Br. J. exp. Path. (1979) 60, 246
RESPONSE OF INTESTINAL GLOBULE LEUCOCYTES IN THE MOUSE DURING A TRICHINELLA SPIRALIS INFECTION AND ITS INDEPENDENCE OF INTESTINAL MAST CELLS E. J. RUITENBERG AND A. ELGERSMA From the Laboratory of Pathology, National Institute of Public Health, 3720 BA Bilthoven, The Netherlands Received for publication November 21, 1978
Summary.-We have previously reported that intestinal mast cells represent a separate population of mast cells which is thymus- (T-) dependent. In this paper we examine whether the appearance of these cells is dependent on thymus-dependent antibodies or thymus serum factor(s). The response of intestinal mast cells and globule leucocytes to a Trichinella spiralis infection was therefore studied in congenitally athymic (nude) mice after treatment with specific anti-T. spiralis hyperimmune serum or normal mouse serum from thymus-bearing litter-mates. However, transfer of both types of serum did not lead to an intestinal mast cell response. It was concluded that the presence of an intact thymus or T-dependent cellular reactions and/or their products are essential for appearance of intestinal mast cells. In contrast infected athymic mice reacted with a minor response of globule leucocytes irrespective of the serum transfer. Occasionally metachromatic intra-epithelially located cells with toluidine-blue-positive granules, believed to be globule leucocytes, showed mitotic figures. Metachromatic cells were observed occasionally within the lumen of the gut. These data were interpreted as supporting the idea that the globule leucocyte is a cell sui generis and independent of the intestinal mast cell.
THE NUMBERS of mast cells and intraepithelially located globule leucocytes in the gut mucosa of various animal species, including laboratory rodents and ruminants, increase during the intestinal phase of nematode infections (Miller and Jarrett, 1971; Kelly and Ogilvie, 1972; Gregg et al., 1978; Murray, Miller and Jarrett, 1968). Both cell types are characterized by intra-cytoplasmatic granules. The function of these cells in the immune expulsion of worms from the gut is still unclear. In a previous study (Ruitenberg and Elgersma, 1976) we reported that intestinal mast cells represent a separate population of mast cells which is thymus(T-) dependent. This conclusion was based on the absence of mast cell and globule leucocyte proliferation in congenitally athymic (nude) BIOLP mice infected with
the nematode Trichinella spiralis. The cellular response was restored after thymus transplantation. This clearly indicated that the thymus plays a crucial role in the appearance of these cell types. Theoretically this may be due to (1) the presence of an intact thymus, (2) thymus serum factor(s), (3) T-dependent serum antibodies (recently Askenase, Haynes and Hayden (1976) reported cutaneous basophil hypersensitivity to be antibodymediated) or (4) T-dependent cellular reactions and/or their products. In this study we investigated the second and third possibilities. The experiments were performed in nude (nu/nu) mice and their heterozygous thymus-bearing (H+/nu) litter mates. T. spiralis adult worms are expelled from the gut in + /nu mice within 10 days after
247
INTESTINAL MAST CELLS AND GLOBULE LEUCOCYTES
infection both in the B 1 OLP (Ruitenberg et al., 1977) and the Balb/c strain (unpublished data). In BIOLP nu/nu mice, adult worms are present until 83 days after infection. This is in keeping with the idea that the immune expulsion is a T-dependent phenomenon. MATERIALS AND METHODS Mice. Male SPF Balb/c nu/nu mice, 8 weeks of age, were obtained from Dr Friis (Gammel Bomholtg,ird Ltd, Aarhus, Denmark). Comparisons were made with age-matched mice heterozygous for the nude gene (+/nu). The animals were kept under laminar flow conditions. Parasite and parasitological methods.-After an acclimatization period of 2 weeks the mice were orally infected, at an age of 10 weeks, with 300 T. spiralis larvae each, using a syringe. The strain of T. spiralis used, their maintenance and infection were as described earlier (Ruitenberg et al., 1977). Histology. The presence of intestinal mast cells and globule leucocytes was studied in a 10cm portion of the jejunum with a Swiss roll technique (Reilly and Kirsner, 1965; Moolenbeek and Ruitenberg, 1977). Tissues were processed as described previously (Ruitenberg and Elgersma, 1976). In brief: tissues were fixed in a mixture of 088% formalin buffered with phosphate-buffered saline (0O01M; pH 7.2) at neutral pH and 40% acetic acid. After overnight fixation at 450, tissues were dehydrated and embedded in Paraplast according to conventional procedures. Sections (5,im thick) were prepared and stained with toluidine-blue at room temperature for 20 min. Toluidine-blue (0-5%o) was dissolved in 20% ethanol. After staining, the sections were washed, dehydrated and mounted with a conventional mounting medium. The number of intestinal mast cells and globule leucocytes characterized by their metachromatic staining in 20 "villus-crypt units" per animal was recorded. A 'villus-crypt unit" represents a portion of gut mucosa, lying between two gland crypts and the tip of the villus above (Jarrett et al., 1968). Experimental design.-In Experlinent 1, +-/nu rnice were examined by necropsy from 0 to 16 days after infection in groups of 5 animals. In Experiment 2, 3 groups of 5 nu/nu mice were examined by necropsy at 7 days after infection. Three groups of 5 nu/nu served as uninfected controls. Two groups, including an infected and an uninfected one, were treated twice with normal Balb/c + /nu serum (NMS), collected from 10-1J-week-old male donors by i.p. injection of 0-5 ml at Days 0 and 5. Two other groups, including an infected and an uninfected
one, were twice treated with hyperimmune serum (HIS), collected from 14-week-old male Balb/c +/nu mice, infected orally with 300 T. spiralis larvae at 10 and 13 weeks of age. An amount of 0 5 ml HIS was injected i.p in the recipient mice at 0 and 5 days after infection. A 72h passive cutaneous anaphylaxis (PCA) reaction performed with HIS yielded a PCA titre of >_1 :40, indicating the presence of specific anti-T. spiralis antibodies. Two other groups, one infected and one uninfected, served as controls for the treatment.
RESULTS In uninfected + /nu mice (Experiment 1) very few intestinal mast cells were present in the villus stroma. They were localized subepithelially or scattered both in the core of the villus, from tip to bottom, and in the lamina propria between the crypts of Lieberkuhn. After infection their number increased but the localization remained the same. Globule leucocytes were present in the uninfected animals and localized in the epithelium of both the crypt and the base of the villus. After infection their number increased rapidly. The cells were now also observed in the epithelium of the mid-villus region and occasionally in the tip region. TABLE I.-Appearance of Intestinal Mast Cells and Globule Leucocytes in the Small Intestine of Male Balb/c + /nu Mice, Various Days after Oral Infection with 300 Trichinella spiralis Larvae Each (Experiment 1) Days after
infec-, tion
0 3 7 10 13 16
Intestinal mast cells
Globule leucocytes -,A__
mean±s.d. Pt
mean ± s.d. *6-6± 1-7
6-2±3-8
117-6±46.3
9 *0.4±0 0-4±0-9
15-0±9-8 20-8±14-0
10-4±5-2
RESPONSE OF INTESTINAL GLOBULE LEUCOCYTES IN THE MOUSE DURING A TRICHINELLA SPIRALIS INFECTION AND ITS INDEPENDENCE OF INTESTINAL MAST CELLS E. J. RUITENBERG AND A. ELGERSMA From the Laboratory of Pathology, National Institute of Public Health, 3720 BA Bilthoven, The Netherlands Received for publication November 21, 1978
Summary.-We have previously reported that intestinal mast cells represent a separate population of mast cells which is thymus- (T-) dependent. In this paper we examine whether the appearance of these cells is dependent on thymus-dependent antibodies or thymus serum factor(s). The response of intestinal mast cells and globule leucocytes to a Trichinella spiralis infection was therefore studied in congenitally athymic (nude) mice after treatment with specific anti-T. spiralis hyperimmune serum or normal mouse serum from thymus-bearing litter-mates. However, transfer of both types of serum did not lead to an intestinal mast cell response. It was concluded that the presence of an intact thymus or T-dependent cellular reactions and/or their products are essential for appearance of intestinal mast cells. In contrast infected athymic mice reacted with a minor response of globule leucocytes irrespective of the serum transfer. Occasionally metachromatic intra-epithelially located cells with toluidine-blue-positive granules, believed to be globule leucocytes, showed mitotic figures. Metachromatic cells were observed occasionally within the lumen of the gut. These data were interpreted as supporting the idea that the globule leucocyte is a cell sui generis and independent of the intestinal mast cell.
THE NUMBERS of mast cells and intraepithelially located globule leucocytes in the gut mucosa of various animal species, including laboratory rodents and ruminants, increase during the intestinal phase of nematode infections (Miller and Jarrett, 1971; Kelly and Ogilvie, 1972; Gregg et al., 1978; Murray, Miller and Jarrett, 1968). Both cell types are characterized by intra-cytoplasmatic granules. The function of these cells in the immune expulsion of worms from the gut is still unclear. In a previous study (Ruitenberg and Elgersma, 1976) we reported that intestinal mast cells represent a separate population of mast cells which is thymus(T-) dependent. This conclusion was based on the absence of mast cell and globule leucocyte proliferation in congenitally athymic (nude) BIOLP mice infected with
the nematode Trichinella spiralis. The cellular response was restored after thymus transplantation. This clearly indicated that the thymus plays a crucial role in the appearance of these cell types. Theoretically this may be due to (1) the presence of an intact thymus, (2) thymus serum factor(s), (3) T-dependent serum antibodies (recently Askenase, Haynes and Hayden (1976) reported cutaneous basophil hypersensitivity to be antibodymediated) or (4) T-dependent cellular reactions and/or their products. In this study we investigated the second and third possibilities. The experiments were performed in nude (nu/nu) mice and their heterozygous thymus-bearing (H+/nu) litter mates. T. spiralis adult worms are expelled from the gut in + /nu mice within 10 days after
247
INTESTINAL MAST CELLS AND GLOBULE LEUCOCYTES
infection both in the B 1 OLP (Ruitenberg et al., 1977) and the Balb/c strain (unpublished data). In BIOLP nu/nu mice, adult worms are present until 83 days after infection. This is in keeping with the idea that the immune expulsion is a T-dependent phenomenon. MATERIALS AND METHODS Mice. Male SPF Balb/c nu/nu mice, 8 weeks of age, were obtained from Dr Friis (Gammel Bomholtg,ird Ltd, Aarhus, Denmark). Comparisons were made with age-matched mice heterozygous for the nude gene (+/nu). The animals were kept under laminar flow conditions. Parasite and parasitological methods.-After an acclimatization period of 2 weeks the mice were orally infected, at an age of 10 weeks, with 300 T. spiralis larvae each, using a syringe. The strain of T. spiralis used, their maintenance and infection were as described earlier (Ruitenberg et al., 1977). Histology. The presence of intestinal mast cells and globule leucocytes was studied in a 10cm portion of the jejunum with a Swiss roll technique (Reilly and Kirsner, 1965; Moolenbeek and Ruitenberg, 1977). Tissues were processed as described previously (Ruitenberg and Elgersma, 1976). In brief: tissues were fixed in a mixture of 088% formalin buffered with phosphate-buffered saline (0O01M; pH 7.2) at neutral pH and 40% acetic acid. After overnight fixation at 450, tissues were dehydrated and embedded in Paraplast according to conventional procedures. Sections (5,im thick) were prepared and stained with toluidine-blue at room temperature for 20 min. Toluidine-blue (0-5%o) was dissolved in 20% ethanol. After staining, the sections were washed, dehydrated and mounted with a conventional mounting medium. The number of intestinal mast cells and globule leucocytes characterized by their metachromatic staining in 20 "villus-crypt units" per animal was recorded. A 'villus-crypt unit" represents a portion of gut mucosa, lying between two gland crypts and the tip of the villus above (Jarrett et al., 1968). Experimental design.-In Experlinent 1, +-/nu rnice were examined by necropsy from 0 to 16 days after infection in groups of 5 animals. In Experiment 2, 3 groups of 5 nu/nu mice were examined by necropsy at 7 days after infection. Three groups of 5 nu/nu served as uninfected controls. Two groups, including an infected and an uninfected one, were treated twice with normal Balb/c + /nu serum (NMS), collected from 10-1J-week-old male donors by i.p. injection of 0-5 ml at Days 0 and 5. Two other groups, including an infected and an uninfected
one, were twice treated with hyperimmune serum (HIS), collected from 14-week-old male Balb/c +/nu mice, infected orally with 300 T. spiralis larvae at 10 and 13 weeks of age. An amount of 0 5 ml HIS was injected i.p in the recipient mice at 0 and 5 days after infection. A 72h passive cutaneous anaphylaxis (PCA) reaction performed with HIS yielded a PCA titre of >_1 :40, indicating the presence of specific anti-T. spiralis antibodies. Two other groups, one infected and one uninfected, served as controls for the treatment.
RESULTS In uninfected + /nu mice (Experiment 1) very few intestinal mast cells were present in the villus stroma. They were localized subepithelially or scattered both in the core of the villus, from tip to bottom, and in the lamina propria between the crypts of Lieberkuhn. After infection their number increased but the localization remained the same. Globule leucocytes were present in the uninfected animals and localized in the epithelium of both the crypt and the base of the villus. After infection their number increased rapidly. The cells were now also observed in the epithelium of the mid-villus region and occasionally in the tip region. TABLE I.-Appearance of Intestinal Mast Cells and Globule Leucocytes in the Small Intestine of Male Balb/c + /nu Mice, Various Days after Oral Infection with 300 Trichinella spiralis Larvae Each (Experiment 1) Days after
infec-, tion
0 3 7 10 13 16
Intestinal mast cells
Globule leucocytes -,A__
mean±s.d. Pt
mean ± s.d. *6-6± 1-7
6-2±3-8
117-6±46.3
9 *0.4±0 0-4±0-9
15-0±9-8 20-8±14-0
10-4±5-2
