adults (P>0l1, Student's t-test) and is similar to results in healthy Europeans reported by others (Jondal et al., 1972). During infection, the percentage of T cells was reduced in association with elevation in percentage of B and 'null' cells. The observed increase during infection in concentration of monocytes cells which also have receptors for C'3 did not account for the observed elevation in percentage EAC rosette-forming cells since monocyte count and EAC rosettes (percentage or concentration) were poorly correlated (r = 0-128; P>0-20; Spearmen rank correlation). Since a decrease in total lymphocytes was observed during infection (Table 1), the concentration of T. B, and 'null' cells was calculated and results are summarized in Table 1 and Fig. 1. These data demonstrate that malaria was associated with a depression in numbers of T cells. The pretreatment concentrations were between 11 and 700 of the post-treatment concentrations when compared in individual patients. In contrast, despite relative alterations in B cells, there was an occasional decrease but no consistent alteration in concentration of B cells. Concentration of 'null' cells varied among patients but generally demonstrated an increase during infection of between 500 and 50000 compared to levels in the same individual after treatment. These observed changes in the lymphocyte subpopulations did not correlate with the age or sex of the patient, or with parasite density, duration of symptoms, or temperature. On the other hand, as suggested in Fig. 1, patients with splenomegaly tended to have lower concentrations of T cells than patients without enlarged spleens (P
Peripheral lymphocyte subpopulations in human fakiparum malaria D. J. WYLER Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases National Institutes of Health, Bethesda, Maryland, U.S.A.
(Received 8 August 1975)
SUMMARY
The concentration of circulating T, B, and 'null' lymphocytes was determined in thirty children and three adults with Plasmodium falciparum infections in West Africa. During infection, both percentage as well as concentration of T cells were decreased as compared to levels following treatment. The percentage but not concentration of B cells was increased. Both percentage and concentration of 'null' cells were increased in malaria. Patients with splenomegaly had the most severe alterations in T-cell number; no other historic or clinical parameter correlated with the degree or pattern of change in circulating lymphocyte subpopulations. These alterations were rapidly reversible after antimalarial treatment and presumably represent the sequestration of T cells in the spleen or other organs.
INTRODUCTION Two subpopulations of lymphocytes have been recognized which have separate but sometimes interacting immunological functions: immunoglobulin-forming cells and their direct precursors (B cells) and thymus-derived lymphocytes (T cells) which are responsible for mediating cellular immune responses and for modulating B-cell function. By light microscopy these subpopulations are morphologically indistinguishable, but they can be identified on the basis oftheir specialized membrane receptors. Human T cells form rosettes (E rosettes) with sheep erythrocytes (SRBC). B cells have surface immunoglobulin and receptors for the third component of complement (C'3). These characteristics of B cells are recognized with fluorescein-conjugated anti-immunoglobulin or SRBC complexed with C'3 which form rosettes (EAC rosettes) around the lymphocytes. Lymphocytes lacking demonstrable receptors are referred to as 'null' cells. Disturbance in the distribution of these subpopulations in peripheral blood has been demonstrated in several diseases (Wybran & Fudenberg, 1973; Williams et al., 1973; Niklasson & Williams, 1974) and may reflect important aspects of the immune response. Although much has been learned about immune mechanisms in rodent and simian malaria which suggests important roles for both T and B cells in the development of protective immunity, less is understood of the immune responses in human infection with Plasmodium falciparum. The production of antibody in malaria and its probable protective role in falciparum infections (Cohen, McGregor & Carrington, 1961) indicates that B-cell activity is important. Peripheral blood T and B lymphocytes from patients with previous falciparum malaria infections proliferate in vitro in response to malaria antigen (Wyler & Oppenheim, 1974; Wyler & Smith, unpublished results), although the protective role of these cells in infection is unknown. In an effort to learn more about these lymphocytes in human malaria, the concentraCorrespondence: Dr D. J. Wyler, Laboratory of Parasitic Diseases, National Institute of Allergy and Diseases, National Institutes of Health, Bethesda, Maryland 20014, U.S.A.
Infectious
471
472
D. J. Wyler
tion of T, B, and 'null' cells in peripheral blood was measured in Africans with P. falciparum infections. It was observed that a marked but rapidly reversible decrease in circulating T cells (occasionally associated with alteration in B cells) and increase in 'null' cells occurred during infection. These alterations may represent non-specific disturbances or could represent redistribution of circulating lymphocytes to lymphoid organs as part of the normal immune response to malaria. MATERIALS AND METHODS Patients. Thirty children (nineteen males, eleven females) age 6 months to 9 years (mean 2J years) and three adults with falciparum malaria were selected at random from the outpatient department of the Medical Research Council Laboratories, Fajara, The Gambia, West Africa, between November 1974 and February 1975 (dry season). Medical histories were obtained and complete physical examinations were performed. Malaria patients with concomitant malnutrition, bacterial or viral infections, or chronic diseases or who had received antimalarial medication or tribal potions were not studied. Packed cell volume (PCV), white blood count (WBC), and differential leucocyte counts were determined. Thick and thin blood smears were stained with Giemsa and parasite density was quantitated by counting the number of parasitized erythrocytes per fifty leucocytes in blood smears and relating this to the WBC (i.e. parasites/mm3 = parasites/50 leucocytes x WBC/50). Patients were treated with oral pyrimethamine (Daraprim, Burroughs Wellcome; 12-5 or 25 mg daily for 1-3 days depending upon age). Four patients did not require treatment but were followed with repeat blood smears. Two patients received intramuscular chloroquine hydrochloride (Aralen, Winthrop; 5 mg base/kg). Nineteen patients returned for follow-up studies 1-43 days (mean: 15-6 days) after treatment. Interim histories, physical examinations and blood tests and smears were again performed, and results suggested that these patients did not represent a clinically selected group. Identification of lymphocyte subpopulations. Heparinized whole blood obtained from jugular or femoral vein in children or antecubital vein in adults was fractionated on Ficoll-Hypaque density gradients according to the method of Boyum (1968). The mononuclear cells were washed three times in Earle's balanced salt solution (BSS), centrifuging each time at 250 g for 10 min at 20'C. Concentration of cells was adjusted to 4 x 106 mononuclear cells/ml in BSS and found to be > 90% viable by Trypan Blue dye exclusion. Rosettes were prepared according to the methods of Jondal, Holm & Wigzell (1972). E rosettes were prepared by incubating at 370C for 5 min 0-25 ml of the mononuclear cell suspension with 0-25 ml of a 0.5% suspension in BSS of thrice-washed SRBC. Cells were centrifuged at 100 g for 5 min at 40C and the supernatant gently replaced by 0 5 ml of SRBC-absorbed, heat-inactivated foetal calf serum (Grand Island Biological Company, Grand Island, New York). Cells were again centrifuged for 5 min at 100 g and placed at 40C overnight. EAC rosettes were prepared from SRBC sensitized with 19S rabbit anti-SRBC antibody using fresh mouse serum as a source of complement. A 0-25-ml aliquot of a 0.5% suspension of the EAC reagent was incubated with 0-25 ml of the mononuclear cell suspension at 37°C for 30 min. All rosettes were gently resuspended and fixed in 1% buffered glutraldehyde (pH 7 0), puddled on glass slides, stained with Giemsa and partially destained with absolute methanol to improve tinctural quality. Slides were coded and read without knowledge of the source of lymphocytes. Four hundred mononuclear cells were counted in oil immersion fields and the percentage of rosette-forming cells determined. The concentration of rosetteforming cells was calculated by multiplying percentage rosette times total concentration of lymphocytes.
RESULTS Parasite densities in thirty patients ranged from 1100 to 334,000 trophozoites/mm3 (geometric mean 24,694). Temperatures (rectal) in these patients ranged from 35-6 to 41O0°C (mean 38 70C) when first seen in the clinic. Ten of the patients were found to have significantly enlarged spleens by examination, with spleen edge palpable between 2 and 8 cm (mean 3 cm) below the left costal margin in the mid-clavicular line. Patients with splenomegaly also tended to have hepatomegaly. In none of the patients were lymphadenopathy, petechiae, rash, or herpes labialis observed. Table 1 summarizes the results of blood tests and rosette assays performed on these patients. Since patients were studied sequentially in relation to their infection, the alterations in values for these parameters during infection is felt to reflect the effects of acute malaria. The proportional distribution of lymphocyte subpopulations in the patients after treatment
e|\o_+',DEi
Peripheral lymphocytes in malaria
"
A
X
|
~~~~~~~00 m t
8 :C C27,
Nt
e < E
+1
.
£
6z
CI
V
~+1
~o
N
.0 ,_
t=oYn
'D: CO:
A
sV
EnX
\
;~~~~~~~~~~~~~C
EE
473
474
D. J. Wyler
was found to be no different from nine randomly studied healthy African adults (P>0l1, Student's t-test) and is similar to results in healthy Europeans reported by others (Jondal et al., 1972). During infection, the percentage of T cells was reduced in association with elevation in percentage of B and 'null' cells. The observed increase during infection in concentration of monocytes cells which also have receptors for C'3 did not account for the observed elevation in percentage EAC rosette-forming cells since monocyte count and EAC rosettes (percentage or concentration) were poorly correlated (r = 0-128; P>0-20; Spearmen rank correlation). Since a decrease in total lymphocytes was observed during infection (Table 1), the concentration of T. B, and 'null' cells was calculated and results are summarized in Table 1 and Fig. 1. These data demonstrate that malaria was associated with a depression in numbers of T cells. The pretreatment concentrations were between 11 and 700 of the post-treatment concentrations when compared in individual patients. In contrast, despite relative alterations in B cells, there was an occasional decrease but no consistent alteration in concentration of B cells. Concentration of 'null' cells varied among patients but generally demonstrated an increase during infection of between 500 and 50000 compared to levels in the same individual after treatment. These observed changes in the lymphocyte subpopulations did not correlate with the age or sex of the patient, or with parasite density, duration of symptoms, or temperature. On the other hand, as suggested in Fig. 1, patients with splenomegaly tended to have lower concentrations of T cells than patients without enlarged spleens (P
