II. Skin Testing and lmmunosuppression of Orally or Subcutaneously Sensitized Mice
CLARENCE E. JONES l Department
College of Veterinary Medicine, Foft Collins, Colorado 80521
JONES, CLARENCE E. 1974. Nernutospiroides dub&: Mechanisms of host immunity II. Skin testing and immunosuppression of orally or SubcutaneousIy sensitized mice. Experimental Parasitology 35, 453-464. The present study was designed to acquire further understanding of the differences in the immune response of mice orally (OS) or subcutaneously (SS) sensitized to Nematospiroides dubius. Two immunosuppressive agents and skin tests were utilized in ,&his, regard. Rabbit antimouse thymus serum (RAMTS) and cyproheptadine (antihistamineantiserotonin) were similarly effective in suppressing the immune response of subcutaneously sensitized mice. When compared to normal rabbit serum and SS control (sensitized, untreated) animals, observations of the intestinal tunica muscularis in the immunosuppressed SS groups revealed granu1omatou.s lesions in which fewer eosinophils enveloped the sequestered parasite. Cyproheptadine was more successful than the other treatments in interrupting the immune expulsion of N. d&us from orally sensitized mice, but this was only at a borderline significance level. The size and intensity of the active cutaneous anaphylactic skin tests in OS mice injected with an adult or larval antigen, was greater than the response elicited in SS mice or uninfected control mice injected with the same preparations. Similarly, the reaotion in subcutaneously sensitized mice exceeded that observed in the noninfected controls. INDEX DESCRIPTORS: Nematospiroides d&us; Oral sensitization; Subcutaneous sensitization; Rabbit antimouse thymus serum; Cyproheptadihe (antihistamine) ; Immunosuppression.
suggested qualitative ‘differences in the immune response of orally or subcutaneously sensitized mice to a challenge infection with Nemutospiroides dubius. The present study was designed to further ilIuminate some of these fundamental differ1974)
1 Present address: ment of Microbiology, cago, Illinois 60637.
920 East 58th Street, DepartUniversity of Chicago, Chi-
453 21pg~cl *
1974 by Academic Press 9 repmduction in any form’
ences through the empIoyment of 2 immunosuppressive agencies and active cutaneous anaphylactic skin tests. MATERIALS
The collection and preservation of infective larvae, parasite recovery and counting techniques, the analysis of d’ata, and the method of oral and subcutaneous sensitization have all been described previously (Jones and Rubin 1974).
of Adult and Larval Antigen
Kent’s ( 1963) method for preparing parasite antigen was utilized to insure that any low molecular weight mast cell degranulators (Uvnas and Wold 1967) would not be present in the test material. One ml of 14 day old N. dubiurr adults was disrupted with a Potter-Elvenhjem tissue grinder, the suspension centrifuged, and the supernatant added to a 1.5 x 90 sephadex G-25 column. At the completion of the run, the tubes corresponding to each peak were pooled, and analyzed for protein content by the Lowery method. Pooled samples were lyophilized, and stored in fair tight vials at -20 C. Larval antigen was prepared in the same fashion except that larval disintegration could only be achieved through sonication ( Bronwill Biosonik IV). The excluded fraction of both larval and adult preparations was analyzed for the presence of antigen by double gel diffusion on microscope slides (Williams and Chase 1971). Skin Tests Skin tests were performed on sensitized mice the day prior to challenge, and on uninfected controls. The method described by Ovary (1958) for active cutaneous anaphylaxis was used. Five one hundredths milliliter (55 pg protein) of larva1 or adult antigen, and 0.05 ml of phosphate buffered saline were introduced into the dermis of the shaven area on the mouse’s back with a bent 27 gauge needle. Immediately after the deposition of the test material, 0.1 ml of 1% Evans blue in sterile physiological saline was injected intravenously. Animals were killed 30 min later, the skin removed, and the intensity of the reaction recorded. Maximum blueing was indicated by + ++ + . The size of the lesions was expressed in millimeters diameter even though in most instances the lesions were not circular.
Preparation and Evaluation of Rabbit Antimouse Thymus Serum (RAMTS) and Cyproheptadine (Antihistamine) The “two pulse” method was utilized to produce the antithymocyte serum (Jooste, et al. 1968). A thymus suspension ( lo*--log cells) from 20 week old, CFW mice was injected intravenously into each of 4 female New Zealand White rabbits (5-7 lbs). RAMTS was collected by cardiac puncture 7 days after the second thymocyte inoculation, The antisera obtained from separate rabbits was pooled, heated at 56 C for 30 min, and absorbed with mouse erythrocytes (1 part cells + 1 part serum) at 37 C for 30 min prior to injecting into recipient mice. The leukoagglutinin and hemagglutinin reciprocal titre of this pooled RAMTS preparation was 256 and 32 respectively. Normal rabbit serum ( NRS ) was also heated at 56 C, but was not absorbed with mouse erythrocytes. The NRS reciprocal leukoagglutinin and hemagglutinin titre was 2 and 0, respectively. Total white cell and differential counts were made on mice receiving RAMTS, NRS, cyproheptadine, and saline. The dose and injection regimen for each of these preparations followed that which was later used in skin grafting, and the experiment designed to suppress the immune response of sensitized animals. Five-tenths milliliter of RAMTS or NRS was injected intraperitoneally into each of 5 mice on Day 0, and 0.3 ml was given on Days 2, 4, 6, 8, and 10. The total dose of cyproheptadine (Peractin) was approximately 6 mg/kg per mouse per day. Two tenths milliliter (0.35 mg/ml suspension) was orally administered to 5 individual mice for 10 consecutive days beginning on Day 0. One milliliter of this suspensionwas also mixed with a small amount of finely ground mouse food and placed in a cage with the same 5 mice, 7-8 hr after individual ‘oral administrations. This process was repeated 7-8 hr later. Food was generally denied the
N. dub&s AND SENSITIZED MICE TABLE Nematospiroides
dubius in CFW Mice. Active Cutaneous Anaphylaxis Skin or Subcutaneously Sensitized Mice and Nonsensitized Controls
Control Oral sensitization Subcutaneous sensitization
Number of mice
of skin re(in mm)
6 6 5
cyproheptadine group except at the time of treatment. Saline was presented in the same manner as cyproheptadine. Mice were bled from the orbital plexus prior to the first injection of each preparation, 24 hr after the first injectimon, and every week thereafter for 2 weeks. The total number ‘of white cells in a given sample was determined by transferring heparinized blood from a capillary tube to a 20 ,~l disposable pipette. This 20 ~1 aliquot was immediately discharged into an appropriate volume ‘of isotonic saline diluent. A coulter counter was then used to estimate the number of white cells per cm3 of blood. Differential counts were made on blood slides stained with Wright’s by the method of Benjamin ( 1961). The skin grafting technique was adopted, with some modii?cation, from Billingham and Medawar ( 1951). Five groups of 5 animals each were included in this experiment: Allograft cyproheptadine; allograft NRS; allograft RAMTS; allograft rejection control; and autograft procedure control. The dosage and schedule of administration of these preparations was similar to that described for blood counts. The exception was that the initial injection occurred the day before skin grafting ( -1). Additionally, cyproheptadine could not be introduced directly into the stomach of grafted mice, and the entire dose was given in ground food.
9.7 19.7 14.2
6.5 17.8 13.4
Intensity of skin reactions Larvae
+ +++ ++
+ +++ ++
Attempts to Suppress Immunity tized Animals
The study was designed to determine whether RAMTS or cyproheptadine could significantly alter the expression of immunity in orally or subcutaneously sensitized mice. The 9 groups in this experiment are listed in Table II. Twelve animals were included in each group. Nine mice were used faorworm counts, and 3 for histopathologic examination. The segments of intestine excised, and the procedure for histopathology have been previously described (Jones and Rubin 1974). RAMTS and NRS were administered 1 day before challenge, and on Days 1, 3, 5, 7, and 9 postchallenge. Cyproheptadine was given on Days 0 through 9. The dosage for each of these preparations was identical to that given for blood count determinations. All animals were killed 10 days after challenge.
Skin Tests Three precipitin bands could be seen when either the adult or larval extraction was reacted with serum from orally sensitized animals. However, only 2 bands formed when adult or larval antigen was reacted with serum from subcutaneously sensitized mice. N
I dik FIG. 1. Total rabbit antimouse
LAST CYPROHEPTADINE TREATMENT
lymphocyte count of saline controls thymus serum, or cyproheptadine.
The results of the skin tests are reported in Table I. Saline evoked a negligible skin response in all animals tested unless the injection was accompanied by hemorrhage. The larval extraction provoked a greater
I LAST RAMTS NRS TREATMENT
reaction than the adults in all groups. The skin lesions in orally sensitized mice were larger and of greater intensity than those observed in subcutaneously sensitized animals or the nonsensitized controls. Skin re-
WORMS WORMS WORMS
2 0’ u
I E 3
FIG. 2. Nemutospiroiah cZubius in CFW mice. Graph comparing the total number of parasites and the percentage of small, medium, and large worms in orally or subcutaneously sensitized mice given normal rabbit serum, rabbit antimouse thymus serum, or cyproheptadine, and untreated controls.
Type I intestinal granuloma. Granuloma composed almost entirely of macrophages. 150.
actions in subcutaneously sensitized were ‘also greater than the controls.
acute process. Rejection usually consisted of a gradu’al and almost imperceptible shrinkage of the graft over a period of 40 Evaluation of RAMTS and Cyproheptadirw days. At the end of 40 days all mice in the allograft rejection control, allograft cyproTwenty-four hours after injection with heptadine, and allograft NRS were consid0.5 ml of RAMTS, CFW mice had a greatly ered rejected. Hair growth in these groups depressed lymphcoyte count (Fig. 1). This was always minimal. In contrast, 2 of the trend continued until Day 7, but by the 3 surviving allograft RAMTS mice were fourth day after the last injection (Day identical to the autograft controls on Day 14)) the peripheral lymphocyte population 40. Hair growth was luxuriant in both these had made a partial recovery. Surprisingly, animals. One of these grafts was greatly the lymphocyte count in NRS animals w’as shrunken by Day 55, and the other by also slightly reduced during the course of Day 60. administration, Cyproheptadine had no apparent effect sonany white blood cell type Attempts to Suppress Immunity in Sensiincluding lymphocytes. The only other tized Animals white blood cell altered by any treatment regimen was the eosinophil. A moderate The suppression of the immune response (5~ normal) increase in peripheral eosino- in morally sensitized (OS) mice was slight phils was observed in RAMTS mice 4 days regardless of the agent used (Table II; after the discontinuation of treatment. Fig. 2). However, worm counts in OS mice It was impossible to assign a precise date given cyproheptadine were greater than the of graft rejection for most ‘of the mice in- controls at (Y< 0.10. RAMTS also enhanced volved in this study. The principal reason the survival and development of Ls larvae was that few grafts were destroyed by an as did NRS.
FIG. 4. Type II intestinal are abundant in the periphery
granuloma. Few granulocytic of the lesion. H&E. X 150.
Both RAMTS and cyproheptadine interrupted the expression of immunity in subcutaneously sensitized (SS) mice. This effect in the cyproheptadine group was especially pronounced. Worm counts in NRS and RAMTS mice were significantly different (01 < 0.025). Three basic types of granulomas could be seen in the wall of the gut of subcutaneous sensitization groups on Day 10 postchallenge. Type I was characteristically seen in the exposure control group in those few lesions that had not been evacuated by the parasite (Fig. 3). These consisted of large worms that had locahzed in the tunica muscularis. These worms may or may not be surrounded by modest numbers of neutrophils. The macrophage was the principal cell type found in the region around the parasite. Few lymphocytes were present. Eosinophils were rarely associated with these lesions. Type I gnanulomas were only occasionally seen in cyproheptadine and RAMTS animals. Type II granulomas were similar to type I, except for the presence
of large numbers of eosinophils in the periphery ‘of the granuloma (Fig. 4). Few (if many) granulmocytic cells were intimately applied to the parasite which was usually quite large in immunosuppressed animals. Type II granulomas were observed primarily in the cyproheptadine and RAMTS groups, and less frequently in NRS and SS controls. Type III could be contrasted from type II by the apposition of considerable numbers of eosinophils (many degenerating) to the sequestered parasite (Fig. 5). This type of lesion was seen with some frequency in SS controls, but also in NRS, cyproheptadine, and to a lesser degree RAMTS mice. In summary, RAMTS and cyproheptadine animals had a greater number of large worms, and these were more frequently associated with a granulomatous inflammatory response that was milder than the SS controls and in which large numbers ‘of eosinophils were not intimately applied to the parasite. The differences among the various oral sensitization groups was not as conspicuous
5. Type III intestinal granuloma. (many degenerating). Large regions of granuIoma. H&E.
Parasite numbers X 150.
as those witnessed in subcutaneous sensitization animals. The primary reason was that greater numbers of active granulomatous lesions were present in SS mice, and this facilitated qualitative comparisons among different groups. Type III granulomas were present in all groups, but were especially numerous in cyproheptadine treated mice. Type II granulomas were seen occasionally in the cyproheptadine and RAMTS animals. DISCUSSION
The greater size and intensity (Table I) of the ‘active cutaneous anaphylactic skin lesions in orally sensitized (OS) animals, when compared to their subcutaneous sensitization ( SS) counterparts, is but another example of the fundamental ‘difference between the 2 sensitization methods. These results may indicate that OS mice have a greater potential than SS mice for immediate hypersensitive expulsion of a challenge infection. This difference in potential could be at least partially responsible for
is encapsulated of eosinophils
by a large disseminated
the patterns of immunity reported in a companion study (Jones and Rubin 1974). This speculation is in agreement with Panter’s ( 1969) contention that immediate hypersensitivity is involved in the elimination of N. dub&s from orally sensitized mice. In the past, immunosuppressive agents have been used by parasitologists with some frequency, to analyze the mechanisms of immunity involved in specific host-parasite models (Campbell et al. 1963; Urquhart et al. 1965; Sharp and Jarrett 1968; Machnicka 1972; Keller and Keist 1972; Okamoto and Koizumi 1972). Recent studies indicate, however, that some of these agents may lack the necessary specificity to be used as an investigative tool in immunity to parasitic diseases. Kelly and Dineen’s ( 1972) work with N. brasiliensis suggests that at least one antihistamine (promethazine) can prevent the successful cellular transfer of immunity from immunized donors to recipients. Gusdon et al. ( 1972) have also found that promethazine
could suppress primary and secondary antibody production, )and *delayed hypersensitivity deveIopment when administered before and during the course of sensitization. Finally, it has been shown that promethazine can significantly diminish the tissue and peripheral blood eosinophilic response in rats infected with Trichinella spiralis (Ismail and Tamrer 1972). Further study is needed, but certainly these results suggest that antihistamines can induce a state of immunosuppression through mechanisms other than competitive inhibition, depletion, or impaired synthesis of biogenie amines. If these observations are ultimately confirmed, promethazine and possibly other antihistamines would no longer be suitable for analysis of immunologic relationships between sensitized animals and specific parasitic organisms (Keller and Ogilvie 1972 ) . In the immunosuppression study, parasites in greater numbers and maturity could be recovered from subcutaneously sensitized mice given RAMTS and cyproheptadine (Table II, Fig. 2). This would seem to indicate that the immunologic disposition of N. dubius within this group is dependent on both sensitized lymphocytes, ‘and tentatively, biogenic amine release. These results are similar to those reported by Rothwell et al. (1971). These investigators were able to show that different antihistamines were immunosuppressive in a host-parasite model ( Trichostrongylus colubriformis in guinea pigs) in which deIayed hypersensitivity is thought to play a vital role (Wagland sand Dineen 1965; Dineen and Wagland 1966; Dineen and Adams 1971). They then proposed that sensitized lymphocytes interacting with antigen could release some “factor” that causes basophils to infiltrate the immunologic tissue locus and subsequently release their biogenic amines. An alternate explanatimon for the current study is that both treatments elicited a marked depression in the eosinophilic response which is thought
to be an essential component of the rejection mechanism in SS mice (Jones and Rubin 1974). Since others have shown that antilymphocyte serum and certain antihistamine-antiserotonin agents can diminish the eosinophil response in rats infected with T. spiralis (Basten and Beeson 1970; Ismail and Tanner 1972), this explanation has a certain amount of appeal and is supported by the histopathologic examination of SS intestine. The granulomatous response observed in the small intestine of SS mice given cyproheptadine and RAMTS was not as severe as that seen in NRS and control animals. The intense eosinophilic halo normally observed around most medium sized parasites on Day 10 postchallenge was also frequently absent. These results indicate that both immunosuppressive agents can inhibit the cellular mechanisms operative in the N. dubius mouse model. Subcutaneously sensitized mice given NRS had slightly greater numbers of parasites than the exposure controls. This may be a result of the apparent antilymphocyte activity of normal rabbit serum (Fig. 1) , or some nonspecific effect of heterologous serum. Since NRS did not prolong allograft survival, the latter conjecture would seem more plausible. The results ‘of immunosuppression of orally sensitized mice are difficult to interpret. However, it appears that the antihistamine preparation was more effective than RAMTS in promoting the survival of the challenge infection. There are two basic reasons for this statement. The first is that OS mice given cyproheptadine had more large and medium worms than animals given RAMTS. Secondly, much of the activity in RAMTS may be attributed to the same nonspecific effect that permitted greater parasite development in the NRS (OS) group. Nevertheless, this experiment would have to be repeated before any definitive conclusions could be reached. In summary, the results of this and the companion paper (Jones and Rubin 1974)
provide considerable evidence that a relationship does exist between the method of “immunization” and the fundamental nature of the subsequent immune response. The removal of N. dubius larvae from subcutaneously sensitized mice seems to be dependent on multiple components of the mouse’s immunol.ogical arsenal. Sensitized lymphocytes, eosinophils, antiworm antibody, and histamine may all be involved in this rejection process. At this point however, the most likely explanation for immune expulsion in OS mice is immediate hypersensitivity. Further study will be necessary before a comprehensive understanding of these phenomena will be possible.
ACKNOWLEDGMENTS The material repotied in this paper was supported financially by an NDEA Title IV grant and the Western Regional Research Project W-102. It is a pleasure to acknowledge the competent technical assistance of Mrs. Glenda Heideman, and Pat McNamara who took the photographs. Mrs. Martha Buderus is to be #thanked for sectioning the histopathology slides, as is Ms. Doralee Grindler for typing the manuscript. The author also wishes to express his appreciation to Dr. Dwayne Hamar for his assistance in preparing and analyzing the parasite antigens. Dr. Robert Phemister and Dr. Roger Jaenke examined selected pathology slides and their contribution is gratefully acknowledged. Thanks are given to Dr. Charles Hibler for his support throughout the study, and to Dr. Elvio Sadun and Dr. N. T. Briggs for their suggestions in preparing the manuscript.
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