Allergic inflammation as a hypothesis for the expulsion of worms from tissues: A review

Allergic inflammation as a hypothesis for the expulsion of worms from tissues: A review

EXPERIMENTAL PARASITOLOGY 37, 251-286 ( 1975) PARASITOLOGICAL Allergic REVIEW Inflammation as a Hypothesis for the Expulsion of Worms from Tissu...

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EXPERIMENTAL

PARASITOLOGY

37, 251-286

( 1975)

PARASITOLOGICAL Allergic

REVIEW

Inflammation as a Hypothesis for the Expulsion of Worms from Tissues: A Review JOHN E. LARSH, JR.

Department

of Parasitology and Laboratory Practice, School of Public University of North Carolina at Chapel Hill, 27514

Health,

AND GEORGE J. RACE Department of Pathology, Baylor University Medical Center, 3500 Gayton and University of Texas Health Science Center, Dallas, Texas, 75246

Acenzce,

LARSH, JOHN E., JR., AND RACE, GEORGE J. 1975. Allergic inflammation as a hypothesis for the expulsion of worms from tissue: A review. Experimental Parasitology 37, 251-266. Introduction; comparison of studies of mice infected with TrichineUa spiralis; discussion; summary; references. INDEX DESCRIPTORS: Parasitological Reviews; Allergy; Helminths, expulsion; Immunity; Mice; Trichinella spiralis; Irradiation, X; Antithymocyte serum; Spleen.

(Dineen et al. 1973; Ogilvie and Jones 1973). On the basis of results from the use of a variety of experimental designs in the

INTRODUCTION

In recent years, interest in the mechanism for expulsion of worms from the intestinal tissues has increased. For example, in their thorough review of work on Nippostrongylus brasiliensis in 1971, Ogilvie and Jones suggested that the mechanism is a “two-step” process: (1) an immunologically specific effect of antibodies on the worms that produces demonstrated damage, followed by (2) an immunologically nonspecific event as part of the inffammatory process that releases amines and directly causes the already damaged worms to be expelled. Later, after successful productions of adoptive immunity by the transfer of sensitized lymphoid cells, this hypothesis was revised so that the second step was suggested to be due to the effect of lymphokines from the transferred cells, which interferes with the metabolism of the worms, thereby causing their expulsion

Trichindlu

model,

it had

much earlier that adverse

changes accompanying

intestinal inflamma-

tion make the injured areas unsuitable persistence Therefore, common

of the there

worms

is not

mechanism

tive in a variety

(Larsh

agreement

for

1963). on a

that might be effec-

of models.

This review

was prepared because of the importance of encouraging more work on the mechanism( s) for expulsion

of worms from all

injured tissues. Aside from advancing knowledge on this facet of parasite immunology, such information should aid in devising better methods of immunodiagnosis, improved means for artificial immunization ( vaccination), and, conceiv251

Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.

spiralis mouse

been suggested

2Jj’,

LARSH

ably, procedures for restoring immunocompetence. For comparison, we will present first in chronological order observations from a series of histopathologic studies with the T. spiralis mouse model. Direct comparisons of studies in this series are possible because the same strain of parasite and host, as well as the same experimental techniques, were used in all instances. For this reason, and in the interest of saving space, photomicrographs from the first paper in the series (Larsh and Race 1954) will be included as points of reference (Figs. l12). After discussion of the hypothesis for expulsion of worms based on the results of these studies, selected hypotheses from studies in other models will be presented and discussed. Thereafter, the review will be concluded with a summary. COMPARISON OF STUDIES OF MICE INFECTED WITH Trichinella

spiralis

1. Observations after an Initial Infection of Mature Mice with 400 Trichinella spiralis Larvae These mice were six months old at the time of infection (Larsh and Race 1954). Sections of intestinal tissue taken 12, 24 (Fig. 2), 48, and 72 hr after infection evidenced no inflammatory response. HOWever, one day later (four days after infection) there was a mild, early reaction adjacent to worms (Fig. 6), and by six days after infection small numbers of neutrophils were noted (Fig. 8), indicating that the acute stage of inflammation had been initiated. This stage reached a zenith on Day 8, when considerable numbers of neutrophils were observed throughout the mucosa and submucosa (Fig. 10). By the tenth day, a subacute or chronic reaction

AND

HACE

was developing, as evidenced by the presence of moderate numbers of plasmacytes, lymphocytes, and macrophages (Fig. 11). This mixed mononuclear exudate still was evident but was diminishing on Day 14 (Fig. 12), the last observation period. Thus, the response to the tissue injury was evident in three distinct stages: (1) a mild, early reaction with few neutrophils, (2) a moderately severe acute reaction with numerous neutrophils that peaked in numbers on Day 8, and (3) a subacute or chronic diminishing reaction characterized by mixed mononuclear cells. 2. Observations in Mature Sensitized Mice after a Challenging Infection with 400 Trichinclla spiralis Larvae These mice, of the same age as those above, had been sensitized at intervals by four previous stimulating infections of 200 larvae each (Larsh and Race 1954). They were challenged with the same batch of larvae and at the same time as the above nonsensitized mice. Three similar stages of were observed: intestinal inflammation (1) a mild, early reaction that developed within the first 12-24 hr after infection when small numbers of neutrophils were noted throughout the mucosa and around the worms (Fig. 1 ), (2) a rapidly developing, severe acute reaction (with the neutrophil as the dominant cell) that reached its peak on the 4th day when a severe, acute panmucosal and submucosal inflammatory reaction was evident (Figs. 35), and (3) a subsiding subacute or chronic reaction characterized by the presence of mixed mononuclear cells (Figs. 7 and 9) that was noted from the fifth to the eighth day, when the last observation was made. Thus, the inflammatory response in the non-

FIGS. l-12. Photomicrographs of sections of the small intestine of mice stained with hematoxylin and eosin (from Larsh and Race 1954 with permission of the Editor, Journal of Infectious Diseases, and the University of Chicago Press). The “nonsensitized” mice were controls given only the challenging infection with TMzineZZa spirulk; the “sensitized” mice had been infected with four prior stimulating infections with T. spiralis before being given the challenging infection.

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AND

WORM

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HYPOTHESIS

1. From a previously sensitized mouse 24 hr after challenge. Note the section of a and only minimal inflammation. X298. FIG. 2. Nonsensitized control mouse one day after infection. Note the worm and relative absence of inflammation. x298. FIG. 3. Sensitized mouse four days after infection, showing an invading adult worm and polymorphonuclear leukocytes throughout the mucosa. x200. FIG. 4. From a sensitized mouse four days after challenge. The cellular infiltration is composed almost entirely of polymorphonuclear leukocytes. X612. Frc.

worm,

253

254

LARSH

AND

RACE

FIG. 5. Sensitized mouse four days after challenging infection. Note the mucosal inflammatory reaction and the submucosal infiltration of plasma cells, lymphocytes and polymorphonuclear leukocytes. X 111. FIG. 6. From a nonsensitized mouse four days after infection. Note the invading adult worm, and the minimal inflammatory reaction. X 162. FIG. 7. From a sensitized mouse six days after challenge. Note the worm sections, and the mixed inflammatory infiltration composed of plasma cells, lymphocytes, macrophages, an d polymorphonuclear leukocytes. X153. FIG. 8. Nonsensitized control mouse six days aftcr infection. The mucosa contains small numbers of polymorphonuclear leukocytes. X 145.

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AND

WORM

EXPULSION

HYPOTHESIS

FIG. 9. Sensitized mouse eight days after infection. The inflammatory reaction is diminished and the predominant cells are mononuclear. X230. FIG. 10. From a nonsensitized mouse eight days after infection. The mucosa and subwith polymorphonuclear leukocytes, mucosa are intensely inflamed, and are infiltrated lymphocytes, and plasma cells. X238. FIG. 11. Nonsensitized control mouse 10 days after infection. The inflammatory reaction is less intense than in Fig. 10. The cellular infiltrate is similarly mixed in type. X174. FIG. 12. From a nonsensitized mouse 14 days after infection. Note the adult worm adjacent to a vein, The cellular infiltration is composed of lymphocytes, plasmacytes, macrophages, and a few polymorphonuclear leukocytes. X136.

2Ti.i

2.56

LARSH

scnsitizcd and sensitized mice was the same, including the cellular components, except for its earlier initiation and greater severity in the sensitized mice. At seven days after challenge, the nonsensitized mice harbored significantly more adult worms in the small intestine than did the sensitized mice; hence, the accelerated and more intense inflammatory response noted in the latter group was correlated with significantly greater worm expulsion and, therefore, with the sensitivity status of these mice at the time of challenge. An earlier study (Larsh et nl. 1952) had shown that nonsensitized mice of similar age expel significant numbers of adult worms between 11 and 14 days after chalsensitized lenge, whereas in previously mice this occurs between five and seven days. Therefore, in both groups the worms were lost a few days after the peak of the acute inflammatory response. This suggested a hypothesis that somehow the inflammation is responsible for the loss of worms, and that the timing and effectiveness of worm expulsion is related to the degree of sensitivity of the host at the time of challenge. Therefore, because young animals, which in most species are not fully immunocompetent, develop sensitivity at a slower rate than do mature ones, we decided to test these suggestions in immature mice. 3. Observntions ufter an Initial Infection of Immature Mice with 300 Trichinella spiralis Larvae These mice were five weeks old at the time of infection (Larsh et al. 1956). There was a significant expulsion of adult worms from the small intestine between 15 and 17 days after infection, or at least three days later than noted in mature nonsensitized mice ( Larsh et al. 1952). Associated with this longer period required for expelling the worms was a corresponding difference in the timing of the inflammatory response in the proximal second one-fourth of the

AND

RACE

small intcstinc. Mild inflammation was first noted seven days after infection, and the acute phase did not reach its peak until 11 days after infection, or three days later than noted in mature nonsensitized mice ( Larsh and Race 1954). There was no detectable difference between the inflammatory response, including the cellular components, in the two groups, except that it was initiated sooner and was more severe in the mature mice. Therefore, the slower and less severe response of the immature mice in all probability was due to the fact that they had not reached full immunocompetence at the time of challenge. It is generally agreed that such animals are incapable of responding to antigenic stimulation as effectively as are fully competent counterparts. The results of the above studies of mature nonsensitized mice, mature sensitized mice mice, and immature nonsensitized were consistent with the hypothesis that the inflammation is responsible for the loss of worms, and that the timing and effectiveness of this loss due to the inflammation is related to the degree of sensitivity of the host at the time of challenge. Therefore, according to this hypothesis, prevention of the inflammatory response or striking inhibition of it should prevent the loss of worms, regardless of the degree of sensitivity established by a previous stimulation( s). For this reason, we decided to test this by the use of sustained cortisone treatment of previously sensitized mice. 4. Observations in Cortisone-Treated, Sensitized Mice after a Challenging Infection with 400 Trichinella spiralis Larvae These mice, five months old at challenge, had been sensitized by three stimulating infections with 200 larvae each at intervals of three weeks (Coker 1956). Three days before challenge and daily until necropsy, they were injected subcutaneously with 0.5 mg of cortisone. At 12 and 24 hr after challenge, the cells in the lamina propria of

ALLERGIC

INFLAMMATION

AND

these mice were almost entirely mononuclear ( plasmacytes, small lymphocytes, and rare macrophages), and neutrophils and eosinophils were rare or absent. Little change was noted on Days 2 and 3, when it was obvious that the cell population was sparse. On Day 4, the villi, by now long and thin and filled with blood, were almost barren of enclosed cells other than the supporting stroma of fibroblasts, other connective tissue, and extensions of the muscularis mucosae. On Days 5, 6, 7, and 9 there was little change from Day 4. Contrasted with these findings, those for the untreated sensitized controls compared favorably to those presented above for mature sensitized mice in the earlier study (Larsh and Race 1954). The main difference was the observation here of infiltrating mononuclear cells (large and small lymphocytes, macrophages, and some plasma cells) at 12 and 24 hr after challenge. This strong suggestion for a delayed hypersensitivity reaction in the areas of antigen deposit was not noted in the earlier work. Perhaps the reason was the fact that in the earlier study the mice had been more strongly sensitized (four vs three stimulating infections ) , which should have resulted in a more effective anamnestic response after challenge. If so, an earlier influx of neutrophils in response to the tissue injury could have masked the early presence of mononuclear cells. Another difference between the observations of untreated, sensitized mice in this and the earlier study was the abundance of eosinophils ( Coker 1956)) which first were noted at I2 hr after challenge, and tended to become more widely dispersed throughout the lamina propria as the reaction developed. This apparent discrepancy probably was due to the use of different staining techniques in the two studies. The use of stains specific for eosinophils, such as Lendrum’s chromotrope 2R, would resolve the problem of differentiating these cells from neutrophils examined

in tissues

under the light microscope.

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257

The complete suppression of the inflammatory response in the cortisone-treated mice was causally related to the persistency of the adult worms. In fact, at seven days after challenge, there was not a significant difference in the numbers of worms harbored by these mice and the nonsensitized controls that had received only the challenging infection, In other words, cortisone had inhibited completely the expression of the sensitivity produced by the stimulating infections administered before exposure to the steroid. Therefore, these results supported the hypothesis that inflammation somehow is responsible for the expulsion of the worms. In the light of present knowledge, it is likely that cortisone did not interfere with #the specific sensitization reaction between T-cells and antigen, but rather affected the macrophages (Weston et al. 1973). That cortisone had a similar effect on the persistence of adult worms in nonsensitized mice, three months old, was shown in our laboratory after daily subcutaneous injections made two days before infection until necropsy (Coker 1955). The cortisone-treated mice harbored significantly more worms than the controls at 14 days after challenge, and at 30 days after infection, the cortisone-treated mice harbored more worms in the small intestine than had been noted in the untreated controls at 14 days. Therefore, it is clear in this instance that cortisone prevented entirely the development of sensitivity. In further attempts to relate the timing and intensity of intestinal inflammation and worm loss with the degree of sensitivity of the animals at the time of challenge, studies were made of mice sensitized with irradiated larvae to eliminate one or more phases of the life cycle. 5. Observations in Mice Sensitized with Irradiated #(7000 R) Larvae of Trichinella spiralis and Challenged with 500 Nonirradiated Larvae These mice were 7.7 months old at the time of challenge ( Larsh et al. 1959). For

2is

LAHSH

sensitization by Phase I worms ( preadults) alone, at intervals they had been given five stimulating infections each with 200 irradiated (7000 R) larvae. At seven days after challenge with normal larvae, these mice harbored more worms (average of 314) than the controls sensitized with nonirradiated larvae (average of 262.3), but the difference is not significant. Studies of tissue sections from the controls sensitized with nonirradiated larvae showed that the inflammatory reactions were similar to those of their counterparts in the first study with mice six months old at challenge (Larsh and Race 1954). However, the reactions were less severe and more time was required for the zenith of the acute phase to be reached (between four and six days after infection vs four days in the earlier study). Also, the initiation of the acute phase was delayed (two days vs 12 hr). The fact ‘that the nonsensitized controls also showed less severe inflammation compared with that in their counterparts in the earlier study, and showed a delay in reaching the zenith of the acute response (between eight and 10 days vs eight days) might suggest that these mice, which were 7.7 months old at challenge, were less immunocompetent than those of six months of age in the earlier study. Other possibilities are that the additional stimulating infection used here (five vs four) partially overwhelmed the immune system, or, alternatively, produced a greater degree of desensitization. In any event, the mice sensitized with irradiated larvae had developed a degree of sensitivity, based after challenge on the numbers of adult worms in the small intestine and larvae in the muscles, that was comparable to that of the controls sensitized with nonirradiated larvae. However, the inflammatory response developed slowly and failed to reach the severity noted in the sensitized controls. Moreover, the zenith of the response was delayed when compared with these controls (between

eight

and

10 days

after

infec-

AND

RACE

tion vs four and six days). There was also a difference when comparisons were made with the nonsensitized controls, Their inflammatory response, as in the instance of the sensitized controls, was less severe than that noted in similar mice six months of age (Larsh and Race 1954), but the zenith of the acute response occurred at about the same time in the two groups of the present study, i.e., between eight and 10 days. In contrast, the mice sensitized with irradiated larvae responded soon after challenge (12 hr) rather than after a lag of several days, but the response developed so slowly that, as noted, neither did it peak until between eight and 10 days. This is interpreted to mean that whereas the infections produced by the irradiated larvae (limited to the Phase I preadults) were immunogenic, based on worm counts after challenge, they were not nearly as effective as those produced by nonirradiated larvae in sensitizing the animals, thereby after challenge resulting in a lesser degree of tissue damage and inflammation. 6. Observations in Mice Sensitized with frradiuted (3.500 R) Larvae of Trichinella spiralis anal Challenged with 200 Nonirradiated Larvae These mice were 7.5 months old at the time of chalIenge (Larsh et al. 1962). For sensitization by Phases I and II (preadults; steriIe adults ) alone, at intervaIs they had been given 4 stimulating infections each with 200 irradiated (3500 R) larvae. After challenge with normal larvae, sections of intestinal tissue of the nonsensitized controls at seven days after infection showed a minimal submucosal inflammatory rcaction, but with sufficient numbers of scattered neutrophils to indicate the initiation of the acute stage. However, although the reaction was similar in all respects, the degree of reaction here at seven days was considerably less than that noted at six days in the nonsensitized mice of the earlier study (Larsh and Race 1954). This

ALLERGIC

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AND

might suggest again that this strain of mice at 7.5 months of age is less immunocompetent than those of six months, but in this instance it is just as likely that the difference in the severity of the response was related to the smaller number of larvae used for challenge (200 vs 400 in the earlier study). In any event, the previously sensitized mice at seven days after challenge were strongly sensitized, based on the number of adult worms recovered at this time, and showed a subacute or chronic inflammatory reaction with the characteristic mixture of mononuclear cells, mostly lymphocytes and plasma cells. Although the reaction at this time was similar to that observed in mice stimulated with nonirradiated larvae (Larsh and Race 1954), in the present instance the reaction in these mice was less intense at seven days after infection than in their counterparts at six days in the earlier study. In addition to the use here of a smaller challenging infection, these results might suggest that larvae irradiated with 3500 R are somewhat less effective in sensitizing mice than nonirradiated ones. When it was shown in our laboratory that whole-body radiation prevented loss of worms from strongly sensitized mice (Yarinsky 1962), it was decided to use this form of treatment to test again the hypothesis that worm loss is due directly to intestinal inflammation. 7. Observations in Mice Sensitized with Zrradiated (3500 R) Larvae of Trichinella spiralis and Then Exposed LOWhole-Body Radiation (450 R) before being Chatlenged with 200 Nonirradiated Larvae These mice were 7.5 months old at the time of challenge (Larsh et al. 1962). Four stimulating infections of 200 irradiated (3500 R) larvae each were given at intervals to limit the development of the worms to Phases I and II. At seven days after challenge ( 15 days after host irradiation), there was little evidence of intestinal in-

WORM

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2.59

flammation in tissues of the sensitized mice even in areas adjacent to the worms. However, on the basis of the presence of a few neutrophils, the reaction was classified as minimal and the one preceding the development of the acute phase. Notwithstanding, the degree of reaction was even less than that observed at this time for the nonsensitized controls given only the challenging infection. These findings correlated with the degree of sensitivity based on the number of adult worms recovered, since there was no significant difference between the two groups. Therefore, it is clear that the treatment had prevented entirely the expression of sensitivity produced in response to the stimulating infections given before the exposure to radiation. This association of persistence of worms in the absence of a normal acute inflammatory response is similar to the above findings in mice suppressed with cortisone after the establishment of sensitivity by several stimulating infections; the association after both treatments supports the view that the inflammation is the direct cause for worm expulsion. After it was shown by us that peritoneal exudate cells from infected donors conferred adoptive immunity upon recipients, it was decided to study the recipients in relation to the association of intestinal inflammation and expulsion of worms. 8. Observations in Recipients Znjected with Peritoneal Exudate Cells from Infected Donors Three Weeks before Challenge with Trichinella spiralis These mice were about 4.5 months old at the time of challenge (Larsh et al 1966). At 24 hr after challenge, there were larger numbers of infiltrating cells, including lymphocytes, than in controls not injected with cells. At four days, it was obvious by the numbers of neutrophils present in the recipients that the acute phase of the inflammatory response was under way; this was not discernible in the controls. By 10

260

LARSH

AND RACE

days, the intestinal mucosa of the recipients was intensely infiltrated with neutrophils, and inflammation was clearly much more severe than in the controls. The latter findings were associated the following day with a significant reduction of worms in the recipients. On the basis of comparison with nonsensitized controls in an carlier study (Larsh and Race 1954), the cellular components were similar, but the intensity of infiltration was considerably less in the present controls, presumably because of the much smaller dose of larvae used for challenge (100 vs 400 in the earlier study). The studies to this point had dealt with mice sensitized with normal or irradiated larvae, or with the recipients of peritoneal exudate cells from infected donors. Therefore, as a further test of the association of intestinal inflammation and worm expulsion, we decided to use mice sensitized artificially by footpad injections of a crude saline extract of larvae mixed in equal volume with Freund’s complete adjuvant. 9. Observations in Mice Artificially Sensitized with a Saline Extract Adigen of Trichinella spiralis and Then Suppressed u?ith Antithymocyte Serum (ATS) before and after Challenge These mice were four months old at the time of challenge (Race et al. 1974). On the day after the second footpad sensitization, they were injected daily with ATS until necropsy. After 16 injections had been made, they were challenged with 100 nonirradiated larvae. At two days after challenge, the tissue sections from these sensitized, suppressed mice indicated clearly that the 18 days of ATS treatment had produced striking depletion of small lymphocytes in the thymus-dependent paracortical areas of intestinal lymph nodes. There also was little or no evidence of an inflammatory response in the mucosa and submucosa even in areas adjacent to worms. At six and 10 days after challenge, there was no change, except that some large mononu-

clear cells and plasma cells were seen adjacent to the worms. Electron microscopy confirmed these observations of H and E sections, and proved the predominance of plasma cells in the intestinal tissue. Again, associated with the absence of an acute inflammatory response was the significant persistence of adult worms in the small intestine (Larsh et al. 1972). On the other hand, the sensitized, nonsuppressed controls and the nonsensitized, nonsuppressed controls showed the expected burden of worms at 11 days after challenge, and the characteristic pattern of intestinal inflammation produced by T. spiralis (Larsh and Race 1954); however, the inflammation was less severe due undoubtedly to the use of a much smaller challenging infection (100 vs 400 in the earlier study). Electron microscopy confirmed the observations made with H and E sections, and revealed by two days the presence of infiltrating plasma and other mononuclear cells in the intestinal tissues of the sensitized, nonsuppressed controls. These observations confirmed earlier ones (Coker 1956; Larsh et al. 1966, 1974a), and are consistent with the view that the early presence of various mononuclear cells was the result of a delayed hypersensitivity reaction in these areas of relrasc of antigen. Moreover, electron microscopy revealed the early presence and persistence of eosinophils (Race et al. 1974), which agrees with the observations of Coker ( 1956). The results with artificially sensitized mice compared favorably with those obtained earlier with mice sensitized with normal larvae; hence, the provocative antigen( s) must have been involved in all instances (Larsh and Weatherly 1974a). The failure of the suppressed mice to expel the worms after challenge and the absence in them of acute intestinal inflammation adds further support for the hypothesis that the inflammation is directly responsible for expulsion of worms. In the final observation to date, it was decided to test the association of inflamma-

ALLEKGlC

INFLAMMAl’ION

ANL)

tion and loss of worms in recipients of spleen cells from donors artificially sensitized as in the previous study and then suppressed with ATS. 10. Observations in Recipients Injected with Spleen Cells from Artificially Sensitized Donors Suppressed with ATS Daily for 16 Days before Collection of the Cells These recipients were four months old at the time of infection (Larsh et al. 1974a). At two days after challenge, light and electron microscopy verified the presence in these recipients only of numerous lymphocytes and large mononuclear cells; hence, the acute inflammatory response had not yet been initiated. On the other hand, the recipients of cells from sensitized, nonsuppressed donors showed clear evidence of this by the presence of moderate numbers of neutrophils. At 10 days after challenge, in the recipients of cells from the sensitized, suppressed donors the severity of the acute inflammation was clearly less than that noted in the tissues of the control recipients. Again, the difference in the severity of this response was associated the next day with the persistence of significantly more worms than in the controls. DISCUSSION

Based on the results of these 10 sets of observations in mice from 8 separate histopathologic reports from our laboratory with the same strain of parasite and host, we draw the following conclusions: (1) there is a close, direct association of a characteristic pattern of acute intestinal inflammation and subsequent expulsion of adult worms of T. spimlis; (2) there is a similar association between the degree of sensitivity of the host at challenge and the timing and intensity of inflammation and loss of worms; (3) th ere is a similar association between the size of the challenging infection and the degree of inflammation; and (4) there is a similar association between the persistence of worms and the absence

WORM

of the

EXPULSION

HYPOTHESIS

characteristic

acute

Xl inflammation.

IncidentaIIy, it has been shown recently that the phospholipase levels in intestinal tissues corresponded strikingly to the degree of inflammation based on the tissue changes reported in 1954 by Larsh and Race (Larsh et aZ. 197413); hence, this might lead to the discovery of the specific factor(s) that accounts for the elimination of the worms. As noted, some of the above observations were made in recipients that had developed adoptive immunity (Larsh et al. 1966, 1974a). This effectiveness of transferred cells also has been demonstrated repeatedly in other studies in our laboratory (Larsh et al. 1964a, 196413, 1969, 1970a, 1970b, 1972). Therefore, we have suggested that delayed hypersensitivity (DH) is causal in the e.xpulsion of the worms (Larsh and Weatherly 1974b, 1975). If one accepts this for the moment, it is noteworthy to recall that the DH reaction has two components: ( 1) an immunologically specific reaction between antigen-sensitive T-cells and the antigen, and (2) an ensuing immunologically nonspecific inflammatory reaction (allergic inflammation) to the tissue injury produced by the interaction of the specific elements in the first component (e.g., see Vassalli and McCluskey 1971). On these bases, the presence of inflammation as demonstrated in the above studies would be totally expected. Also, as noted, when inflammation was prevented in previously sensitized mice by use of treatments with cortisone, whole-body radiation, or ATS, the worms persisted in numbers comparable to those in challenged, nonsensitized controls. It is now known that cortisone interferes with DH by affecting macrophages (Weston et al. 1973), whole-body radiation by destroying nonspecific bone marrow precursors (Visakorpi 1972; Volkman and Collins 1968), and ATS by causing the destruction of T-cells (Lance 1970). Therefore, treatments known to abolish DH reactions precluded the ensuing tissue injury that follows normally and thereby

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precluded the nonspecific inflammatory response. Since the adult worms expelled from inflamed areas moved to, and established in, lower levels of the intestine, and because inflammation produced by other agents caused a significant degree of expulsion of T. spiralis (Larsh 1967), we have postulated that changes in the inflamed tissues are detrimental to the persistence of the worms (Larsh 1967; Larsh and Weatherly 1974b, 1975). In summary our complete hypothesis for the mechanism of expulsion of adult T. spiralis is: ( 1) an immunologically specific DH reaction between antigen-sensitive Tcells and antigen that results in tissue injury, followed by (2) an ensuing immunologically nonspecific inflammatory reaction to the injury that results in tissue changes unfavorable to maintenance of the worms. It should be added that one such tissue change, viz, acidosis, has been shown to be detrimental to these worms (Castro et al. 1973). In any event, it is obvious that this hypothesis is totally consistent with events accepted to occur in the DH reaction. Recent findings in another laboratory with this model strongly support our suggestion for the mechanism involved in expulsion of the worms (Walls et al. 1973). In these studies of “deprived” CBA/H mice (thymectomized, exposed to 850 R whole-body radiation, and injected intravenously with syngeneic bone marrow cells), it was noted #that the inflammatory response in the small intestine and in skeletal muscles was “negligible.” In fact, only small numbers of eosinophils were observed in the intestinal tissues lo-15 days after infection, whereas within three to 10 days mild submucosal inflammation was noted in controls, and there were infiltrations of neutrophils, eosinophils, lymphocytes, and macrophages. On the basis that there is no generalized depression in the inflammatory response of deprived animals, such as to oxazolone (Davies et al. 1969), these workers concluded that the striking interference with it in this instance was

AND RACE

due to T-cell deficiency. Associated with the negligible inflammatory response in the small intestine was the persistence of adult worms in the mucosa for as long as 38 days, and a fourfold greater number of larvae in the muscles at 70 days than in controls. A different mechanism has been suggested for the expulsion of Nippostrongylus brasiliensis from the small intestine of rats, and that for expulsion of Trichotirongylus colubriformis from this area in guinea pigs. In the instance of N. brasiZiensis, the mechanism was suggested to be diphasic: (1) an immunologically specific effect of antibodies on the worms that produces demonstrated damage, and th,en (2) the release of lymphokines from T-cells that has a direct effect on the metabolism of the worms and thereby causes their expulsion (Dineen et al. 1973; Ogilvie and Jones 1973 ) . For T. cokrbriformis, it was suggested that the mechanism is as follows: (1) an immunologically specific interaction between T-cells and antigen, which triggers myeloid involvement ( eosinophils and basophils ) and the release of amines, and (2) an immunologically nonspecific event, whereby directly or indirectly the action of the amines from these myeloid cells causes the rejection of the worms (Rothwell et aZ. 1971; Rothwell and Dinecn 1972 ) . Although excellent studies have resulted in data to indicate unquestionably the role of DH (generally considered by cellular immunologists to be synonymous with “cell-mediated immunity;” e.g., see Bernhard et al. 1973) as one of the two components in the mechanism of expulsion in both of these models, it is surprising that resulting local tissue injury and allergic inflammation were not mentioned specifically as having a possible role. In the instance of the N. brasiliensis hypothesis, the answer seems to lie in the assumption that transferred memory T-cells play no role for foul duys while damaging antibodies are pre-

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AND

paring the worms for expulsion. On the other hand, in the instance of the T. cdubriformk hypothesis, a different role is speculated for the T-cells, i.e., they initiate the mechanism by reacting with antigen before the second step can ensue. As reported elsewhere (Larsh and Weatherly 1975), the various results reported from recent studies with these two models can be explained logically by application of the hypothesis presented above for the T. ~idis model. One might argue that such an analogy is open to question because different host species are involved. However, it should be noted that recent evidence showed that the staining characteristics, distribution, and biologic actions of a set of lymphocytes in the rat corresponded exactly with T-cells in the mouse (Balch and Feldman 1974), hence a direct comparison with N. bmsiliensis is possible. The failure to mention the possible involvement of intestinal inflammation in the N. brasiliensis model is even more surprising when the results of the classic histopathologic studies reported 35 years ago by Taliaferro and Sarles (1939) are recalled. The timing of the initiation of inflammation after challenge, and the time required to reach the zenith of the acute response, differed in rats given a primary immune” infection and in “moderately counterparts. The cellular components of the response were the same in both groups, but inflammatory changes were evident sooner, were more intense, and involved more hematogenous cells in the previously infected rats. Therefore, the expulsion of transplanted worms (damaged by antibodies before transfer from donors) within five days after transfer of sensitized cells (Dineen et al. 1973) could be accounted for logically by the effects of inflammation, since by 3.5 days after challenge of “moderately immune” rats, intense inflammatory reactions were evident (Taliaferro and Sarles 1939). Therefore, in the interest of time in proving the mechanism( s ) for expulsion of

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worms, we believe it would be worthwhile to test our hypothesis in a wide variety of models that involve worms in tissues. The fact that it fits well the results of studies of Fasciala hepatica in the livers of mice (Lang 1967; Lang et al. 1967) and rabbits (Dodd and Nuallain 1969) might suggest that expulsion from all inflamed tissues has a common mechanism. After all, ‘the DH response and the resulting local changes can occur in any tissue after contact of T-cells with the provocative antigen. SUMMARY

It was first suggested 20 years ago from histopathologic studies of mice that intestinal inflammation is the direct cause for the expulsion of adult worms of T. spiralis ( Larsh and Race 1954). Over the years, a series of histopathologic studies with a variety of different experimental designs has been carried out to test this hypothesis (Larsh et al. 1956; Coker 1956; Larsh et al. 1959, 1962, 1966, 1974a; Race et al. 1974). The results in all instances are considered here to be consistent with this hypothesis. Moreover, we concluded from the results that: ( 1) there is a close, direct association between the degree of sensitivity of the host at challenge and the timing and intensity of inflammation and loss of worms; (2) there is a similar association between the size of the challenging infection and the degree of resulting inflammation; and (3) there is a similar association between the persistence of worms and the absence of the characteristic acute inflammation. Because lymphoid cells transferred from sensitized donors produced adoptive immunity in recipients (Larsh et al. 1964a, 1964b, 1966, 1969, 1970a, 1970b, 1972, 1974a), we suggest that delayed hypersensitivity ( DH) is causally related to the expulsion of T. spidis. In view of this, it is important to recall that the DH reaction has two components: ( 1) an immunologically specific reaction between antigensensitive T-cells and the antigen, followed by (2) an ensuing immunologically non-

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specific inflammatory reaction (allergic inflammation) to the tissue injury produced by interaction of the specific elements in the first component (e.g., see Vassalli and McCluskey, 1971). Therefore, if one accepts the first step of this characteristic reaction to be responsible as the specific basis for #the production of adoptive immunity, it follows logically that inflammation would be the sequential event. In this light, the above hypothesis that expulsion of the worms is due directly to this inflammation takes on added meaning. We suggest that the worms are expelled by this nonspecific tissue phenomenon, because of local changes in the injured areas that become unsuitable for their continued persistence. Different mechanisms for the expulsion of N. brmilierwis (Dineen et a,l. 1973; Ogilvie and Jones 1973) and T. colubriformis (Rothwell et al. 1971; Rothwell and Dineen 1972) have been proposed. These are presented, and we ‘suggest, on the basis of a different role proposed for transferred memory T-cells in the two models, that the proposals are open to question, and that the mechanisms can be explained equally well by our proposed hypothesis. We conclude this review with a plea that, in the interest of time in proving the mechanism( s ) for expulsion of worms from all injured tissues, the hypothesis presented here for expulsion of T. ,spidis be thoroughly tested in other models. ACKNOWLEDGMENTS Some of our work reported here was supported in part by Grant AI-10671 from NIAID. REFERENCES BALCH, C. M., ANU FELD~~AN, J. D. 1974. Thymus dependent (T) lymphocytes in the rat. Journul of Immunology 112, 79-86. BEHNHARD, J. D., ROSENFELD, S. S., ANU KLEIN, E. 1973. Blocking of delayed hypersensitivity by humoral antibody in an in uiuo mouse transfer assay using sensitized guinea pig cells. Cell&r Inmunology 8, 408-412.

CASTRO, G. A., COTTER, M. V., FERGUSON, J. D., AND GORDEN, C. W. 1973. Trichinosis: physiologic factors possibly altering the course of infection. Journal of Parasitology 59, 26& 276. COKER, C. M. 1955. Effects of cortisone on Trichinda spiralis infections in non-immunized mice. Journal of Parasitology 41, 498504. COKER, C. M. 1956. Cellular factors in acquired immunity to Trichinella spiralis, as indicated by cortisone treatment of mice. Journal of Infectious Diseases 98, 187-197. DAVIES, A. J. S., CARTER, R. L., LEUCHARS, E., AND WALLIS, V. 1969. The morphology of immune reactions in normal, thymectomized and reconstituted mice. II. The response to oxazolone. Immunology 17, 111-126. DINEEIL’, J. K., OGILVIE, B. M., AND KELLY, J. D. 1973. Expulsion of Nippostrongylus brasiliensis from the intestine of rats. Collaboration between humoral and cellular components of the immune response. Immunology 24, 467475. DODD, K., AND NUALLAIN, T. 0. 1969. Effect of antilymphocytic sera on the histopathology of Fasciolu hepatica infestation in rabbits. Journal of Pathology 99, 335-337. LAA-CE, E. M. 1970. The selective action of antilymphocyte serum on recirculating lymphocytes: A review of the evidence and alternatives. Clinical ancl Experimental Immunology 6,789-802. LANG, B. Z. 1967. Host-parasite relationships of Fascioku hepotica in the white mouse. II. Studies on acquired immunity. Journal of Purusitology 53, 21-30. LAX, B. Z., LARSH, J. E., JR., WEATHERLY, N. F., AXI) GOULSON, H. T. 1967. Demonstration of immunity to Fasciola hepatica in recipient mice given peritoneal exudate cells. Journal of Parasitology 53, 208-209. LAHSH, J, E., JR. 1963. Experimental trichiniasis. In “Advances in Parasitology” (Ben Dawes, Ed.), Vol. 1. pp. 213-286. Academic Press, London. LARSH, J. E., JR. 1967. The present understanding of the mechanism of immunity to TrichineZZa spirulis. American Journal of Trolli& Al&itine and Hygiene 16, 123-132. LARSM, J. E., JH., AND RACE, G. J. 1954. A histopathologic study of the anterior small intestine of immunized and nonimmunized mice infected with Trichinellu spirdis. Jotrrnal of Infectious Diseases 94, 262-272. J. E., JR., AND WEATHERLY, N. F. 1974a. Studies on clelayed (cellular) hypersensitivity jn mice infected wit11 Trichkelln spiralis. IX.

LARSH,

ALLERGIC

INFLAMMATION

AND

Delayed dermal sensitivity in artificially sensitized donors. JournaZ of ParasitoZogy 60, 93-98. LARSH, J. E., JR., AND WEATHER~Y, N. F. I974b. Cell-mediated immunity in certain parasitic infections. Current Topics in Microbiology and Immunology 67, 113-137. LARSH, J. E., JR., AND WEATHERLY, N. F. 1975. Cell-mediated immunity against certain parasitic worms. In “Advances in Parasitology” (Ben Dawes, Ed.), Vol. 13. Academic Press, London ( in press ). LARSH, J. E., JR., GILCHRIST, H. B., AND GREENBERG, B. G. 1952. A study of the distribution and longevity of adult TrichineZZu spiraZis in immunized and non-immunized mice. Journal of the Elisha Mitch.eZZ Scientific Society 68, 1-11. LARSH, J. E., JR., RACE, G. J., AND JEFFRIES,W. B. 1956. The association in young mice of intestinal inflammation and the loss of adult worms following an initial infection with TrichineZZa spiralis. Journal of infectious Diseases 99, LARSH, J. E., 1959. A munized spiraZis.

63-71. JR., RACE, G. J., AND GOULSON, H. T. histopathologic study of mice imwith irradiated larvae of Trichine?Za Journal of Infectious Diseases 104,

156-163. LARSH, J. E., JR., RACE, G. J., AND YARINSKY, A. 1962. A histopathologic study in mice immunized against TrichineZZa spiralis and exposed to total-body X-irradiation. American JournaZ of TropicaZ Medicine and Hygiene 11, 633640. GOULSON, H. T., AND LARSH, J. E., JR., WEATHERLY, N. F. 1964a. Studies on delayed (cellular) hypersensitivity in mice infected with Trichz’nella spiralis. II. Transfer of peritoneal exudate cells. Journal of PaTasitology 56,496-498. GOULSON, H. T., AND LARSH, J. E., JR., WEATHERLY, N. F. 196413. Studies on delayed (cellular) hypersensitivity in mice infected with TrichineZZa spiralis. I. Transfer of lymph node cells. Journal of the Elisha Mitchell

Scientific

Society 80, 133-135.

LARSH, J. E., JR., RACE, G. J., GOULSON, H. T., AND WEATHERLY, N. F. 1966. Studies on delayed (cellular) hypersensitivity in mice infected with TrichineZZa spirazis. III. Serologic and histopathologic findings in recipients given peritoneal exudate cells. JournuZ of ParasitoZogy 52, 146-156. LARSH, J. E., JR., GOULSON, H. T., WEATHERLY, N. F., AND CHAFFEE, E. F. 1969. Studies on delayed (cellular) hypersensitivity in mice infected with Trichinello spiralis. IV. Arti-

WORM

EXPULSION

HYPOTHESIS

265

ficial sensitization of donors. Journal of Parasitology 55, 726-729. LARSH, J. E., JR., GOULSON, H. T., WEATHERLY, N. F., AND CHAFFEE, E. F. 1970a. Studies on delayed (cellular) hypersensitivity in mice infected with TrichineZk spirazis. V. Tests in recipients injected with donor spleen cells I, 3, 7, 14, or 21 days before infection. JournaZ of Parasitology 56, 978-981. LARSH, J. E., JR., GOULSON, H. T., WEATNERLY, N. F., AND CHAFFEE, E. F. 1970b. Studies on delayed ( cellular ) hypersensitivity in mice infected with TrichineZZa spiralis. VI. Results in recipients injected with antiserum or “freeze-thaw” spleen cells. Journal of ParasitoZogy 56, 1206-1209. LARSH, J. E., JR., WEATHERLY, N. F., GOWLSON, H. T., AND CHAFFEE, E. F. 1972. Studies on delayed (cellular) hypersensitivity in mice infected with TrichineZZa spiralis. VII. The effect of ATS injections on the numbers of adult worms recovered after challenge. Journal of Parasitology 58, 1052-1060. LARSH, J. E., JR., RACE, G. J., MARTIN, J. H., AND WEATHERLY, N. F. 1974a. Studies on delayed (cellular) hypersensitivity in mice infected with Trichinella spiralis. VIII. Serologic and histopathologic responses of recipients injected with spleen cells from donors suppressed with ATS. Journal of Parasitology 60, 99-109. LARSH, J. E., JR., OITOLENGHI, A., AND WEATHERLY, N. F. 1974b. TrichineZZa spiralis: phospholipase in challenged mice and rats. Experimental ParasitoZogy 36, 299-306. OGILVIE, B. M., AND JONES, V. E. 1971. NippostrongyZus brasdiensis: A review of immunity and the host/parasite relationship in the rat. ExperimentaZ ParasitoZogy 29, 138-177. OGILVIE, B. M., AND JONES, V. E. 1973. Immunity in the parasitic relationship between helminths and hosts. Progress in AZZergy 17, 93-144. RACE, G. J., LARSH, J. E., JR., MARTIN, J. H., AND WEATHERLY, N. F. 1974. Light and electron microscopy of the intestinal tissue of mice parasitized by TrichineZZa spiralis. In “Trichinellosis, Proceedings of the Third International Conference on Trichinellosis.” (C. W. Kim, Ed.), pp. 75-100. Intext, New York. ROTHWELL, T. L. W., AND DINEEN, J. K. 1972. Cellular reactions in guinea pigs following primary and challenge infection with TrichostrongyZus coZubriformis with special reference to the roles played by eosinophils and basophils in rejection of the parasite. Immunology 22,733-745. ROTHWELL, T. L. W., DINEEN, J. K., AND LOVE, R. J. 1971. The role of pharmacologically active

%ii(i

LARSH

amines in resistance to colubriformis in the guinea 21,925-938. TALIAFERRO, W.

H.,

Trichostrongylus pig. Immunology

AND SARLES, M.

P.

1939.

The cellular reactions in the skin, lungs and intestine of normal and immune rats after infection with Nippostrongylus muris. Journal of Infectious Diseases 64, 157-192. VASSALLI, P., AND MCCLUSKEY, R. T.

1971.

De-

layed hypersensitivity. In “Inflammation, Immunity and Hypersensitivity.” ( H. Z. Movat, Ed.), pp. 179-234. Harper and Row, New York. VISAKORPI, R. 1972. Effect of irradiation on established delayed hypersensitivity. Acta PathoZogy and Microbiology of Scandinazjia Sect. B 80,788-794.

AND

RACE

VOLKhlAN, A., AND COLLINS, F. M. 1968.

Recovery

of delayed-type hypersensitivity in mice following suppressive doses of X-radiation. Journal of Immunology 101, 846-859. WALLS,

R. S., CAHTER, R. L., LEUCHARS, E., AND

DAVIES, A. J. S. 1973.

The immnnopathology of trichiniasis in T-cell deficient mice. Clinical Experimentul Immunology 13, 231-242. WESTON, W.

L.,

G.

1973.

G.

cellnlar

CLAMAN,

Site

of

N., AND KRUEGER,

H.

action

Journal

immunity.

of of

cortisol

in

Immtinology

110, 880-883. YAHINSKY, A. 1962.

on

the

Trichinellu Mitchell

The

immonity

influence of mice

spiralis. Scientific

Journal Society

of X-irradiation to

infection of

the

78, 2943.

with Elisha