KINETICS OF TRYPANOSOMA CRUZI DESTRUCTION IN THE MOUSE SPLEEN

By sequentially and histologically studying the evolution of a virulent Y strain infection in young mice, two striking features were observed: a) the progressive increase of macrophage parasitism in the spleen followed by a massive destruction of both macrophages and parasites at the height of infection, which coincided with a sudden drop in parasitemia and which often preceded animal death; b) when mounting splenic parasitism was abrogated by chemotherapy, infected animals survived for prolonged periods of time, splenic parasitism completely disappearing, while chronic infection followed its course. Thus, the dramatic and massive parasite destruction represents a transition from susceptibility to immunity. Although several well-conducted sequential histological studies of the spleen in acute T. cruzi infection with the Y strain have been made, most of the characteristics of this transitional phase here considered have been overlooked 5 9 .

During infection of mice with a virulent Y strain of Trypanosoma cruzi, macrophages appear as the predominant site of parasite multiplication.This macrophagotropism is a prominent feature of Type 1 strains, which include the Y strain and the Peruvian strain as prototypes 1 .
By sequentially and histologically studying the evolution of a virulent Y strain infection in young mice, two striking features were observed: a) the progressive increase of macrophage parasitism in the spleen followed by a massive destruction of both macrophages and parasites at the height of infection, which coincided with a sudden drop in parasitemia and which often preceded animal death; b) when mounting splenic parasitism was abrogated by chemotherapy, infected animals survived for prolonged periods of time, splenic parasitism completely disappearing, while chronic infection followed its course.Thus, the dramatic and massive parasite destruction represents a transition from susceptibility to immunity.Although several well-conducted sequential histological studies of the spleen in acute T. cruzi infection with the Y strain have been made, most of the characteristics of this transitional phase here considered have been overlooked 2 5 9 10 .
The present work attempts to investigate by light and electron microscopy the splenic changes leading or preceding the transition from susceptibility to resistance during a virulent T. cruzi infection in vivo.

MATERIAL AND METHODS
White swiss mice, 10/12g, of both sexes were intraperitoneally injected with 1,000 to 20,000 blood forms of T. cruzi in eight series of experiments, each involving about 50 animals.Parasitemia was evaluated daily for the first five groups and, for the other three groups, every other day, starting on day 5 th .Mortality was very high.Even with small inoculum (5,000 trypomastigotes) mortality was total by the 10-11 th day, eventually reaching the 15 th /18 th day in those receiving the smallest inoculum (1,000 trypomastigotes).Animals found dead were discarded.Following inoculation the animals were sacrificed on the 5 th , 7 th , 10 th , 12 th , 14 th , 15 th and 18 th day.
To avoid high mortality, a group of 95 mice infected with 2,000 to 5,000 trypomastigotes was treated on the 7 th day of infection with 50mg/kbw of benznidazole daily, during 5 days.They were sacrificed at different periods of time, up to 30 days after infection.Parasitemia was considerably reduced after treatment and remained low thereafter (Figure 1) and the animals presented normal development with no signs of disease.Sacrificed animals were submitted to complete autopsies.The spleen was weighted.Fragments of the spleen, liver, heart and  Parasitemia was performed in a drop of blood obtained from the mouse tail, covered with a cover slip and immediately examined with 40x objective x 10x ocular.One-hundred fields were counted and results were expressed as averages (Figure 1).

RESULTS
Parasitemic curves are shown in Figure 1.At light microscopy parasitism of splenic macrophages was detected on day 5 th in isolated cells present specially within the marginal zone of the lymph follicles.Occasionally, macrophages in the red pulp also appeared parasitized at this time.Parasitism became more evident from the 7 th day on.Amastigotes in cells of the white pulp appeared later (15 th day).Progressive accumulation of parasitized cells in the spleen was followed at first by relatively mild changes in splenic histology.Only after the 7 th day of infection, three main changes became progressively more constant: hyperplasia of non-parasitized macrophages, depletion of lymphoid cells in the red pulp and activation of germinal centers of the white pulp.Depletion of lymphocytes was probably due to apoptosis, since many of them exhibited cytoplasmic condensation, nuclear clumping, picnosis and cariorrhexis.Non-parasitized macrophages, presented clear and enlarged cytoplasm, tending toward an epithelioid appearance.The center of the lymphoid follicles and the marginal perifollicular zones were expanded and the latter gradually fused with the red pulp.Dark dots, probably nuclear fragments, were frequently found within phagocytes in the center of the white pulp.After the 7 th day of infection a few, isolated, parasitized macrophages disintegrated and the liberated amastigotes were seen within the shrunken and vacuolated macrophage cytoplasm or free in the interstitial tissue.This was regularly accompanied by an influx of polymorphonuclear neutrophils, which sometimes were seen with phagocytized amastigotes (Figure 2).Macrophage parasitism, lymphoid-cell depletion and macrophage hyperplasia became accentuated in the following days.However, the most striking change was observed around the 12 th -15 th days when intracellular parasitism was at its highest point.Then, all parasitized cells appeared swollen, fragmented, containing disintegrating parasites (Figure 3).No polymorphonuclear leukocytes were seen at this time, when a single parasitized macrophage could ever be detected, even after a thorough search of the histological sections.As indicated before these changes were coincident with a sharp decline in parasitemic curve and this event was regularly followed by the death of the animal.
Macrophages in the liver (Kupffer cells) also contained amastigotes and revealed the same changes observed in splenic macrophages (Figure 4).However, sections of the myocardium showed well preserved parasites within myocytes at the same time parasitized cells in t h e s p l e e n a n d l i v e r w e r e u n d e rg o i n g destruction (Figure 4).Some animals were sacrificed next day after parasitemic drop and so the degradative changes could be analyzed by electron microscopy.
Then, every intra and extra-cellular parasites showed evidences of degradation (Figure 5).Amastigotes were found within the cytoplasm of disintegrating macrophages or free in the interstitial tissue.In both cases they exhibited nuclear and cytoplasmic degenerative alterations.They usually presented an irregular outline and shrunken, dark, vacuolated cytoplasm and nuclear picnosis or cariorrhexis (Figure 6).The kinetoplast/mitochondrial complexes were markedly swollen and/or vacuolated.All parasitized macrophages were partially or totally destroyed, but the non-parasitized ones appeared normal.Frequently aggregated and partially degranulated platelets were observed within splenic capillaries (Figure 7), which showed excessive signs of pinocytosis and irregular vacuolization.Sometimes cells with the characteristics of myofibroblasts (fusiform cells with dark submembranar contractile apparatus) exhibited focal proliferation, forming clusters in the middle or in close proximity to disintegrated macrophages.(Figure 8).
Two animals survived up to the 18 th day of infection, 4 days following parasitemic drop.One was agonizing, but the other seemed in fair condition.No parasites were seen in their spleens, but numerous viable macrophages containing nuclear and other debris within the cytoplasm were observed.Lymphoid-cell depletion was not a striking feature as in the previous cases.Accumulation of plasmocytoid and other basophilic lymphoid cells appeared at several scattered foci within the red pulp.Focal accumulation of macrophages, sometimes with a micro-granulomatous structure, was also observed.The marginal zone and the lymph follicles became more apparent, suggesting a returning to the normal splenic structure.However, a few clear, empty areas were present within the red pulp, suggesting the dropping out of splenic cells.
The spleens taken from treated mice were essentially within normal limits and no parasites were seen within macrophages or elsewhere in the spleen after 5 days of

DISCUSSION
Sequential observation of splenic changes during a virulent T. cruzi infection in the mouse revealed that parasitism of splenic macrophages progressed up to a point of massive involvement and then, suddenly, there was a diffuse destruction of parasitized cells and parasites.Significance of this finding involves immunologic and pathogenetic a s p e c t s o f g r e a t i m p o r t a n c e .I m m u n e destruction of infected cells probably means the expression of foreign antigen(s) in the macrophage external membranes.Such antigens are then a target to sensitized cytotoxic lymphocytes, specific antibodies, and/or antibody-mediated cell citotoxicity.Why the destruction occurred suddenly is a matter probably related to the reaching of an equivalence or critic point amongst immunologic factors 15 .
This would mean that the host became strongly immune, but paradoxically this transition is often fatal, probably due to the liberation of toxic products by the disintegrating cells.One of such product could well be TNF (tumor necrosis factors), since it is synthesized in great amount by macrophages parasitized by T. cruzi 13 and has been hold responsible for generalized toxic symptoms and even shock 10 .E v e n t u a l d i s i n t e g r a t i o n o f p a r a s i t i z e d macrophages occurring before the episode of massive cell destruction may depend on a different pathogenesis, since the host reaction was then limited to a focal infiltration by polymorphonuclear leukocytes.Similar findings observed in the connective tissue of mice have been correlated with local antigen-antibody reaction and hypocomplementemia 12 .
Macrophages are probably the first cells to be infected by T. cruzi.Trypomastigotes entering or being phagocytized by naive macrophages readly escape from the phagocytic vacuole and get into the cell cytosol, where they successively multiply as amastigotes.However, if macrophages are stimulated by sensitized T-cell lymphocyte soluble products, they become able to rapidly destroy internalized trypomastigotes 6 7 .These early events are crucial and may determined the outcome of the infection 14 .Suppressive chemotherapy accelerates this transitional phase and thus allows for a direct passage to the chronic stage, as has been demonstrated here and by others 3 .Probably, the elimination of parasites from the spleen would expose adequate antigens to the immune system and/or would interferes with the generation of suppressive splenic cells or other suppressive factors 4 .
Findings observed in two animals that apparently survived the critical period of massive parasitized-cell destruction are suggestive that reconstitution of splenic structure may be quite r a p i d a n d c o m p l e t e .T h a t c a p a c i t y f o r regeneration shown by the spleen is in keeping with observations made in other lymphoid tissues 8 .

Figure 1 -
Figure 1 -Parasitemic curves: a) animals inoculated with 20,000 trypomastigotes; b) shows a somewhat less severe course seen in animals inoculated with 4,000 trypomastigotes; c) it demonstrates the effect of treatment, which was administered from the 7th up to the 12th day of infection.

Figure 2 -
Figure 2 -Infiltration of polymorphonuclear neutrophil leukocytes around disintegrating parasitized macrophages (small arrows) as observed at the 7 th day of infection.Heavily infected but well preserved macrophages are also present (larger arrows).Hematoxylin & Eosin, 250X.

Figure 3 -
Figure 3 -Aspect of the red splenic pulp during the phase of massive cellular destruction.Parasitized macrophages appear swollen, pale and vacuolated.Internalized parasites exhibit irregularities in nuclear size and staining (arrows) Hematoxylin & Eosin, 400X.

Figure 4 -
Figure 4 -Parasitized macrophages inside the hepatic sinusoids show disintegration in a similar way and at the same time as the splenic macrophages during the phase of massive cell destruction (arrow).Hematoxylin & Eosin, 160X.

Figure 5 -
Figure 5 -Section of the myocardium showing a large well preserved amastigote collection inside a myocyte.There is no reaction within the myocardial area seen in this picture.Hematoxylin & Eosin, 160X.

Figure 6 -
Figure 6 -Electron micrograph showing disintegrating amastigotes within a vacuolated splenic cell cytoplasm.The parasites exhibit an irregular outline, condensation and vacuolation of the cytoplasm, tumefaction of the mitochondrial-kinetoplasm apparatus and picnosis (arrows).7,000X.