The Schistosoma japonicum Egg Granuloma Kenneth S. Warren, MD, Dov L. Boros, MD, Le Minh Hang, MD, and Adel A. F. Mahmoud, MD
Although Schistosoma japonicum egg granulomas are generally considered to be similar to those of S. mansoni (which are largely immunologic reactions of the delayed hycpersensitivity type) there are suggestions that the histopathology and perhaps the etiology of the lesions are different. In mice with light S. japonicum infections. at 5 weeks after infection (2 weeks after egg production began), the livers contained 36.000 eggs each. but there was no reaction to the eggs. nor any evridence of hepatosplenic disease. By 6 weeks, large abscesses replete with eosinophils occurred around some of the eggs. and there was periportal inflammation consisting predominantly of plasma cells. From this time on. major lesions occurred mainly around large aggregates of eggs. and there was hepatosplenomegaly and portal hypertension. Living S. japonicum eggs injected into the pulmonary microvasculature of mice did not evoke significant granulomatous reactions on either primary or secondary exposure. Even when the eggs were injected into the lungs of infected animals, which had large granulomas around egg aggregates in the liver, little or no inflammatory reaction was seen around the eggs distributed singly- throughout the pulmonary -essels. When the priming dose of eggs or soluble egg antigens was injected subcutaneously with or without complete Freund's adjuvant. significant granuloma formation occurred around eggs subsequently injected into the lungs. On the basis, therefore, of differences in the parasite factor (eggs) and host factors (histopathology and responses to routes of injection) it is suggested that the immunologic factors responsible for granuloma formation around S. mansoni and S. japonicum eggs may differ significantly. (Am J Pathol 80:279-294. 1975)
TEXTBOOKS OF CLINICAL PARASITOLOGY, tropical medicine, liver disease, and pathology make little or no distinction among schistosomiasis japonica. mansoni, and haematobia with respect to pathogenesis and clinical disease. -4 The one exception is the greater severity ascribed to S. japonicum infection because of higher ouitput of eggs by the worms.5 There are actuallv numerous other differences among the infections stuch as size, degree of aggregation of the eggs, and their tendency to calcify .6 8 It has also been stuggested in the literature that S. japonicum eggs produice more severe lesions than those of S. mansoni and S. haematobium.5 Carefuil studies of the pathogenesis of schistosomiasis mansoni in experimental animals have shown that the determining factor in the development of hepatosplenic disease is the host granulomatouis response From the Division of Geographic Nledicine. Department of \Medicine. Case Western Resenre Universitv and University Hospitals. Cleseland. Ohio Suipported by Grant AI-31814-06 and Contract PH4:3-67-7.36 from the US-Japan Cooperativ.e Mledical Science Program administered by the National Institiite of Allerg- and Infectiouis Diseases. National Instittutes of Health Accepted for ptublication March 17. 1973 Address reprint requiests to Dr Kenneth S. Warren. Division of Geographic \Medicince. W\earn Research Bujilding. University Hospitals. Cleveland. OH 44106 279
280
WARREN ET AL
American Journal of Pathology
to the schistosome eggs trapped in the liver and that this response is largely an immunologic reaction of delayed hypersensitivity type.9'1 Recent comparative sttudies of schistosomiasis mansoni, haematobia, and japonica, however, suggest that the histopathology of the lesions in the livers of animals infected with S. japonicum may differ from that seen with the other two schistosomes. 12-15 When the lung injection techniquie of von Lichtenberg was used,'6 a major difference in the host response to S. japonicum eggs became apparent.17 While injection of S. mansoni and S. haematobium eggs into the pulmonary microvasculature revealed a sensitization phenomenon that was relatively specific, S. japonicum eggs did not evoke a significant granulomatouis reaction on primary injection nor did prior exposure to eggs result in a secondary reaction.17 In the present study a detailed examination of the early response to S. japonicum eggs in the livers of infected animals was made, and the reactions to eggs injected into the pulmonary arterioles were studied further. On the basis of differences in histopathology, kinetics, and response to the rouite of injection of the antigens, it appears that the host reaction to S. japonicum eggs is ftundamentally different from that to S. mansoni or S. haematobium eggs. Materials and Methods Most of the animals uised in these stuidies were obtained from the Mluselum of Zoology of the University of Michigan. Swiss albino female mice, 18 to 22 g in weight, were infected with cercariae of a Philippine strain of S. japonicum obtained from infected Oncomelania quadrasi snails. The mice were anesthetized by an intraperitoneal injection of sodiuim pentobarbital, their abdomens shaved and moistened with spring water, and 10 cercariae placed on the skin of each animal. At 4, 5, 6, 8, and 10 weeks after exposuire, groulps of 10 mice chosen at random were weighed and anesthetized as described above. The peritoneal cavity was opened, a needle connected to a pressture transduicer was inserted into the portal vein, and the pressuire was recorded. When the needle was removed from the vein, blood was collected in a heparinized capillary ttube for microhematocrit determination. The thoracic cavity was then opened, and the esophagtus examined for the presence of varices. The liver and spleen were removed and weighed; a small portion of the liver was preserved in 10%O bhiffered formalin for sectioning and staining with hematoxylin and eosin and with Archer's modification of the Leishman stain for eosinophils 18; the remainder was weighed again and frozen for stubseqtuent counting of eggs as previoulsly described."9 The 5-g-thick liver sections from each animal were examined for the following characteristics: ntumber of eggs and percent egg aggregates (3 or more eggs in a groulp); stage of matturity of eggs as described recently by von Lichtenberg et al.13; presence of grantulomas and the types of cells comprising them; degree of necrosis and fibrosis in the lesions; and degree of periportal inflammation and the cells involved. Necrosis, fibrosis, and periportal inflammation were scored in each section on the basis of 0 to 3+. For precisely timed stuidies of granuiloma formation, the techniquie developed by von Lichtenberg was titilized 16: schistosome eggs were isolated from the livers of n-iice 20 exposed 7 weeks previouisly to 50 cercariae of S. japonicum of either a Philippine or Celebes strain, the latter of which was sttudied at NAMRU-2 in Taiwan with the aid of Dr. John H. Cross, Jr. Eggs were then injected intravenouisly via a tail vein into the ptulmonary
Vol. 80. No. 2 August 1975
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microvasculature of young female mice At sarious time periods thereafter. randomly chosen grouips of mice u-ere anesthetized. 1 ml of 10%- buiffered formalin injected intratracheallv. and the ltngs removed and placed in a container of the same solultion Sections from each ltung. 5-M-thick and at least 2.30 p apart. u ere stained X-ith hematox%lin and eosin or u-ith the Archer modification of the Leishman stain " and examined for the presence of eggs The size of each egg. incluiding the reaction arouind it, was determined by measturing tuso diameters at right angles to each other %sith a Vicker's AEI image-splittin,g eyepiece For each experimental grouip the mean diameter of at least 25 lesions was calcuilated. and the sections searched for a lesion representatixe of the mean. u hich was marked and then photographed Initial stuidies in olx ed injection of 2000 eggs of the Philippine strain into prex iousl tinexposed. uininfected mice and the remoxal of ltungs at 3. 6, 12. 24. 48. 96. and 192 houirs Next, grotups of mice x-ere injected intraperitoneally wvith large numbers of eggs of either the Philippine or Celebes strain of S. japonicum 2 weeks prior to intravenouis injection of eggs, Ilngs \-ere removed at 8 days Stuidies \-ere then performed by the intravenous injection of eggs into grouips of mice u-ith nattural infections one grouip was exposed to 10 cercariae of a Philippine strain. eggs u-ere injected at 7 weeks of infection and the ltngs removed 8 davs later: the other grouip Alas exposed to 3 cercariae of a Celebes strain. eggs mvere injected at 1 3 .5 andi xweeks into infected mice (bisexuial infectionegs in liver! and matching controls utinisexuial infection-no eggs in liver and the lulngs u-ere remoxved S davs later Soluible egg antigens (SEA) w-ere prepared from S. japonicum eggs by homogenization and uiltracentriftugation as presiously described for S. mansoni.1' Protein concentration (Lowry) was 230 ug ml. w-hich u-as equixalent to 50.000 eggs ml U sing the SEA. and also intact eggs. mice x-ere exposed to the follou-ing sensitizing regimens: SEA t10.000 egg equiivalentsi intraperitoneally. stibeitaneously. and suibcuitaneously uwith complete Freuind's adjutvant: eggs suibeutaneously (3000) and eggs suibeutaneouisly with complete Freuind's adjuv ant Tu-o weeks later. 2000 intact eggs u-ere injected intravenously. and the Ilungs were removed 8 days thereafter
Results Eggs in the Liver in Early Infections
In mice infected wvith S. japonicum the worms begin to produce eggs at abouit 3 -eeks. By 4 weeks the average liver egg couints totalled almost 8,000, buit there -as no granuloma formation, no significant enlargement of the liver or spleen, nor any increase in portal pressture (Table 1). Even at 3 weeks, when verx high mean egg couints were seen (36,000). granuiloma formation wvas minimal. as wvere signs of disease (Table 1). By 6 weeks, sporadic large necrotic granuilomas were seen, and the onset of significant hepatomegaly, splenomegaly and portal hypertension wvas obser-ed (Table 1). Bx 8 and 10 Xweeks, grantilomas became more ty-pical in appearance wvith less necrosis and more histiocvtes, fibrosis ensuied, and hepatosplenic schistosomiasis developed fullx (Table 1). Careftul examination of the histopathologv of the livers at 4 weeks revealed no inflammation Xw-hatsoever around the eggs, almost all of w-hich wvere in the earliest stages of embryonic development (Table 2, Figture 1). At 3 weeks. 2 wveeks after egg production had begun, only minimal grantilomas consisting largely of eosinophils were seen (Table 2. Figture
282
American Journal of Pathology
WARREN ET AL
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WARREN ET AL
American Journal of Pathology
Table 3-Granuloma Formation Around Schistosoma japonicum Eggs at Different Time Periods After Their Intravenous Injection Into the Pulmonary Arterioles of Mice Time after egg injection (hrs)
Mean granuloma diameter ± SE*
0 3 6 12 24 48 96 192
47±1.0 46±1.7 51 ±1.8 54+1.8 52±0.9 59 ± 3.2 53±2.7
(I)
51 ± 2.8
* Four mice/25 granulomas at each time period.
2). The eggs had advanced in development but were still largely immature. Mild periportal inflammation made up principally of histiocytes was observed. At 6 weeks some of the eggs had matured, and large granuilomas first appeared. Many of these lesions, which were unique to this time period, displayed severe necrosis and abscess-like agglomerations of polymorphontuclear cells which were largely eosinophils (Table 2, Figure 3). Periportal inflammation was more pronounced and, strikingly, included large ntumbers of plasma cells (Table 2, Figure 4). At 8 and 10 weeks the eggs were largely mature, and by 10 weeks many had reached the degenerate stage (Table 2). The granulomas, which showed far less necrosis, still consisted largely of eosinophils, but on a base of histiocytes and fibroblasts, and fibrosis was becoming more and more evident (Figure 5). The massive grantulomas typical of S. japonicum were found around the large aggregates of eggs which made up 20 to 30% of the egg grotups seen in the tisstue sections (Table 2). Marked periportal inflammation was seen, and at this time the cells consisted largely of eosinophils (Table 2, Figuire 6). Eggs Injected into the Pulmonary Microvasculature
Exceedingly little inflammatory activity was observed arouind S. japonicum eggs injected into the pulmonary microvasculature when stuidied at frequent intervals from 3 hours to 8 days after egg injection (Table 3). Exposure to massive numbers of eggs by intraperitoneal injection 2 weeks prior to the intravenous injection of eggs was associated with little or no increase in the host granulomatouis response to the eggs (Table 4). Finally, when living eggs isolated from the livers of mice infected for 7 weeks were injected into the pulmonary microvasculatture of mice with natuiral infections at the point of peak granuloma formation in the liver (7
Vol. 80. No. 2 August 1975
SCHISTOSOMA GRANULOMAS
285
to 8 wveeks), little granulomatous inflammation wvas seen in the Ilungs (Table 4). This experiment was repeated in mice each exposed to three cercariae of a Celebes strain of S. japonicum. Approximately half of the animals developed one pair of worms with attendant egg produiction; the remainder had uinisexual infections. Using the latter as controls, granuloma formation around eggs injected into the lung arterioles wvas compared at 2, 4, 6, and 8 weeks of infection with negative results (Textfigture 1). Further studies were then planned with adjuvants in an attempt to induce granulomatous hypersensitivity around the eggs injected into the lungs. When either SEA or intact eggs were emtulsified with complete Freund's adjuvant and injected subcutaneously, marked granuloma formation occurred around eggs subsequently injected into the lungs (Table 3, Figures 7-9). Significant granuloma formation also occuirred in control mice which had been sensitized by the subcutaneous injection of SEA or eggs alone (Table 3, Figures 7-9). It should be noted that in a previous experiment in which SEA in complete Freund's adjuvant was injected intraperitoneallv bv mistake, there was no significant granuiloma formation around eggs in the lungs. Careful histologic examination of the lesions around the eggs injected into the lungs revealed that the majority of the cells w-ere polymorphonuclear, and on staining with both hematoxx lin and eosin and Archer stains, thex appeared to be virtually all eosinophils. Table 4-Granuloma Formation Around Schistosoma japonicum Eggs Eight Days After Their Intravenous Injection Into the Pulmonary Arterioles of Mice Previously Exposed to Eggs No. of mice/No. of granulomas measured
Mean granuloma diameter ± SE u)
Experiment 1 -intraperitoneal injection of eggs of the Celebes strain of S. japonicum 2 weeks prior to intravenous injection Control 6/75 64 ± 2.2 5,000 eggs IP 6/81 67 ± 1.6 50,000 eggs IP 6/87 58 ± 1.9 Experiment 2-intraperitoneal injection of eggs of the Philippine strain of S. japonicum 2 weeks prior to intravenous injection Control 4/25 59 t 2.4 5,000 eggs IP 4/29 72 ± 7.2 10,000eggslP 4/25 61 s5.4
Experiment 3-exposure to 10 cercariae of the Philippine strain of S. japonicum 8 weeks previously (egg production begins at 3 weeks) Control Infected IP
=
intraperitoneally.
4/25 4/25
56 s 2.3 72 ±6.6
WARREN ET AL
286
American Journal of Pathology TEXT-F I(G.
w
I=
w
cr +1
25 20
U' P i _
-
Although Schistosoma japonicum egg granulomas are generally considered to be similar to those of S. mansoni (which are largely immunologic reactions of the delayed hycpersensitivity type) there are suggestions that the histopathology and perhaps the etiology of the lesions are different. In mice with light S. japonicum infections. at 5 weeks after infection (2 weeks after egg production began), the livers contained 36.000 eggs each. but there was no reaction to the eggs. nor any evridence of hepatosplenic disease. By 6 weeks, large abscesses replete with eosinophils occurred around some of the eggs. and there was periportal inflammation consisting predominantly of plasma cells. From this time on. major lesions occurred mainly around large aggregates of eggs. and there was hepatosplenomegaly and portal hypertension. Living S. japonicum eggs injected into the pulmonary microvasculature of mice did not evoke significant granulomatous reactions on either primary or secondary exposure. Even when the eggs were injected into the lungs of infected animals, which had large granulomas around egg aggregates in the liver, little or no inflammatory reaction was seen around the eggs distributed singly- throughout the pulmonary -essels. When the priming dose of eggs or soluble egg antigens was injected subcutaneously with or without complete Freund's adjuvant. significant granuloma formation occurred around eggs subsequently injected into the lungs. On the basis, therefore, of differences in the parasite factor (eggs) and host factors (histopathology and responses to routes of injection) it is suggested that the immunologic factors responsible for granuloma formation around S. mansoni and S. japonicum eggs may differ significantly. (Am J Pathol 80:279-294. 1975)
TEXTBOOKS OF CLINICAL PARASITOLOGY, tropical medicine, liver disease, and pathology make little or no distinction among schistosomiasis japonica. mansoni, and haematobia with respect to pathogenesis and clinical disease. -4 The one exception is the greater severity ascribed to S. japonicum infection because of higher ouitput of eggs by the worms.5 There are actuallv numerous other differences among the infections stuch as size, degree of aggregation of the eggs, and their tendency to calcify .6 8 It has also been stuggested in the literature that S. japonicum eggs produice more severe lesions than those of S. mansoni and S. haematobium.5 Carefuil studies of the pathogenesis of schistosomiasis mansoni in experimental animals have shown that the determining factor in the development of hepatosplenic disease is the host granulomatouis response From the Division of Geographic Nledicine. Department of \Medicine. Case Western Resenre Universitv and University Hospitals. Cleseland. Ohio Suipported by Grant AI-31814-06 and Contract PH4:3-67-7.36 from the US-Japan Cooperativ.e Mledical Science Program administered by the National Institiite of Allerg- and Infectiouis Diseases. National Instittutes of Health Accepted for ptublication March 17. 1973 Address reprint requiests to Dr Kenneth S. Warren. Division of Geographic \Medicince. W\earn Research Bujilding. University Hospitals. Cleveland. OH 44106 279
280
WARREN ET AL
American Journal of Pathology
to the schistosome eggs trapped in the liver and that this response is largely an immunologic reaction of delayed hypersensitivity type.9'1 Recent comparative sttudies of schistosomiasis mansoni, haematobia, and japonica, however, suggest that the histopathology of the lesions in the livers of animals infected with S. japonicum may differ from that seen with the other two schistosomes. 12-15 When the lung injection techniquie of von Lichtenberg was used,'6 a major difference in the host response to S. japonicum eggs became apparent.17 While injection of S. mansoni and S. haematobium eggs into the pulmonary microvasculature revealed a sensitization phenomenon that was relatively specific, S. japonicum eggs did not evoke a significant granulomatouis reaction on primary injection nor did prior exposure to eggs result in a secondary reaction.17 In the present study a detailed examination of the early response to S. japonicum eggs in the livers of infected animals was made, and the reactions to eggs injected into the pulmonary arterioles were studied further. On the basis of differences in histopathology, kinetics, and response to the rouite of injection of the antigens, it appears that the host reaction to S. japonicum eggs is ftundamentally different from that to S. mansoni or S. haematobium eggs. Materials and Methods Most of the animals uised in these stuidies were obtained from the Mluselum of Zoology of the University of Michigan. Swiss albino female mice, 18 to 22 g in weight, were infected with cercariae of a Philippine strain of S. japonicum obtained from infected Oncomelania quadrasi snails. The mice were anesthetized by an intraperitoneal injection of sodiuim pentobarbital, their abdomens shaved and moistened with spring water, and 10 cercariae placed on the skin of each animal. At 4, 5, 6, 8, and 10 weeks after exposuire, groulps of 10 mice chosen at random were weighed and anesthetized as described above. The peritoneal cavity was opened, a needle connected to a pressture transduicer was inserted into the portal vein, and the pressuire was recorded. When the needle was removed from the vein, blood was collected in a heparinized capillary ttube for microhematocrit determination. The thoracic cavity was then opened, and the esophagtus examined for the presence of varices. The liver and spleen were removed and weighed; a small portion of the liver was preserved in 10%O bhiffered formalin for sectioning and staining with hematoxylin and eosin and with Archer's modification of the Leishman stain for eosinophils 18; the remainder was weighed again and frozen for stubseqtuent counting of eggs as previoulsly described."9 The 5-g-thick liver sections from each animal were examined for the following characteristics: ntumber of eggs and percent egg aggregates (3 or more eggs in a groulp); stage of matturity of eggs as described recently by von Lichtenberg et al.13; presence of grantulomas and the types of cells comprising them; degree of necrosis and fibrosis in the lesions; and degree of periportal inflammation and the cells involved. Necrosis, fibrosis, and periportal inflammation were scored in each section on the basis of 0 to 3+. For precisely timed stuidies of granuiloma formation, the techniquie developed by von Lichtenberg was titilized 16: schistosome eggs were isolated from the livers of n-iice 20 exposed 7 weeks previouisly to 50 cercariae of S. japonicum of either a Philippine or Celebes strain, the latter of which was sttudied at NAMRU-2 in Taiwan with the aid of Dr. John H. Cross, Jr. Eggs were then injected intravenouisly via a tail vein into the ptulmonary
Vol. 80. No. 2 August 1975
SCHISTOSOMA GRANULOMAS
281
microvasculature of young female mice At sarious time periods thereafter. randomly chosen grouips of mice u-ere anesthetized. 1 ml of 10%- buiffered formalin injected intratracheallv. and the ltngs removed and placed in a container of the same solultion Sections from each ltung. 5-M-thick and at least 2.30 p apart. u ere stained X-ith hematox%lin and eosin or u-ith the Archer modification of the Leishman stain " and examined for the presence of eggs The size of each egg. incluiding the reaction arouind it, was determined by measturing tuso diameters at right angles to each other %sith a Vicker's AEI image-splittin,g eyepiece For each experimental grouip the mean diameter of at least 25 lesions was calcuilated. and the sections searched for a lesion representatixe of the mean. u hich was marked and then photographed Initial stuidies in olx ed injection of 2000 eggs of the Philippine strain into prex iousl tinexposed. uininfected mice and the remoxal of ltungs at 3. 6, 12. 24. 48. 96. and 192 houirs Next, grotups of mice x-ere injected intraperitoneally wvith large numbers of eggs of either the Philippine or Celebes strain of S. japonicum 2 weeks prior to intravenouis injection of eggs, Ilngs \-ere removed at 8 days Stuidies \-ere then performed by the intravenous injection of eggs into grouips of mice u-ith nattural infections one grouip was exposed to 10 cercariae of a Philippine strain. eggs u-ere injected at 7 weeks of infection and the ltngs removed 8 davs later: the other grouip Alas exposed to 3 cercariae of a Celebes strain. eggs mvere injected at 1 3 .5 andi xweeks into infected mice (bisexuial infectionegs in liver! and matching controls utinisexuial infection-no eggs in liver and the lulngs u-ere remoxved S davs later Soluible egg antigens (SEA) w-ere prepared from S. japonicum eggs by homogenization and uiltracentriftugation as presiously described for S. mansoni.1' Protein concentration (Lowry) was 230 ug ml. w-hich u-as equixalent to 50.000 eggs ml U sing the SEA. and also intact eggs. mice x-ere exposed to the follou-ing sensitizing regimens: SEA t10.000 egg equiivalentsi intraperitoneally. stibeitaneously. and suibcuitaneously uwith complete Freuind's adjutvant: eggs suibeutaneously (3000) and eggs suibeutaneouisly with complete Freuind's adjuv ant Tu-o weeks later. 2000 intact eggs u-ere injected intravenously. and the Ilungs were removed 8 days thereafter
Results Eggs in the Liver in Early Infections
In mice infected wvith S. japonicum the worms begin to produce eggs at abouit 3 -eeks. By 4 weeks the average liver egg couints totalled almost 8,000, buit there -as no granuloma formation, no significant enlargement of the liver or spleen, nor any increase in portal pressture (Table 1). Even at 3 weeks, when verx high mean egg couints were seen (36,000). granuiloma formation wvas minimal. as wvere signs of disease (Table 1). By 6 weeks, sporadic large necrotic granuilomas were seen, and the onset of significant hepatomegaly, splenomegaly and portal hypertension wvas obser-ed (Table 1). Bx 8 and 10 Xweeks, grantilomas became more ty-pical in appearance wvith less necrosis and more histiocvtes, fibrosis ensuied, and hepatosplenic schistosomiasis developed fullx (Table 1). Careftul examination of the histopathologv of the livers at 4 weeks revealed no inflammation Xw-hatsoever around the eggs, almost all of w-hich wvere in the earliest stages of embryonic development (Table 2, Figture 1). At 3 weeks. 2 wveeks after egg production had begun, only minimal grantilomas consisting largely of eosinophils were seen (Table 2. Figture
282
American Journal of Pathology
WARREN ET AL
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Vol. 80, No. 2 August 1975
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284
WARREN ET AL
American Journal of Pathology
Table 3-Granuloma Formation Around Schistosoma japonicum Eggs at Different Time Periods After Their Intravenous Injection Into the Pulmonary Arterioles of Mice Time after egg injection (hrs)
Mean granuloma diameter ± SE*
0 3 6 12 24 48 96 192
47±1.0 46±1.7 51 ±1.8 54+1.8 52±0.9 59 ± 3.2 53±2.7
(I)
51 ± 2.8
* Four mice/25 granulomas at each time period.
2). The eggs had advanced in development but were still largely immature. Mild periportal inflammation made up principally of histiocytes was observed. At 6 weeks some of the eggs had matured, and large granuilomas first appeared. Many of these lesions, which were unique to this time period, displayed severe necrosis and abscess-like agglomerations of polymorphontuclear cells which were largely eosinophils (Table 2, Figure 3). Periportal inflammation was more pronounced and, strikingly, included large ntumbers of plasma cells (Table 2, Figure 4). At 8 and 10 weeks the eggs were largely mature, and by 10 weeks many had reached the degenerate stage (Table 2). The granulomas, which showed far less necrosis, still consisted largely of eosinophils, but on a base of histiocytes and fibroblasts, and fibrosis was becoming more and more evident (Figure 5). The massive grantulomas typical of S. japonicum were found around the large aggregates of eggs which made up 20 to 30% of the egg grotups seen in the tisstue sections (Table 2). Marked periportal inflammation was seen, and at this time the cells consisted largely of eosinophils (Table 2, Figuire 6). Eggs Injected into the Pulmonary Microvasculature
Exceedingly little inflammatory activity was observed arouind S. japonicum eggs injected into the pulmonary microvasculature when stuidied at frequent intervals from 3 hours to 8 days after egg injection (Table 3). Exposure to massive numbers of eggs by intraperitoneal injection 2 weeks prior to the intravenous injection of eggs was associated with little or no increase in the host granulomatouis response to the eggs (Table 4). Finally, when living eggs isolated from the livers of mice infected for 7 weeks were injected into the pulmonary microvasculatture of mice with natuiral infections at the point of peak granuloma formation in the liver (7
Vol. 80. No. 2 August 1975
SCHISTOSOMA GRANULOMAS
285
to 8 wveeks), little granulomatous inflammation wvas seen in the Ilungs (Table 4). This experiment was repeated in mice each exposed to three cercariae of a Celebes strain of S. japonicum. Approximately half of the animals developed one pair of worms with attendant egg produiction; the remainder had uinisexual infections. Using the latter as controls, granuloma formation around eggs injected into the lung arterioles wvas compared at 2, 4, 6, and 8 weeks of infection with negative results (Textfigture 1). Further studies were then planned with adjuvants in an attempt to induce granulomatous hypersensitivity around the eggs injected into the lungs. When either SEA or intact eggs were emtulsified with complete Freund's adjuvant and injected subcutaneously, marked granuloma formation occurred around eggs subsequently injected into the lungs (Table 3, Figures 7-9). Significant granuloma formation also occuirred in control mice which had been sensitized by the subcutaneous injection of SEA or eggs alone (Table 3, Figures 7-9). It should be noted that in a previous experiment in which SEA in complete Freund's adjuvant was injected intraperitoneallv bv mistake, there was no significant granuiloma formation around eggs in the lungs. Careful histologic examination of the lesions around the eggs injected into the lungs revealed that the majority of the cells w-ere polymorphonuclear, and on staining with both hematoxx lin and eosin and Archer stains, thex appeared to be virtually all eosinophils. Table 4-Granuloma Formation Around Schistosoma japonicum Eggs Eight Days After Their Intravenous Injection Into the Pulmonary Arterioles of Mice Previously Exposed to Eggs No. of mice/No. of granulomas measured
Mean granuloma diameter ± SE u)
Experiment 1 -intraperitoneal injection of eggs of the Celebes strain of S. japonicum 2 weeks prior to intravenous injection Control 6/75 64 ± 2.2 5,000 eggs IP 6/81 67 ± 1.6 50,000 eggs IP 6/87 58 ± 1.9 Experiment 2-intraperitoneal injection of eggs of the Philippine strain of S. japonicum 2 weeks prior to intravenous injection Control 4/25 59 t 2.4 5,000 eggs IP 4/29 72 ± 7.2 10,000eggslP 4/25 61 s5.4
Experiment 3-exposure to 10 cercariae of the Philippine strain of S. japonicum 8 weeks previously (egg production begins at 3 weeks) Control Infected IP
=
intraperitoneally.
4/25 4/25
56 s 2.3 72 ±6.6
WARREN ET AL
286
American Journal of Pathology TEXT-F I(G.
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