Journal of the Egyptian Society of Parasitology, Vol. 39, No. 2, August2009

J. Egypt. Soc. Parasitol., 39 (2), 2009: 1 - 10

COMPARATIVE STUDY BETWEEN NONLETHAL AND LETHAL STRAINS OFPLASMODIUM YOELII WITH REFERENCE TO ITS IMMUNOLOGICAL ASPECT

By

HANAA Y. BAKIR, FATMA G. SAYED, SALMA A. RAHMAN,

AMANY I. HAMZA, ABEER E. MAHMOUD, LAMIA A. GALAL

AND RASHA A. ATTIA

Department of Parasitology, Faculty of Medicine, AssiutUniversity, Assiut, Egypt

Abstract

Innate immunity has an important role inthe protection against malaria.Toclarify its effect on nonlethal and lethal strain ofPlasmodium yoelii,comparison between two groups of C57BL/6 mice infected with 104 parasitized RBCs was performed. Liver and spleen mononuclear cells were isolated and analyzed by flowcytometry.The parasite appeared in blood on day 3 in both strains,with non lethal infection parasitemia reached a peak of60%on day 14 and mice completely recovered, while in lethal infection parasitemia was 80% on day 7 and mice succumbed to death.In non lethal strain,mice became anemic and the hematocrit percentage returned to its normal value during recovery, while in the lethal strain mice were severely anemic before death. The major expanding cells were found to be TCRIntermediate (TCRint) cells, mainly NK1.1-subset, these TCRint cells were distinguished from conventional T cells of thymic origin. CD4- CD8- cells increased in both strains. During malarial infection, the population of conventional T cells did not increase and usually associated with thymic atrophy.The present results showed that TCRint cells were intimately associated with the protection against malarial infection in both non lethal and lethal strains but the mice died in lethal infection due to the massive destruction of red blood cells leading to fatal anemia.

Abbreviations:TCRint cells: intermediate T cell receptor cells; TCRhi cells: high T cell receptor cells; NKT cells: natural killer T cells.

Keywords: Malaria,Plasmodium yoelii, Intermediate T cell, B6 mice

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Introduction

Many authors characterized the nature of intermediate T cell receptor (TCRint) in the liver of mice in terms of phenotype (Abo et al., 1991; Okuyama et al., 1992;Crispe and Mehal,1996;Ohteki and MacDonald, 1996;Murakami and Paul, 1998), morphology (Watanabe et al., 1995), and function(Sato etal., 1995; Kawachiet al., 1995; Watanabeet al., 1996). TCRint cells in mice can be identified by low density of TCR–CD3 complex on their surface and comprise both NK1.1+subset (i.e. NKT cells) and NK1.1- subset at a ratio of 1:1 (Watanabe etal., 1995). TCRint cells are mainly present in liver and are generated by an alternative intrathymic pathway (in case of NK1.1+ subset) or possibly by an extrathymic pathway of T cell differentiation in liver (especially in case of NK1.1- subset)(Lantz and Bendelac,1994;Bendelac,1995a,b; MacDonald et al., 1995; Kawachiet al., 1995;Watanabe et al., 1995,1996).

It is very important to determine how the immune system was modulated during malarial infection in humans and animals. In mice, the unconventional T cells such as NK1.1-TCRint cells and NK1.1+TCRint cells (i.e., NKT cells); have been reported to be the major population among expanding lymphocytes (Pied et al., 2000; Molano et al., 2000, Gonzalez-Aseguinolaza et al., 2000). On the other hand, conventional T cells (i.e., thymus-derived T cells) did not seem to respond significantly to malarial infection because they were slightly suppressed in parallel with thymic involution (Mannoor et al., 2002).

One of the prominent features ascribed to TCRint cells is self-reactivity against abnormal self cells,therefore it is expected that these TCRint cells might be associated with the surveillance of abnormal self cells (e.g., self-cells infected with intracellular pathogens) (Ohtsuka et al., 1995). Therefore, it might be difficult for conventional T cells to recognize pathogens which are present in erythrocytes, at least during blood stageof malarial infection.

The objective of the present study was based on the fact that malaria parasites are intracellular pathogens, CD56 + T and CD57 + T cells (T cells which express NK cell markers)are abundant in the liver but are absent in the thymus of humans and theyexpand in the peripheral blood of patients with Plasmodium falciparum and P. vivax(Takiiet al., 1994; Okadaet al., 1995).

This study aimedto demonstrate how the immune system modulates itself during the malarial course by comparison between two strains of P. yoelii (non lethal and lethal) regarding the kinetics of parasite, the level of anemia and the flowcytometric changes after infection.

Materials and Methods

ThirtyC57BL/6(B6) mice at the age of8-10 weeks were used for these experiments; the micewere maintained at the animal facility of AssiutUniversityunder specific pathogen-freeconditions. Fifteen mice were used for nonlethal experiment (G1)and other fifteenwere used for lethal experiment (G2).P. yoelii17XNL(nonlethal strain) and 17XL (lethal strain)were obtained from NiigataUniversity(Niigata, Japan). Parasites were maintainedby routine passages in mice. Mice were infected byintra-peritoneal injection with 104 parasitized erythrocytes per mouse. Parasitemia of blood erythrocytes was observed by Giemsa staining every 2 or 3 days and mice were sacrificed at the indicated days after infection.Lymphocytes were obtained from the liver and spleen of control and infected mice (Watanabe et al.,1995).

Hepatic mononuclear cells were isolated by a previously described method (Miyaji etal., 2002). Briefly, the liver was removed, pressed through 200-gauge stainless steel mesh, and suspended in Eagle’s MEM (Nissui Pharmaceutical Co., Tokyo, Japan) supplemented with 5 mM Hepes and 2% heat-inactivated newborn calf serum. After being washed once with the medium, the cells were fractionated by centrifugation in 15 ml of 35% Percoll solution (Pharmacia Fine Chemicals, Piscataway, NJ) for 15 min at 2000 rpm. The pellet was resuspended in erythrocyte lysing solution (155 mM NH4Cl + 10 mM KHCO3+1 mM EDTA-Na+170 mM Tris, pH 7.3). The splenocytes were obtained by forcing spleen through 200-gauge stainless steel mesh. Splenocytes were used after erythrocyte lysing.

FITC-, PE-, of mAbs were used and biotinconjugatedreagents were developed with TRI-COLOURconjugatedstreptavidin (Caltag Lab., San Francisco,CA) (Watanabe etal., 1995). The mAbs used here included anti-CD3 (145-2C11), anti-IL-2R (TM-b1), anti-NK1.1 (PK136), anti-CD4 (PM4-5), and anti-CD8 (53-6.7) antibodies (PharMingen, San Diego, CA). Cellswere analyzed by FACScan (Becton–Dickinson Co.,Mountain View, CA). To prevent nonspecific binding ofmAbs, CD16/32 (2.4G2) (PharMingen) was added beforestaining with labeled mAb. Dead cells were excludedby forward scatter, side scatter, and propidiumiodide gating.

Statistical analysis: Data were analyzed by one factor analysis of variance (ANOVA) or student t- test.

Results

Theparasite appeared in the blood of both non lethal and lethal strain in day 3, parasitemia ofthe first group reached a peak around 60% on day 14 and started to recoveron day 21 with a complete recovery on day 25. While in the second group the parasitemia was very high on day 7 and mice died around day 10 (Fig.1A) (P< 0.05). Hematocrit percentage was also compared between the two groups. In nonlethal strain, the mice became anemic at the peakand theHematocrit percentagereturned to normal during recovery. However,in lethal strainthe mice became severely anemic and died probably from anemia (Fig.1B) (P< 0.05).

The number of lymphocytes yielded by the liver and spleen during malarial infection was compared between the two strains (Fig.2) (P< 0.05). The number of lymphocytes in the liver and spleen greatly increased especially duringthe recovery phase in nonlethal strain more than in lethal strain(Fig.2).Acute phase was on day 7 and recovery phase was on day 21 in nonlethalstrain while in lethal strain acute phase was on day 3 and severe phase was on day 7.

Phenotypic characterization of liver and spleen lymphocytes during malarial infection:To identify lymphocyte subsets, two-colour staining for CD3 and IL-2R was conducted (Fig. 3A). NK cells were identified as CD3-IL-2R+, CD3int cells (or extrathymic T cells) as CD3intIL-2R+, and TCRhigh cells (or conventional T cells) as CD3highIL-2R-. In the liver of all tested mice, CD3int cells began to expand from acute phase to recovery and severe phase in both nonlethal and lethal strain. However, this response was more prominent in nonlethal strain (indicated by arrows). The staining change of spleen lymphocytes was minimal.

TCRint cells comprised both a NK1.1+ subset (i.e., NKT cells) and NK1.1-subset, so we examined how these cells expanded during malarial infection through a two-colour staining of liver and spleen lymphocytes for CD3 and NK1.1 (Fig.3B). Among total CD3int cells, the majority was estimated to be NK1.1-cells although the number of NKT cells showedno big difference in both stains throughout the infection.

Two-colour staining for CD4 and CD8 was conducted (Fig.4). The most prominent result from this experiment was the existence of double-negative (DN) CD4-CD8- cells among T cells. The total proportion of T cells was greater than the sum proportion of CD4+ and CD8+ cells in the liver of all tested mice after malarial infection. This result revealed that the majority of extrathymic T cells (i.e., CD3int cells) which appeared as a result of malarial infection were DNCD4-8-.

Discussion

The present study demonstrated how the immune system modulates itself during the course of malarial infection by comparing twostrains of P. yoelii (non lethal and lethal). The parasite kinetics were observed every other day after infection, the course was long with nonlethal strain and mice recovered, while in lethal strain the whole course took from 7 to 10 days as the parasitemia was very high and the mice died. This result agreed with that of Weerasinghe et al. (2001). Also, in the present investigationhematocrit percentage showed that the mice were severely anemic at the peak andthe percentageof hematocritreturned to its normal value during recoveryin non lethal infection, onthe contrary mice died from severe anemia due to hemolysis of large numbers of RBCsin lethal infection.

It was reported (Weerasinghe et al., 2001; Mannoor et al., 2001; Mannoor et al., 2002; Halder et al., 2003) that the major malaria-infectious reactions (i.e., hepatomegaly, splenomegaly) and an increase in the number of lymphocytes in the liver and spleen even during the blood stageof malarial infection. In this study, the number of lymphocytes in the liver and spleen greatly increased especially during the recovery phase in nonlethal strain more than in the lethal one.

Many investigators elucidated that the protection against malaria was mediated by TCRint(Mannoor et al., 2002; Weerasinghe et al., 2001; Mannoor et al., 2001; Halder et al., 2003); their results are in a good agreement with our results. In the present study, the major subset of TCRint cells were NK1.1- cells with the double-negative CD4– CD8– phenotype in the liver of both non lethal and lethal strains. Although we emphasize the importance of TCRint cells for the protection against blood-stage (merozoite) of malarial infection, we do not deny the possibility that some other lymphocytes, such as conventional T and B cells are also associated with the protection against the hepatic stage (sporozoite) of malarial infection.

Depending on the investigators, there has been some controversy, especially as to the function of NKT cells (Molano et al., 2000; Gonzalez-Aseguinolaza et al., 2000).Immunomodulation during malarial infection was reported in a similar experiment (liver-stage infection) demonstrating that DN CD4-8- subset increased in the liver of malaria-infected mice. However NK1.1-TCRint cells in the liver were not identified (Pied et al., 2000).

Thepresent result showed that the changes happening in the immune system of both strains were almost the same starting earlier in the lethal than in the non lethal strain despite the aggressiveness of the lethal one. Theprotection was limited in the lethal strain and this may be due to the hemolysis of a huge number of red blood cells manifested by severe anemia followed by the death of mice within 10 days. This period was not long enough for the immune system to work in full capacity together with the lack of new red blood cellsgeneration.In contrast to the non lethal strain where there was a great expansionof intermediate cells throughout the course of infection and the hematocrit percentage returned to its normal value during recovery.

Conclusion

The TCRint cells in the liver and other organs were intensively activated in number during malarial infection. The protection against malaria is an event of innate immunity mediated by TCRint cells in both non lethal and lethal strain ofP. yoelii. But the severity of anemia in the lethal strain leads to deathwithout giving chance to the immune system to gain its full capacity.

References

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Figure legends

Figure1: Kineticsof malarial infection in B6 mice. (A) Recovery from infection in nonlethal strain and mice death in lethal strain. (B) Hematocrit percentages in nonlethal and lethal stains of P. yoelii. Acute phase on day 7 and recovery phase onday 21 in non lethal strain. In lethal strain, acute phase on 3rdday and severe phase on7th day(P< 0.05).

Figure2: Time kinetics of the number of lymphocytes yielded by liver and spleen in control and infected mice with nonlethal and lethal strain (P< 0.05).

Figure3:Expansion of CD3int cells in liver and spleen of mice infected with non lethal and lethal strain ofP. yoelii; (A) two-colour stain of lymphocytes for CD3 andIL-2R; (B) two-colour stain for CD3 and NK1.1 in liver and spleen. Numbers in figure indicate percentages of fluorescence-positive cellsin corresponding areas. Data are representative of three experiments.

Figure4: Identification of CD4 CD8 T cells and their phenotypes. Two-colour stain for CD4 CD8 in the liver and spleen during malarial infection. Numbers in figure represent percentages of fluorescence-positive cells in corresponding areas. Representative results of three experiments are depicted.

الملخص العربى

دراسة مقارنة بين النوع غير المميت و المميت من طفيل بلازموديم يولى من الناحية المناعية

د.هناء يوسف بكيير,ا.د. فاطمة جلال سيد, ا.د.سلمى محمد عبد الرحمن, ا.د.أمانى إبراهييم حمزة, د.عبير السيد محمود, د.لمياء أحمد عبد العزيز ود.رشا عبد المنعم حسن عطية

قسم الطفيليات- كلية الطب- جامعة أسيوط

الأهداف: مقارنة النوع الغير مميت و المميت من طفيلبلازموديم يولى من الناحية المناعية ودور المناعة الطبيعية في الحماية من الملاريا

الطريقة: تم اختيار مجموعتين من فئران التجارب بي 6 وتم عدوتها عن طريق الغشاء البريتونى بحوالى عشرة الاف من خلايا الدم الحمراء المحتوية على طفيل بلازموديم يولى (النوع غير المميت والنوع المميت) وتم عزل الخلايا وحيدة النواة من الكبد و الطحال ثم تحليلها بواسطة الفلوسيتومترى.

النتائج: وجد ان كلا النوعين يظهر فى الدم فى اليوم الثالث من العدوى وأثناء العدوى بالنوع غير المميت من الطفيل وجد انه يظهر فى اعلى معدلاته 60% في اليوم الرابع عشر من العدوي و يتم الشفاء التام للفئران. ولكن في النوع المميت من الطفيل تصل الإصابة 80% في اليوم السابع وتتدهور حالة الفئران المصابة حتى الموت.

فى حالة العدوى بالنوع غير المميت من الطفيل وجد ان الفئران اصيبت بالانيميا عند ظهور اعلى معدل للطفيل فى الدم ثم عادت نسبة الهيماتوكريت الى معدلها الطبيعى عند شفاء الفئران ولكن فى حالة العدوى بالنوع المميت من الطفيل اصيبت الفئران بانيميا حادة قبل الموت.

وجد ان الخلايا السائدة فى هذا التحليل عبارة عن خلايا تى. سى. أر. متوسطة الكثافة و الخلايا الرئيسية في هذه الخلايا إن. ك. 1.1 السالبة و يمكن تمييز هذه الخلايا عن خلايا التيى التقليدية الخارجة من غدة الثايمث.

لوحظ أيضاً ازدياد عدد خلايا سى.دي.4 و سي.دى.8 السالبة أثناء العدوى بالنوع غير المميت و النوع المميت من الطفيل.

أثناء الإصابة بمرض الملاريا يلاحظ عدم ازدياد الخلايا التقليدية و يكون المرض عادة مصاحباً بضمور فى غدة الثايمث.

الخلاصة: و هذه النتائج الحالية ترجح ان ظهور خلايا تى. سى. أر. متوسطة الكثافة يكون مصاحبا للحماية ضد مرض الملاريا بنوعية ولكن موت الفئران كان نتيجة الانيميا الحادة المصاحبة للمرض فى حالة الطفيل المميت.

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