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Peripheral blood white cell responses during Angiostrongylus cantonensis infection in rats
InternationalJournalfor Pamitology Voll4, No. 2, pp. m-21 Printedin GreatBritain.
PERIPHERAL
002&7519/84$3.00+0.00 PergamonPressLtd. 0 1984Awndim Societyfor Pamitology
1,1984.
BLOOD WHITE CELL RESPONSES DURING CANTONENSIS INFECTION IN RATS
ANGIOSTRONGYLUS
W. K. YONG*and C. DOBSON Department of Parasitology, University of Queensland, St. Lucia, Brisbane, Queensland, 4067, Australia (Received 26 May 1983) Abstract-Yom W. K. and Jhaso~ C. 1984. Peripheral blood white cell responses during Angiostrongyhu cantonensb infections in rats. Interntionol Journal for Parasitology 14: 207-211. Changes in white blood ceu (WBC) populations and their proliferative responses to phytohaemagglutinin (PHA) and parastte antigens in virro were studied in rats gtven one to three concurrent infections with Angiostrongylus cantonensis. WBC counts were elevated following infection; these changes were augmented following each successive reinfection. The WBC response could be partitioned into variations in the numbers of four major cell types. There was a loss of lymphocytes from the circulation after infection or reinfection followed by an increase in circulating lymphocytes when the parasite migrated to the lungs and matured. An eosinophilia was observed in all rats immediately after infection which was enhanced successively after each reinfection. The monocyte populations increased in a similar, but less obvious manner, to the eosinophil leucocytes. Neutrophil leucocytes increased after infection, but the numbers declined after reinfection. All rats given one to three infections showed a neutrophilia late in the experiment. A reversal in the neutrophil leucocyte-lymphocyte ratio was observed after each infection. A peak response in the proliferation of peripheral blood lymphocytes in vitro to PHA preceded and exceeded that stimulated by A. cantonensis antigen. These responses were interpreted as the dissemination of uncommitted thymus-dependent lymphocytes involved in the induction of antigen sensitized memory cells released following the protective immune reaction. The degree of lymphocyte responsiveness to mitogens correlated with the numbers of these cells circulating at each time interval. The relationships between in vitro lymphocyte responses and protective immunity in the rat against A. cuntonensis are discussed.
INDEX KEY WORDS: Rats; Angiostrongylw cantonen& haemagglutinin; white blood cells; eosinophil and neutrophil INTRODUCTION INFECTIVE Angiostrongylus
cantonensis third-stage larvae are eaten, migrate to, and develop in, the brain
of rats; young adult worms then move to the lungs where they mature, and live a relatively long life in the pulmonary arteries (Mackerras & Sandars, 1955; Bhaibulaya, 1975; Yong & Dobson, 1982a). Rats become immune to A. cantonensis (Heyneman & Lim, 1965) and produce both sensitized lymphocytes and antibodies (Kamiya, 1970; Yoshimura & Soulsby, 1976) which passively immunize recipient rats (Yong & Dobson, 1982b, c). Changes in the serum anti-A. cantonensis titres closely relate to the various developmental phases of the worm in rats after single and multiple infections (Au & Ko, 1979; Yong & Dobson, 1982b). The present experiments investigated changes in the populations of circulating WBC and the in vitro replicative responsiveness of peripheral blood lymphocytes from rats concurrently infected up to *Present address: Regional Veterinary Laboratory, P.O. Box 406, Hamilton, Victoria, 3300, Australia.
lymphocyte transformation; leucocytes; monocytes.
three times with A. cantonensis
phyto-
to parasite antigens
and PHA. MATERIALS AND METHODS
Hosts. Line-bred 4-month-old DA strain Rattus norvegicus, from CSIRO, Long Pocket Laboratory, Brisbane, were used as the definitive host for Angiostrongylus cuntonensis. The intermediate host was a land snail of the genus Helicarion, which was bred and maintained in our laboratory. Parasite. A. cantonensis was maintained by serial passage in DA rats and laboratory reared Helicarion sp. snails as described by Bhaibulaya (1975). Parasitological techniques. Procedures for infecting rats with A. cuntonensis and for their necropsy are described in Yong & Dobson (1982a). Blood. All rats were bled by cardiac puncture under light ether anaesthesia. Total WBC counts were done using a Coulter Counter (Model ZF, Coulter Electronics Ltd., England) with Isoton (Coulter) as diluent, after lysing erythrocytes with Zaponin (Coulter). Differential leucocyte counts were made from Giemsa-stained thin smears of fresh blood. In vitro peripheral blood cultures. A modification of the whole blood culture method described by Han & Pauly 207
208
1.1.~. VOL.14.1984
W. IS. YONGand C. DOBSON
(1972)was used. Heparinized cardiac rat blood was diluted 1:60 with sterile RPM1 1640 culture medium @Grand isiand Biological Company, New York, U.S.A.) supplemented with 2% heat-inactivated foetal calf serum, 200 units heparin, 100 units ml-’ sodium penicillin and 1OOng ml-’ streptomycin sulphate (Commonweahh Serum Laboratories, Melbourne). A thin blood film was also made with this blood. Aliquots of 2 ml diluted whole blood cell suspension was dispensed into nine sterile 13 x 100 mm Pyrex tubes and tightly stoppered with sterile neoprene bungs. The remaining diluted blood was used for total WBC counts. PHA (Difco, Detroit, U.S.A.) (20 ph 0.01 mi-’ RPM1 1640) was dispensed into each of three cultures, adult A. cuntonensis antigen (Yong & Dobson, 1982b) (20 pg O.Ol-’ RPM1 1640) was added to each of three other cultures. Three untreated cultures were retained as controls. The cultures were vortex-mixed and incubated upright at 37°C and mixed every 12 h until the cells were harvested 4 days later. Each culture received 37 kB tritiated thymidine; specific activity 74 GBq mmol -?
at intervals as before. All rats were killed 120 days after the co~encement of the experiment, worms were recovered and counted. Statistical analyses were done where appropriate using Student’s Ytest. RESULTS
The development and numbers of A. cantoneMs recovered from rats with 1, 2 and 3 concurrent infections are given in Yong & Dobson (1983). Briefly, fewer A. cantonensis derived from secondary or tertiary infections survived to the adult stage (6 and 2 worms in the second and the third infections respectively) than those derived from primary infection (26 worms). However, the total size of the concurrent adult worm populations remained the same as that from a primary infection.
Pattern of circulating white blood cells in rats infected with A. cantonensis (Radiochemical Centre, Amersham, England) dispensed in The cell counts for infected rats are given as either 0.05 ml RPM1 1640 16 h before harvesting when the positive or negative changes from the mean cell cultures were stopped and the red blood cells lysed by counts of non-infected rats done at the same time. adding two drops of Zaponin to each culture. The remaining white cells were filtered on to Whatman GF 81 The total WBC counts from infected rats were greater than those from non-infected rats. They glass fibre pads (W. & R. Balston Ltd., England), washed increased after infection, particularly after the with 30 mi 0.15 M saline, bleached with 2 ml hydrogen peroxide and dehydrated with 10 ml methyl alcohol. Each juvenile worms migrated from the brain 35 days until pad was placed in a 12 x 47 mm glass pill vial (Australian 60 days after infection @ < 0.01) (Fig. 1A). The Glass Manufacturers Company, Sydney) and dried for 6 h WBC counts declined thereafter to stabilize above at 37°C. Scintillation fluid (2 ml) containing 6.0 g PPO and the non-infection control level 75 days after infec0.2 g POPOP 1-l toluene was added to each vial, stoppered, tion. The WBC count declined shortly after second placed in scintillation vials and counted for 20 min at 2% and third reinfection, but soon returned to normal preset error in a Beckman LS-100-C scintillation counter (Beckman Instruments Inc., California, U.S.A.) after 4 h levels and was elevated at the time the parasite returned to the lungs following reinfection (Fig. dark adaptation. The radioactivity in counts min-’ for each 1A). sample was converted to disintegrations min-’ lo6 lymphocytes-’ (d.p.m.) and the lymphocyte profiferative response, There was an initial loss of circulating lymphocytes induced by worm antigen or PHA, was expressed as transfollowing A, cantonensis infections in rats after 1, 2 formation ratios (T.R.). and 3 infections. In each case this was followed by an d.p.m. antigen stimulated cultures elevation of lymphocyte numbers when the juvenile T.R. = worms migrated to the lungs from 35 days after d.p.m. unstimulated cultures Experimentaldesign. DA strain rats were infected with 50 infection. A further depletion of circulating lymphoA. cantonensisthird-stage larvae at intervals of 40 days on cytes occurred 70 days after a primary infection to three occasions. E&h@,rats were infected with A. cantonensis levels below those in non-infected rats. Reinfection accentuated the patterns of lymphocyte traffic in the larvae and 20 were kept as uninfected controls. Sterile cardiac blood was taken at intervals from 10 rats in the circulation displayed during primary infections. By infected and non-infected groups. All rats in each group the end of the experiment the twice reinfected rats were bled and no animal was bled on two successive showed greatly elevated lymtihocyte counts (p < occasions. The blood was used for in vitro lymphocyte O+OOl)(Fig. 1B). cultivations, to assess total WBC numbers and to differenCnculating monocyte numbers were elevated tiate WBC types. Forty days after the primary infection during primary, secondary and tertiary infections in 20 rats were retained in the primary infection group and the rest were reinfected with 50 A. canfonensislarvae. A rats; reinfection accentuated the increase in numbers of monocytes (Fig. 1C). further 40 rats were given primary infections from the same batch of infective larvae and a further 10 rats were included The pattern of circulating neutrophil leucocytes among the non-infected controls. Rats from all groups during primary infections generally has a bimodal (continuing primary infections, new primary infections, distribution. They increased early in the primary secondary infections, and non-infected controls) were bled infections but the counts returned to control levels 45 at intervals for the same reasons as before. When the original days after infection until about 90 days when they primary infection had run 80 days, rats with secondary were again elevated. Neutrophil leucocyte counts infections, and 20 of the second group of rats with primary were also elevated in reinfected rats but in each case infections were retained and the remainder were reinfected there was a loss of cells from the blood after rewith 50 A. cantonensis larvae. Ten further non-infected infection (Fig. 1D). control rats were added to the experiment and 20 additional rats were given primary infections. Ail these rats were bled A single infection with A. cantonensis larvae
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proliferation to PHA preceded that induced by worm antigen and occurred when the adult worms left the brain for the lungs (3540 days). The lymphocyte response to PHA again became elevated about 100 days after infection but the response against worm antigen did not (Fig. 2). The proliferation of rat peripheral blood lymphocytes induced by parasite antigen was depressed immediately, and up to 20 days after reinfection but thereafter increased to a peak after 40 days. These variations in the lymphocyte proliferative responses were more accentuated after a third compared with those after a second infection. The in vitro responses of blood lymphocytes taken at different times from reinfected rats to PHA were similar to but preceded those induced by parasite antigen (Fig. 2). DISCUSSION
The WBC count increased in all the infected rats studied, however, in prolonged infections without reinfection it returned to levels similar to that in uninfected rats. increased WBC levels are common in mammals infected with metazoan parasites
10
DAYS AFTER
INFECTION
FIG. 1. Changes in (A) total white blood cells, (B) lymphocytes, (C) monocytes, (D) neutrophil leucocytes and (E) eosinophil leucocytes in peripheral blood of rats given a ) and two (O---O) and three infections of 50 Angiostntngyltls cantonensis larvae. Each point represents the differences
in the mean cell number of an average six infected and noninfected animals caused a rapid increase in the numbers of eosinophil leucocytes in the blood; reinfection greatly accentuated this response giving a distinct bimodal pattern. Eosinophil leucocyte count ‘eturnec to the control values in prolonged primary infections (Fig. 1E). No differences were detected in the basophn leucocyte counts of rats with primary and multiple concurrent infections compared with uninfected rats. In vitro responses of ~eripherai lymphocytes from rats infected with A. cantonensis Transformation ratios (TR) for cultured lymphocytes from the three successive primary infections in rats were not different and the data were pooled. Similar compilations were made for data from rats given two secondary infections. The in vitro proliferation of rat peripheral blood lymphocytes to parasite antigens (Fig. 2A) and PHA (Fig. 2B) was greatly depressed immediately after, and up to 10 days following infection with A. ca~tone~s~, but thereafter increased between 10 and 35 days after infection. The peak of lymphocyte
FIG. 2. The transformation ratios of lymphocytes from rats and two (0 - - - 0) and three given a primary (@ -------a) concurrent infections of 50 Angiostrongyhs (A-A) cantonensis larvae when cultured in vitro with (A) adult A. cantonerd antigen and (B) phytohaemaggiutinin. Hatched horizontai bar represents transformation ratios (mean it LE.) of iymphocytes from non-infected rats (average six animals at each point).
210
W. K.
YONG
and G.
(Charleston, 1964, Cypess, 1972). The mitigation of A. cantonensis to the brain and lungs involves tissue damage and inflammation which would account for the elevated traffic in neutrophil leucocytes following primary infection. Dobson (1968) and Cypess (1972) related similar accumulations of neutrophil leucocytes in the blood of rodents infected with other nematode parasites to the induction of inflammatory reactions. The less marked neutrophilia following reinfection probably related to the involvement of these cells in antibody mediated protective immune reactions. Indeed, whenever the blood lymphocyte count and in vitro response of these cells to parasite mitogens was high the neutrophil leucocyte count was low and vice versa, Larsh (1967) showed a similar reversal in lymphocyte-neutrophil leucocyte ratio together with elevated WBC in mice infected with Trichinella spiralis. Marked eosinophilia, particularly in the cerebrospinal fluid, is characteristic of A. cantonens~ infections in rodents and man (Ottolenghi, Weatherly, Kocan & Larsh, 1977; Au & Ko, 1979). In the present infections eosinophilia developed progressively following infection and reinfection; it was bimodal and related to the tissue and adult phase of each infection. Eosinophil leucocytes are believed to function in protective immunity against metazoan parasites. They may proliferate in association with thymus-processed lymphocytes and be activated by antibody- and lymphocyte-antigen reactions (Walls, Basten, Leuchars & Davies, 1971; Rothwell & Dineen, 1972). In this study, the numbers of eosinophi1 leucocytes were consistently high throughout with peak responses denoting the developmental and reproductive stages of the parasite life cycle. The primary eosinophil leucocyte peak occurred when sensitized circulating lymphocytes which proliferated in vitro in response to PHA and parasite antigen left the blood. It is possible therefore that the eosinophil leucocyte proliferates and responds to both the cellular and antibody products of thymus dependent immunological responses. Protective immunity against A. cantonensis has powerful antibodymediated and less efficient lymphocyte-mediated components (Yong & Dobson, 1982d). Sensitized T lymphocytes and their lymphokine products have been previously implicated in the induction by helminths and their eggs of peripheral blood eosinophilia (Colley, 1973; Phillips, DiConza, Gold & Reid, 1977). Thus eosinophilia occurred in these rats when A. cantonensis was highly immunogenic as it developed and when it reproduced and subsided thereafter when the major reproductive effort and antigen production of the parasite declined. Traffic in eosinophil leucocytes could also relate to parasite immunosuppressive activities which have been attributed to A. cantonensis by Yoshimura & Soulsby (1976). ‘1ne m vitro replicative responses of the peripheral lymphocytes to parasite antigens declined dramati-
D~BSON
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cally immediately following each infection which indicated either a marked increase in the migration of lymphocytes from the circulation or a change in their function. A similar change in the activities of peripheral lymphocytes has been observed by Dobson & Soulsby (1974) in guinea pigs infected with T~ichost~o~gyl~s co~~briformis. It is probably that both nonspecifically active (Naor & Sulitzeanu, 1967; Mandel & Byrt, 1971) and specifically sensitized lymphocytes were attracted to the invading A. cantonensis larvae at these times. Yong & Dobson (1982b) showed that anti-A. cantonensk serum antibodies were also depleted in these animals at this time and attributed this to the manifestation of antibodyantigen reactions involved in protective immunity, which supports this view. It is interesting that the circulating eosinophil leucocyte count peaked at this time which suggests that eosinophil leucocyte proliferation may be a response to these reactions and may not have had an immediate involvement in the protective immune reaction (Speirs, Speirs & Ponzio, 1974). Elevated antiparasite lymphocyte TR were observed after the adult worms had migrated from the brain to the lungs 40-65 days after infection or reinfection. Yoshimura & Soulsby (1976) demonstrated a similar peak response from rat peripheral blood lymphocytes to A. cantonensis antigens 70 days after infection. Dobson & Soulsby (1974) also found that the major response of infected guinea pig peripheral blood lymphocytes to T. colubriformis antigens related to the appearance of the adult parasite. The proliferation of lymphocytes in the presence of sensitizing antigens has been interpreted as a memory response (Dutton, 1967; Vischer & Stastny, 1967). Dobson & Soulsby (1974) suggested that the elevation of the peripheral blood lymphocyte response following infection represented the parenteral dissemination of such memory cells. The present results can be interpreted in the same way because lymph~~e proliferation accelerated and generated greater intensity with each reinfection. Moreover the dynamics of the serum anti-A. cantonensis antibody titres corresponded to those for the anti-parasite lymphocyte TR (Yong & Dobson, 1982b). The activities of these circulating cells also suggest that protective immunity acted largely against the larval stages of A. Cantonese in rats. This conclusion is supported by experiments which showed that protective immunity could be passively transferred with immune lymph node cells and serum (Yong & Dobson, 1982b, c). PHA responsive lymphocytes comprise a population of uncommitted thymus-dependent cells capable of being stimulated to transform by and to react with specific antigen (Weber, 1967). The appearance of elevated numbers of these cells in the circulation soon after each infection suggests that they were involved in the induction and potentiation of the protective immune response. There was a
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Celi responses to A. can~o~ens~ in rats
dynamic relationship between the presence of PHA and A. cantonerds antigen responsive lymphocytes in the blood of infected rats suggesting that the circulation of uncommitted cells preceded that of similar sensitized thymus dependent lymphocytes res~ns~bIe for the dissemination of immunologic~ memory. Acknowledgements-This work was supported by a grant from the Australian Research Grants Committee. The authors wish to thank Miss W. C. Huf for excellent typing.
REFERENCES
Au A.C.S. & Ko R. C. 1979. Changes in worm burden, haematological and serological responses in rats after cantonensis single and multiple Angiostrongylus infections. Zeitschriftfiir Parasitenkunde 58: 233-242. BHAIBULAYA M. 1975. Comparative studies on the life history of Angi~trongylus ma~kerrasae Bhaibulaya, 1968 and Angiostrongylas cantonensis (Chen, 1935). International Journalfor Parasitology 5: 7-20. CHARLESTON W. A. G. 1964. Comparison of blood and bone marrow changes in lambs with haemonchosis and in Iambs repeatedly bled. Journal of Comparative Pathology 74: 223-240. COLLEY D. G, 1973. Eosinophils and immune mechanisms. 1. Eosinophil stimulation promoter (ESP): A lymphokine induced by specific antigen or phytohemagglutinin. Journal of Immunology 110: 1419-1423. CYPSSSR. H. 1972. Blood cell count changes associated with immunity to Nematospiroides dubius. Journal of Parasitology 58: 563-566. DOBSON C. 1968. Haematological changes induced in laboratory rats by larval Amplicaecum robertsi Sprent & Mines, 1960. Australian Journal of Biological Sciences 21: 341-350. DOBSONC. & SOULSBY E. J. L. 1974. Lymphoid kinetics in guinea pigs infected with Trichostrongylus colubriformis: tritiated thymidine uptake in gut and allied lymphoid tissue, humoral IgE and haemagglutinating antibody responses, delayed hypersensitivity reactions and in vitro lymphocyte transformations during primary infections. Experimental Parasitofogy 35: 16-34. DUTTONR. W. 1967. In vitro studies of immunological responses of lymphoid cells, Advances in Immunology 6: 253-336. HAN T. & PAULYJ. 1972. Simplified whole blood method for evaluation in vitro lymphocyte reactivity of Clinical and Experimental laboratory animals. ~mmunolo~ 11: 137-142. HEYNEMAN D. & LIM B. L. 1965. Prolonged survival in rats immunized by a small number of low-level doses of Angiostrongylus cantonensis and challenged with a lethal level of infective larvae. Medical Journal of Malaya 20: 162-163.
KAM~YAM. 1970. Haemagglutination test in rats infected with Angiostron~l~ Cantonese. Southeast Asian Journal of Tropical Medicine and Public Health 1: 570-571. LARSH J. E., Jr. 1967. The present understanding of the mechanism of immunity to Trichinella spiralis. American Journal of TropicalMedicine andHygiene 16: 123-132. MACKERRAS M. J. & SANDARSD. F. 1955. The life-history of the rat lungworm, Angiostron~l~ canfonensis
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Australian @hen) (Nematoda : Metastrongylidae). Journal of Zoology 3: l-21. MANDELT. & BYRTP. 1971. The uptake of antigens and non-antigenic markers by mouse spleen and peritoneal cells. In: The Role of Lymphocytes and Macrophages in the immunological Response, Xl11 international Congress of Haematology, Munich, August 2-8, 1970 (Edited by DUMO~~ D. C.), pp. 38-42. Springer, Berlin. NAOR D. L SULITZEANU D. 1967. Binding of radioiodinated bovine serum albumin to mouse spleen cells. Nature (London) 214: 687-688. OTTOLENGHI A., WEATHERLY N. F., KOCANA. A. & LARSH J. E., Jr. 1977. Angiostrongylus cantonensis: Phospholipase in nonsensitized and sensitized rats after chahenge. Infection andimmunity 15: 13-18. P~LLIPS S. M., DICONZAJ. J., GOLD J. A. & REID W. A. 1977. Schistosomiasis in the congenitally athymic (nude) of eosinophilia, mouse: 1. Thymic dependency granuloma formation and host morbidity. Journal of Immunology 118: 594-599. ROTHWEUT. L. W. t DWEEN J. K. 1972. CeIlular reactions in guinea pigs following primary and challenge infections with Trichostrongylus colubriformis with special reference to the roles played by eosinophils and basophils in rejection of the parasite. Immunology 22: 733-745. SPEIRSR. S., SPEIRSE. E. & PONZION. M. 1974. Eosinophi& in humoral and cell-mediated responses. In: Developments in Lymphoid Cell Biofogy (Edited by JOTTLIEB A. A.), pp. 51-73. CRC Press, Cleveland. VISCH~RT. L. & STASTNY P. 1967. Time of appearance and distribution of cells capable of secondary immune response following primary immunization. Immunology 12: 675-687. WALLSR. S., BASTENA., LEUCHARSE. & DAVIESA. J. S. 1971. Mechanisms for eosinophilic and neutrophilic leucocytoses. British MedicaI Journal 3: 157-159. WEBERW. T. 1967. The response to phytohemagglutinin by lymphocytes from the spieen, thymus and bursa of Fabricius of chickens. Experimental Cell Research 46: 464-466. YONG W. K. & DOBSON C. 1982a. Population dynamics of Angiostrongylus cantonensis during primary infections in rats. Parasitology 85: 399-409. YONG W. K. & DOBSON C. 1982b. Antibody responses in rats infected with Angiostrongyl~ cantonens~ and the passive transfer of protective immunity with immune serum. ZeitschriftfiirParasitenkunde 67: 329-336. YONG W. K. & DOBSON C. 1982~. The passive transfer of protective immunity against Angiostrongylus cantonensis with immune lymph node cells from different Iymphoid tissues in rats. International Journal for Parasitology 12: 423-425. YONG W. K. & DOBSON C. 1982d. Passive immunity in rats infected with Angiostrongylus cantonensis: Interactions between syngeneic immune serum and sensitized lymph node cells. Zeitschriftftir Parasitenkunde 68: 87-92. MONO w. g, & DOnsON C. 1983. ImmUnOlOgiC~ regukition
of Angiostrongylus cantonensis infection in rats: modulation of population density and enhanced parasite growth following one or two su~rimposed infections. Journai of Helmintholo~ 57: 155-165. YOSHIMURA K. & SOULSBY E. J. L. 1976. Angiostrongylus cantonensis: Lymphoid cell responsiveness and antibody production in rats. American Journal of Tropical Medicine and Hygiene 25: 99-107.
PERIPHERAL
002&7519/84$3.00+0.00 PergamonPressLtd. 0 1984Awndim Societyfor Pamitology
1,1984.
BLOOD WHITE CELL RESPONSES DURING CANTONENSIS INFECTION IN RATS
ANGIOSTRONGYLUS
W. K. YONG*and C. DOBSON Department of Parasitology, University of Queensland, St. Lucia, Brisbane, Queensland, 4067, Australia (Received 26 May 1983) Abstract-Yom W. K. and Jhaso~ C. 1984. Peripheral blood white cell responses during Angiostrongyhu cantonensb infections in rats. Interntionol Journal for Parasitology 14: 207-211. Changes in white blood ceu (WBC) populations and their proliferative responses to phytohaemagglutinin (PHA) and parastte antigens in virro were studied in rats gtven one to three concurrent infections with Angiostrongylus cantonensis. WBC counts were elevated following infection; these changes were augmented following each successive reinfection. The WBC response could be partitioned into variations in the numbers of four major cell types. There was a loss of lymphocytes from the circulation after infection or reinfection followed by an increase in circulating lymphocytes when the parasite migrated to the lungs and matured. An eosinophilia was observed in all rats immediately after infection which was enhanced successively after each reinfection. The monocyte populations increased in a similar, but less obvious manner, to the eosinophil leucocytes. Neutrophil leucocytes increased after infection, but the numbers declined after reinfection. All rats given one to three infections showed a neutrophilia late in the experiment. A reversal in the neutrophil leucocyte-lymphocyte ratio was observed after each infection. A peak response in the proliferation of peripheral blood lymphocytes in vitro to PHA preceded and exceeded that stimulated by A. cantonensis antigen. These responses were interpreted as the dissemination of uncommitted thymus-dependent lymphocytes involved in the induction of antigen sensitized memory cells released following the protective immune reaction. The degree of lymphocyte responsiveness to mitogens correlated with the numbers of these cells circulating at each time interval. The relationships between in vitro lymphocyte responses and protective immunity in the rat against A. cuntonensis are discussed.
INDEX KEY WORDS: Rats; Angiostrongylw cantonen& haemagglutinin; white blood cells; eosinophil and neutrophil INTRODUCTION INFECTIVE Angiostrongylus
cantonensis third-stage larvae are eaten, migrate to, and develop in, the brain
of rats; young adult worms then move to the lungs where they mature, and live a relatively long life in the pulmonary arteries (Mackerras & Sandars, 1955; Bhaibulaya, 1975; Yong & Dobson, 1982a). Rats become immune to A. cantonensis (Heyneman & Lim, 1965) and produce both sensitized lymphocytes and antibodies (Kamiya, 1970; Yoshimura & Soulsby, 1976) which passively immunize recipient rats (Yong & Dobson, 1982b, c). Changes in the serum anti-A. cantonensis titres closely relate to the various developmental phases of the worm in rats after single and multiple infections (Au & Ko, 1979; Yong & Dobson, 1982b). The present experiments investigated changes in the populations of circulating WBC and the in vitro replicative responsiveness of peripheral blood lymphocytes from rats concurrently infected up to *Present address: Regional Veterinary Laboratory, P.O. Box 406, Hamilton, Victoria, 3300, Australia.
lymphocyte transformation; leucocytes; monocytes.
three times with A. cantonensis
phyto-
to parasite antigens
and PHA. MATERIALS AND METHODS
Hosts. Line-bred 4-month-old DA strain Rattus norvegicus, from CSIRO, Long Pocket Laboratory, Brisbane, were used as the definitive host for Angiostrongylus cuntonensis. The intermediate host was a land snail of the genus Helicarion, which was bred and maintained in our laboratory. Parasite. A. cantonensis was maintained by serial passage in DA rats and laboratory reared Helicarion sp. snails as described by Bhaibulaya (1975). Parasitological techniques. Procedures for infecting rats with A. cuntonensis and for their necropsy are described in Yong & Dobson (1982a). Blood. All rats were bled by cardiac puncture under light ether anaesthesia. Total WBC counts were done using a Coulter Counter (Model ZF, Coulter Electronics Ltd., England) with Isoton (Coulter) as diluent, after lysing erythrocytes with Zaponin (Coulter). Differential leucocyte counts were made from Giemsa-stained thin smears of fresh blood. In vitro peripheral blood cultures. A modification of the whole blood culture method described by Han & Pauly 207
208
1.1.~. VOL.14.1984
W. IS. YONGand C. DOBSON
(1972)was used. Heparinized cardiac rat blood was diluted 1:60 with sterile RPM1 1640 culture medium @Grand isiand Biological Company, New York, U.S.A.) supplemented with 2% heat-inactivated foetal calf serum, 200 units heparin, 100 units ml-’ sodium penicillin and 1OOng ml-’ streptomycin sulphate (Commonweahh Serum Laboratories, Melbourne). A thin blood film was also made with this blood. Aliquots of 2 ml diluted whole blood cell suspension was dispensed into nine sterile 13 x 100 mm Pyrex tubes and tightly stoppered with sterile neoprene bungs. The remaining diluted blood was used for total WBC counts. PHA (Difco, Detroit, U.S.A.) (20 ph 0.01 mi-’ RPM1 1640) was dispensed into each of three cultures, adult A. cuntonensis antigen (Yong & Dobson, 1982b) (20 pg O.Ol-’ RPM1 1640) was added to each of three other cultures. Three untreated cultures were retained as controls. The cultures were vortex-mixed and incubated upright at 37°C and mixed every 12 h until the cells were harvested 4 days later. Each culture received 37 kB tritiated thymidine; specific activity 74 GBq mmol -?
at intervals as before. All rats were killed 120 days after the co~encement of the experiment, worms were recovered and counted. Statistical analyses were done where appropriate using Student’s Ytest. RESULTS
The development and numbers of A. cantoneMs recovered from rats with 1, 2 and 3 concurrent infections are given in Yong & Dobson (1983). Briefly, fewer A. cantonensis derived from secondary or tertiary infections survived to the adult stage (6 and 2 worms in the second and the third infections respectively) than those derived from primary infection (26 worms). However, the total size of the concurrent adult worm populations remained the same as that from a primary infection.
Pattern of circulating white blood cells in rats infected with A. cantonensis (Radiochemical Centre, Amersham, England) dispensed in The cell counts for infected rats are given as either 0.05 ml RPM1 1640 16 h before harvesting when the positive or negative changes from the mean cell cultures were stopped and the red blood cells lysed by counts of non-infected rats done at the same time. adding two drops of Zaponin to each culture. The remaining white cells were filtered on to Whatman GF 81 The total WBC counts from infected rats were greater than those from non-infected rats. They glass fibre pads (W. & R. Balston Ltd., England), washed increased after infection, particularly after the with 30 mi 0.15 M saline, bleached with 2 ml hydrogen peroxide and dehydrated with 10 ml methyl alcohol. Each juvenile worms migrated from the brain 35 days until pad was placed in a 12 x 47 mm glass pill vial (Australian 60 days after infection @ < 0.01) (Fig. 1A). The Glass Manufacturers Company, Sydney) and dried for 6 h WBC counts declined thereafter to stabilize above at 37°C. Scintillation fluid (2 ml) containing 6.0 g PPO and the non-infection control level 75 days after infec0.2 g POPOP 1-l toluene was added to each vial, stoppered, tion. The WBC count declined shortly after second placed in scintillation vials and counted for 20 min at 2% and third reinfection, but soon returned to normal preset error in a Beckman LS-100-C scintillation counter (Beckman Instruments Inc., California, U.S.A.) after 4 h levels and was elevated at the time the parasite returned to the lungs following reinfection (Fig. dark adaptation. The radioactivity in counts min-’ for each 1A). sample was converted to disintegrations min-’ lo6 lymphocytes-’ (d.p.m.) and the lymphocyte profiferative response, There was an initial loss of circulating lymphocytes induced by worm antigen or PHA, was expressed as transfollowing A, cantonensis infections in rats after 1, 2 formation ratios (T.R.). and 3 infections. In each case this was followed by an d.p.m. antigen stimulated cultures elevation of lymphocyte numbers when the juvenile T.R. = worms migrated to the lungs from 35 days after d.p.m. unstimulated cultures Experimentaldesign. DA strain rats were infected with 50 infection. A further depletion of circulating lymphoA. cantonensisthird-stage larvae at intervals of 40 days on cytes occurred 70 days after a primary infection to three occasions. E&h@,rats were infected with A. cantonensis levels below those in non-infected rats. Reinfection accentuated the patterns of lymphocyte traffic in the larvae and 20 were kept as uninfected controls. Sterile cardiac blood was taken at intervals from 10 rats in the circulation displayed during primary infections. By infected and non-infected groups. All rats in each group the end of the experiment the twice reinfected rats were bled and no animal was bled on two successive showed greatly elevated lymtihocyte counts (p < occasions. The blood was used for in vitro lymphocyte O+OOl)(Fig. 1B). cultivations, to assess total WBC numbers and to differenCnculating monocyte numbers were elevated tiate WBC types. Forty days after the primary infection during primary, secondary and tertiary infections in 20 rats were retained in the primary infection group and the rest were reinfected with 50 A. canfonensislarvae. A rats; reinfection accentuated the increase in numbers of monocytes (Fig. 1C). further 40 rats were given primary infections from the same batch of infective larvae and a further 10 rats were included The pattern of circulating neutrophil leucocytes among the non-infected controls. Rats from all groups during primary infections generally has a bimodal (continuing primary infections, new primary infections, distribution. They increased early in the primary secondary infections, and non-infected controls) were bled infections but the counts returned to control levels 45 at intervals for the same reasons as before. When the original days after infection until about 90 days when they primary infection had run 80 days, rats with secondary were again elevated. Neutrophil leucocyte counts infections, and 20 of the second group of rats with primary were also elevated in reinfected rats but in each case infections were retained and the remainder were reinfected there was a loss of cells from the blood after rewith 50 A. cantonensis larvae. Ten further non-infected infection (Fig. 1D). control rats were added to the experiment and 20 additional rats were given primary infections. Ail these rats were bled A single infection with A. cantonensis larvae
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proliferation to PHA preceded that induced by worm antigen and occurred when the adult worms left the brain for the lungs (3540 days). The lymphocyte response to PHA again became elevated about 100 days after infection but the response against worm antigen did not (Fig. 2). The proliferation of rat peripheral blood lymphocytes induced by parasite antigen was depressed immediately, and up to 20 days after reinfection but thereafter increased to a peak after 40 days. These variations in the lymphocyte proliferative responses were more accentuated after a third compared with those after a second infection. The in vitro responses of blood lymphocytes taken at different times from reinfected rats to PHA were similar to but preceded those induced by parasite antigen (Fig. 2). DISCUSSION
The WBC count increased in all the infected rats studied, however, in prolonged infections without reinfection it returned to levels similar to that in uninfected rats. increased WBC levels are common in mammals infected with metazoan parasites
10
DAYS AFTER
INFECTION
FIG. 1. Changes in (A) total white blood cells, (B) lymphocytes, (C) monocytes, (D) neutrophil leucocytes and (E) eosinophil leucocytes in peripheral blood of rats given a ) and two (O---O) and three infections of 50 Angiostntngyltls cantonensis larvae. Each point represents the differences
in the mean cell number of an average six infected and noninfected animals caused a rapid increase in the numbers of eosinophil leucocytes in the blood; reinfection greatly accentuated this response giving a distinct bimodal pattern. Eosinophil leucocyte count ‘eturnec to the control values in prolonged primary infections (Fig. 1E). No differences were detected in the basophn leucocyte counts of rats with primary and multiple concurrent infections compared with uninfected rats. In vitro responses of ~eripherai lymphocytes from rats infected with A. cantonensis Transformation ratios (TR) for cultured lymphocytes from the three successive primary infections in rats were not different and the data were pooled. Similar compilations were made for data from rats given two secondary infections. The in vitro proliferation of rat peripheral blood lymphocytes to parasite antigens (Fig. 2A) and PHA (Fig. 2B) was greatly depressed immediately after, and up to 10 days following infection with A. ca~tone~s~, but thereafter increased between 10 and 35 days after infection. The peak of lymphocyte
FIG. 2. The transformation ratios of lymphocytes from rats and two (0 - - - 0) and three given a primary (@ -------a) concurrent infections of 50 Angiostrongyhs (A-A) cantonensis larvae when cultured in vitro with (A) adult A. cantonerd antigen and (B) phytohaemaggiutinin. Hatched horizontai bar represents transformation ratios (mean it LE.) of iymphocytes from non-infected rats (average six animals at each point).
210
W. K.
YONG
and G.
(Charleston, 1964, Cypess, 1972). The mitigation of A. cantonensis to the brain and lungs involves tissue damage and inflammation which would account for the elevated traffic in neutrophil leucocytes following primary infection. Dobson (1968) and Cypess (1972) related similar accumulations of neutrophil leucocytes in the blood of rodents infected with other nematode parasites to the induction of inflammatory reactions. The less marked neutrophilia following reinfection probably related to the involvement of these cells in antibody mediated protective immune reactions. Indeed, whenever the blood lymphocyte count and in vitro response of these cells to parasite mitogens was high the neutrophil leucocyte count was low and vice versa, Larsh (1967) showed a similar reversal in lymphocyte-neutrophil leucocyte ratio together with elevated WBC in mice infected with Trichinella spiralis. Marked eosinophilia, particularly in the cerebrospinal fluid, is characteristic of A. cantonens~ infections in rodents and man (Ottolenghi, Weatherly, Kocan & Larsh, 1977; Au & Ko, 1979). In the present infections eosinophilia developed progressively following infection and reinfection; it was bimodal and related to the tissue and adult phase of each infection. Eosinophil leucocytes are believed to function in protective immunity against metazoan parasites. They may proliferate in association with thymus-processed lymphocytes and be activated by antibody- and lymphocyte-antigen reactions (Walls, Basten, Leuchars & Davies, 1971; Rothwell & Dineen, 1972). In this study, the numbers of eosinophi1 leucocytes were consistently high throughout with peak responses denoting the developmental and reproductive stages of the parasite life cycle. The primary eosinophil leucocyte peak occurred when sensitized circulating lymphocytes which proliferated in vitro in response to PHA and parasite antigen left the blood. It is possible therefore that the eosinophil leucocyte proliferates and responds to both the cellular and antibody products of thymus dependent immunological responses. Protective immunity against A. cantonensis has powerful antibodymediated and less efficient lymphocyte-mediated components (Yong & Dobson, 1982d). Sensitized T lymphocytes and their lymphokine products have been previously implicated in the induction by helminths and their eggs of peripheral blood eosinophilia (Colley, 1973; Phillips, DiConza, Gold & Reid, 1977). Thus eosinophilia occurred in these rats when A. cantonensis was highly immunogenic as it developed and when it reproduced and subsided thereafter when the major reproductive effort and antigen production of the parasite declined. Traffic in eosinophil leucocytes could also relate to parasite immunosuppressive activities which have been attributed to A. cantonensis by Yoshimura & Soulsby (1976). ‘1ne m vitro replicative responses of the peripheral lymphocytes to parasite antigens declined dramati-
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cally immediately following each infection which indicated either a marked increase in the migration of lymphocytes from the circulation or a change in their function. A similar change in the activities of peripheral lymphocytes has been observed by Dobson & Soulsby (1974) in guinea pigs infected with T~ichost~o~gyl~s co~~briformis. It is probably that both nonspecifically active (Naor & Sulitzeanu, 1967; Mandel & Byrt, 1971) and specifically sensitized lymphocytes were attracted to the invading A. cantonensis larvae at these times. Yong & Dobson (1982b) showed that anti-A. cantonensk serum antibodies were also depleted in these animals at this time and attributed this to the manifestation of antibodyantigen reactions involved in protective immunity, which supports this view. It is interesting that the circulating eosinophil leucocyte count peaked at this time which suggests that eosinophil leucocyte proliferation may be a response to these reactions and may not have had an immediate involvement in the protective immune reaction (Speirs, Speirs & Ponzio, 1974). Elevated antiparasite lymphocyte TR were observed after the adult worms had migrated from the brain to the lungs 40-65 days after infection or reinfection. Yoshimura & Soulsby (1976) demonstrated a similar peak response from rat peripheral blood lymphocytes to A. cantonensis antigens 70 days after infection. Dobson & Soulsby (1974) also found that the major response of infected guinea pig peripheral blood lymphocytes to T. colubriformis antigens related to the appearance of the adult parasite. The proliferation of lymphocytes in the presence of sensitizing antigens has been interpreted as a memory response (Dutton, 1967; Vischer & Stastny, 1967). Dobson & Soulsby (1974) suggested that the elevation of the peripheral blood lymphocyte response following infection represented the parenteral dissemination of such memory cells. The present results can be interpreted in the same way because lymph~~e proliferation accelerated and generated greater intensity with each reinfection. Moreover the dynamics of the serum anti-A. cantonensis antibody titres corresponded to those for the anti-parasite lymphocyte TR (Yong & Dobson, 1982b). The activities of these circulating cells also suggest that protective immunity acted largely against the larval stages of A. Cantonese in rats. This conclusion is supported by experiments which showed that protective immunity could be passively transferred with immune lymph node cells and serum (Yong & Dobson, 1982b, c). PHA responsive lymphocytes comprise a population of uncommitted thymus-dependent cells capable of being stimulated to transform by and to react with specific antigen (Weber, 1967). The appearance of elevated numbers of these cells in the circulation soon after each infection suggests that they were involved in the induction and potentiation of the protective immune response. There was a
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dynamic relationship between the presence of PHA and A. cantonerds antigen responsive lymphocytes in the blood of infected rats suggesting that the circulation of uncommitted cells preceded that of similar sensitized thymus dependent lymphocytes res~ns~bIe for the dissemination of immunologic~ memory. Acknowledgements-This work was supported by a grant from the Australian Research Grants Committee. The authors wish to thank Miss W. C. Huf for excellent typing.
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