Immune-complexes-mediated evasion of Plasmodium knowlesi from destruction by macrophages

Acta Troptca. 46(1989)239-247 Elsevier

239

ATP 00032

Immune-complexes-mediated evasion of Plasmodium knowlesi from destruction by macrophages* P r a t i Pal S l n g h a n d G . P

Dutta

Dtvtslon o[ Mlcrobtologv, Central Drug Research lnstttute, Lucknow, India (Recewed 13 June 1988, revised versmn recewed 12 December 1988, accepted 7 February 1989)

The role of immune-complexes m the evasion of Pla~modtum knowlesz from destruction by macrophages was stud~ed m vitro Incubation of macrophages w~th immune-complexes, prepared either by m~xmg total parasite anngens soluble in culture medmm wRh normal or immune monkey serum, or by polyethylene glycol preclpRatlon of serum from monkeys acutely infected with P knowle~t, significantly reduced both the pool size of the macrophages that bound parasmzed erythrocytes, and the number of parasmzed erythrocytes bound per macrophage Parasitized erythrocytes w~th mature sch~zonts were mvarlably preferred over those containing rings These observations appear to m&cate that during P kno~4le~t mfect~on m rhesus monkeys, immune-complexes may inhibit the bmdmg of parasttlzed erythrocytes with mononuclear phagocytes and thus may enable them to evade the destructive mechanisms mounted by the host Key words Plasmodmm know'lest, Macrophage, Immune-complex-medmtedevasmn, Parasite destruction

Introduction

The strategies by which malaria parasites evade the various destructive mechanisms mounted by the host are not fully understood (reviewed by Howard and Barnwell, 1983, Howard, 1987) During murlne (June et al, 1979), avian (Sonl and Cox, 1975), simian (Ward and Conran, 1966, Shephard et al, 1982) and human (Houba et al, 1976) malarlas, the presence of immune-complexes (IC) formed by antigens and antibodies m the plasma has been confirmed, but their precise function(s) remains unknown Brown and Kreler (1982), using Plasmodmm berghel/rat model, reported the inhibition of the an vitro antibody-mediated bmdlng of parasites to normal peritoneal macrophages, by mixtures of soluble P berghet antigens with serum, and by IC precipitated from the acute-phase serum (APS) of infected rats Packer and Kreler (1986), using the same model also demonstrated the inhibition of the phagocytosls of parasitized erythrocytes (PE) by peritoneal macrophages, pretreated with APS IC from mice infected with P berghet also Inhibited the in vitro phagocytosts of erythrocytes by macrophages (Shear et al, 1979), but had no effect *Commumcatlon Number 4268, Central Drug Research Institute, Lucknow, India

Correspondence address Dr Pratl Pal Slngh, Dwlslon of Microbiology, Central Drug Research Institute, P O Box No 173, Lucknog-226001, India 0001-706X/89/$03 50 @ 1989 Elsevier Science Pubhshers B V (Biomedical Division)

240 on their attachment (Shear, 1984) However, there appears to be no report regarding the effect of IC on the In vitro binding (attachment) of P knowlest PE to monkey macrophages In th~s communication, we present evidence to suggest that IC prepared either by mixing P knowlesl total parasite antigens soluble in culture medium (SA) and serum, or those precipitated from APS of P knowles~ infected monkeys, inhibited the binding of PE to monkey macrophages, in vitro

Materials and Methods

Antmals Rhesus monkeys (Macaca mulatta) of either sex, weighing 5-7 kg were used They were X-ray examined and tuberculin tested, and were housed in the Instltute's primate house, under fluorescent illumination from 7 00 h to 19 00 h This photocycle synchronized the infection, and upto 100% mature schlzonts could be obtained between 6 00 h and 8 00 h

Parastte Plasmodtum knowlesz (W 1 strain) obtained through the courtesy of Prof P C C G a r n h a m , U K , was used Infections were lnmated by intravenous inoculation of the infected blood from a donor monkey (1 x 10 6 parasitized red blood cells) or by cryopreserved parasites Parasltaemm (%) was m o m t o r e d by counting the number of infected erythrocytes per 1 x 1 0 4 erythrocytes in a Glemsa-stamed thin blood film PE were harvested from the blood of infected monkeys (40-60% parasltaemla with mature schIzonts), collected m cold acid-citrate-dextrose saline, after centrlfugatlon at 750 x g at 4°C for 12 mln The buffy coat was removed, and the upper brown layer containing PE was aspirated off PE were washed with cold Hanks balanced salt solution (HBSS) and resuspended in culture m e d m m at 6 x 107 PE per ml Sera, SA and IC Sera both normal monkey serum (NMS) or P knowlesl immune monkey serum (IMS) were obtained from the venous blood Monkeys were immunized by repeatedly infecting with P knowle~t, and cure (Dutta and Slngh, 1980) Prior to use sera were heat-inactivated for 30 mln at 56°C and stored at - 2 0 ° C APS was collected from monkeys having imtlal 5 0 to 30 0% P knowlest refection, and stored frozen The SA and IC were prepared according to Brown and Kreler (1982) Erythrocyte membrane-free P knowlesl schlzonts (0 5× 10 l°, Sadun and Gore, 1968) were ultrasonicated for 10 s in 2 0 ml D M E M and centrifuged at 4°C (3300 x g, 15 mm) The clear supernatant containing SA (2200 ~tg plasmodml protein per ml) was removed and stored at - 70°C The IC were prepared by either mixing SA with N M S or IMS (1 3) or by polyethylene glycol (PEG) precipitation of APS Equal volumes of APS and 3 5% P E G (tool wt 6000) in borate buffer (0 1 M, p H 8 5) were mixed and allowed to react for 18 h at 4~'C (Crelghton et a l , 1973) The IC were separated by centrlfugataon, dlahzed against HBSS and stored at 4=C

241

Macrophages Macrophages were either blood monocyte-derlved (Rldel et a l , 1986) or from spleen Mononuclear phagocytes were obtained from the E D T A treated monkey blood by density gradient centnfugatlon (400 x g, 35 rain) These cells were grown in Dulbecco modified Eagle medium ( D M E M ) supplemented with 0 01 M HEPES, 100 gg per ml gentamycln and 10% NMS, complete D M E M ( C D M E M ) , at 37°C in 5% COz-aIr atmosphere, for 72 h in T-25 tissue culture flasks The non-adherent cells were then removed by washing with warm (37°C) C D M E M , and the remaining adherent cells were relncubated in C D M E M for 1 week The monocyte-derlved macrophages were harvested by detaching them with sterile rubber scrapper The young macrophages were washed 3 times with C D M E M and resuspended in the same medium at a cell density of 1 x 105 cells per ml The splenic macrophages were harvested from the aseptically removed spleen of the monkeys The cell suspension was prepared by finely mincing the spleen tissue in C D M E M (Langhorne et a l , 1979) and the adherent macrophages were obtained by allowing the cells to attach wlth the plastic surface of T-25 flask at 37°C for 2 h in 5% CO/-alr atmosphere The non-adherent cells were removed by washing, and the adherent macrophages were harvested by scrapping, and resuspended in C D M E M (1 x 105 cells per ml) The macrophages obtained from either source were 95% viable, as assessed by Trypan Blue dye exclusion test 3 ml of cell suspension was transferred to each 35 m m plasnc petrldlsh The cells were incubated at 37°C and used in the assays, immediately

Phagocyttc assay For the determination of the effect of IC on the binding of PE (ring or schlzont) to macrophages, 3 x 105 macrophages (blood monocyte-derlved or splemc) grown in petridlshes were incubated with 2 0 ml IC suspension or serum for 30 mln at 4°C After incubation, 3 x 107 P E m 0 5 ml C D M E M were added to each culture and the petrldlshes were again incubated at 4°C for 15 mln The monolayers were then washed with C D M E M containing 10 -1 M sodium azlde to stop the further internalization of surface bound PE by the macrophages All treatments were run In triplicate Following the assay, the petrldlshes were gently washed with HBSS to remove any unbound PE, a drop of foetal bovine serum was placed on the monolayers, and the dishes were air dlred The cells were fixed with absolute methanol and stained with Glesma Approximately 500 macrophages per petrldlsh were examined under the 100 x objective lens of a microscope, and the number of macrophages with attached PE, and the numbers of PE bound per macrophage were recorded The data was calculated as the mean of the percentage_+ standard deviation (SD) based on triplicate cultures Student t-test was used for the statistical analysis and p < 0 05 was considered significant The 'Inhibition Index' was calculated by dividing the number of PE bound in the absence of SA or IC, by the number of PE bound in the presence of SA or IC

Results

Effect o[ IC prepared by mtxmg SA and serum on the bmdmg o[ PE to blood monoo'te-derlved o; splemc rna~rophages When blood monocyte-dertved macrophages treated with N M S or IMS were used m the assays (Table 1), ~t was observed that IMS treatment resulted both in the maximum percentage of macrophages that bound PE (42 _+6 and 71 _+ 12 for PE with rings and schlzonts, respectively), and in the m a x i m u m number of PE (180+34 for PE with rings and 268 + 4 4 with schtzonts) bmdlng per macrophage When macrophages were treated with N M S and SA, the binding was reduced as compared to N M S alone, but the reductxon was not yet slgmficant However, when macrophages were treated with IMS and SA, the binding was reduced stgnlficantly, as compared to IMS alone, but it was still higher or equal to that observed by N M S and SA Almost similar results were obtained when splenic macrophages were used xn the assays (Table 2) In all the experiments PE wtth schtzonts were preferred over those containing rings It appeared that the treatment of macrophages with IC, especmlly those with IMS, significantly reduced both the percentage of macrophages that bound PE as well as the number of PE bound per macrophage

E/]ect o! IC prepared by ptec;pttatton oJ APS by PEG on the bmdmg oJ PE to blood monocyte-dertved or splenic macrophages Sera from animals acutely infected with malaria are known to contain IC which can be precipitated by P E G (Crelghton et a l , 1973) D a t a in Table 3 show that, unlike the IC prepared by SA and NMS, these IC in the presence of N M S had no effect on the blndmg of PE However, in the presence of IMS these IC significantly reduced both the total number of macrophages binding PE as well as the number of PE per macrophage When macrophages were treated wtth IC alone, only a few bound more TABLE 1 Effect of soluble P Anowl,os; antigens and serum on the binding of PE to blood rnonocyte-denved

macrophages Treatment

Normal monkey serum b Normal monkey s e r u m + b soluble anhgens

of PE bound per 500 macrophages"

Inhibition mdex

binding PE" Ring

Schlzont

Ring

Ring

Schlzont

15_+6

23_+9

54_+ 13

70,+18

3

25

8_+3

12,+4

18_+5

24_+7 67

70

o o macrophages

No

Schlzont

Immune monkey serum ~

42_+6

71 ,+ 12

180+34

268 _+44

Immune monkey serum + h soluble anhgens

11 _+2

15 _+_5

27 ,+ 3

38_+5

"Mean _+standard dexlatlon of tnphcate cultures bValues not significantly &fferent (p > 0 05) "Values slgmficantly &fferent (p <"0 05)

243 TABLE 2 Effect of soluble

P knowleslantigens and serum on the binding of PE to splenic macrophages

Treatment

% macrophages binding PE"

No of PE bound per 500 macrophages"

Inhibition index

Ring

Schlzont

Ring

Ring

Schlzont

Normal monkey serum b

12_+5

22_+8

53_+ 15

75_+ 19

31

28

Normal monkey serum b + soluble antigens

4+2

8+3

17_+5

26_+6

Immune monkey serum ~

49_+7

77_+16

204_+38

340_+51

78

82

Immune monkey serum b + soluble antigens

17_+3

18_+3

26_+4

41 _+7

Schlzont

aMean _+standard deviation of triplicate cultures UValues not significantly different (p > 0 05) ~Values significantly &fferent (p < 0 05) TABLE 3 Effect of the PEG-precipitated immune-complexes from APS and serum on the binding of PE to blood monocyte-derlved macrophages Treatment

% macrophages binding PE"

No of PE bound per 500 macrophages"

Inhibition index

Ring

Schlzont

Ring

Schlzont

Ring

Schlzont

Normal monkey serum b

20+5

36+ II

61 _+ 17

109+29

10

11

Normal monkey serum b + PEG precipitated IC

17_+4

33+9

56_+14

94_+25

Immune monkey serum c

39+8

84_+18

202_+30

366_+47

42

51

Immune monkey serum b + PEG precipitated IC

14+4

22_+6

48+ 11

71 + 18

3+ 1

7+2

l I _+4

16_+6

PEG precipitated IC

"Mean _+standard deviation of tnphcate cultures bValues not slgmficantly different (p > 0 05) Walues significantly different (p < 0 05) t h a n o n e P E T h e s e I C w h e n t e s t e d o n s p l e n i c m a c r o p h a g e s ( T a b l e 4) s h o w e d r e s u l t s similar to those obtained by using blood monocyte-derIved macrophages, suggesting t h a t m a c r o p h a g e s f r o m d i f f e r e n t a n a t o m i c a l sites a n d i n r e l a t i v e l y d i f f e r e n t s t a g e s o f maturation responded to IC, similarly In these experiments too, PE with sch~zonts were preferred by macrophages

Effect of IC prepared with SA and serum on the bmdmg of opsomzed PE to blood monocyte-dertved macrophages T h e o b j e c t t v e o f t h e s e e x p e r i m e n t s w a s t o s t u d y t h e effect o f o p s o n l z a t l o n o f P E o n their uptake by macrophages, pretreated with NMS or IMS alone or their complexes

244 TABLE 4 Effect ot PEG-precipitated lmmune-comple\es from APS and serum on the binding of PE to splenic macrophages Treatment

% macrophages binding PE"

No of PE bound per 500 macrophages"

Inhibition lnde×

Ring

Ring

Schlzont

Ring

Schlzont

10

10

38

50

Schlzont

Normal monke;y serum b

18-+7

37+13

72+14

127_+26

Normal monkey serum" + PEG precipitated IC

14+5

2 9 + 10

6 6 + 12

119+22

Immune monkey serum ~

44--1i

97_+21

218_+33

382±54

I m m u n e monkey serum" + PEG precipitated IC

I I +_2

26-+7

56-+8

75+_ 16

2_+ 1

5-+2

13_+4

21 -+5

PEG precipitated IC

~Mean ± btandard de,¢latlon of trlphcate cultures bValues not significantly different (p > 0 05) Walues slgmficantly different ( p < 0 05)

with SA (Table 5) Results very clearly showed that opsonlzed PE were always a preferred target by the macrophages, particularly for those pretreated with NMS or NMS and SA Exposure of macrophages to IC containing NMS had no effect on the binding of non-opsonlzed PE However, IC exerted significant inhibitory effect on the IMS-medlated binding, irrespective of the presence of IMS in IC or on the PE surface (due to opsonlzatlon) Since apparently no difference in the binding of PE by blood monocyte-derlved or splemc macrophages was observed in the previous experiments, data with only blood monocyte-denved macrophages are presented Discussion

Macrophage hyperplasla and the presence of normal and infected red cells m the mononuclear phagocytes of the malarious monkeys strongly suggests their role m parasite ehmlnatlon (Tahaferro and Connan, 1936, Langhorne et al, 1979, Barnwell et al, 1983) Any mechanism that may interfere with the interaction between parasites and macrophages, might eventually prevent or reduce the clearance of the parasites by the host In this study we have demonstrated that P knowlesl derived IC can inhibit the binding of PE to macrophages, in vitro These results thus do not appear to be In agreement with those of Shear (1984), who observed no inhibition of the attachment of mouse erythrocytes coated with IgG or complement (C), and sheep erythrocytes coated with IgM and C to macrophages, and for which we have no explanation We have not determined the composition of IC Plasmo&al components, IgG, IgM and C have been reported in the malarious IC (Brown and Krexer, 1982) However, Shear (1984) could not detect C in the IC from malarious mice Based on these reports we can expect our IC to contain plasmodial components, IgG, IgM and C, despite hypocomplementemta durmg malarial Infections (Hussaln et al, 1983, Phanuphak et al, 1985)

Normal monkey serum Normal monkey serum I m m u n e monkey serum I m m u n e monkey serum

Normal monkey serum ~ + soluble antigens

I m m u n e monkey serum b

Immune monkey serum ° + soluble antigens

Immune monkey serum c

Immune monkey serum" + soluble antigens

~Mean __+standard devlahon of tnphcate cultures bValues not significantly different ( p > 0 05) Walues slgmficantly different (p < 0 05)

I m m u n e monkey serum I m m u n e monkey serum

Normal monkey serum c

27_+4

40_+6

20+3

33_+6

31 + 6

45+7

14+07

16+ 1

Normal monkey serum Normal monkey serum

Normal monkey serum b

Ring

54+6

78_+9

44+5

65+8

71 + 7

9 6 + 1!

28+2

32+3

Schlzont

% macrophages binding PE PE

Normal monkey serum b + soluble antigens

Macrophages

Pretreatment

163+28

277+33

93_+21

158+27

217+32

310_+39

54_+4

66+5

Ring

243_+37

424+47

142+21

257+29

384+42

572+66

80+7

106__+ I1

Schlzont

N o of PE bound" per 500 macrophages

17

17

14

12

Ring

17

18

14

13

Schlzont

Inhlbmon index

Effect of soluble P kno~tle~l anhgens and serum on the b m d m g of opsomzed PE to blood monocyte-derlved macrophages

TABLE 5

246

The mechanism of PE binding to macrophages still remains unresolved Macrophages are known to possess Fc-receptors (FcR) and C-receptors (C3R) on their surface membrane (Lay and Nussenzwelg, 1968), which mediate the binding of IgGand C-coated particles to macrophages (Mantovanl et a l , 1972) Mediation of FcR in the attachment and phagocytosls of PE has been demonstrated (Shear et a l , 1979, Shear, 1984), however, the role of C3R in the blndmg of PE to macrophages seems unlikely, smce the presence of C3b on P E t s still a matter of conjecture (Shear et a l , 1979, Topley et al., 1973, Abdalla et a l , 1983) Thus in our studies too, the PE would have bound macrophages through FcR, and the 1C, though they might have contained both IgG and C, would have blocked (mainly) the FcR mediated binding This may be further supported from our observations wherein PE with schlzonts were invariably preferred by the macrophages P knowlesl antigens are known to occur mamly on the surface of PE containing schlzonts (reviewed by Howard and Barnwell, 1983) These parasite derived antigens after reacting with cytophthc or opsontc antibodies present in malarious plasma or IMS, may cross-link macrophage surface FcR to effictently promote the clearance of the opsonized PE (Leslie, 1985) Macrophages from two different anatomical sources were used in this study The rationale for thts stems from the fact that in the spleen, a major site of retlculoendothellal cells, macrophages are confined in an envtronment where other lmmunocompetent cells like T- and B-cells are also present These cells are known to influence the functions of macrophages Also, these macrophages are in the terminal stages of their maturation and differentiation On the other hand, macrophages derived from blood monocytes are comparatively recently derived from the bone marrow and thus may &ffer from splemc macrophages in thetr various functions Based on these considerations it was considered expedient to use macrophages from at least two different anatomical sources, such as these In our stu&es, however, both types of macrophages behaved almost similarly Since the presence of IC in the plasma of monkeys infected with P knowlest is well estabhshed, our results indicate that they may lnhtblt the uptake of PE by mononuclear phagocytes during P knowlesl infection and thus may impede the clearance of parasites by the host

Acknowledgments We are grateful to Prof B N Dhawan, FNA, present Director, and to Dr M M Dhar, F N A , former Director, for their help and continued encouragement during the course of this study One of us (P P S) is thankful to the Council of Scientific and Industrial Research, New Delhi, for his placement in Scientist Pool

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