0 1992 Hanvood Academic Publishers GmbH Printed in the United States
Autoimmunity. 1992, Vol. 14, pp. 137-142 Reprints available directly from the publisher Photocopying permitted by license only
A STUDY OF MAST CELLS IN AUTOIMMUNE NZB/W F1 MICE: POSSIBLE RELATIONSHIP BETWEEN MAST CELLS AND INCREASED VASCULAR PERMEABILITY IN THE THYMUS OF NZB/W F1 MICE JUN OHMORI and MASAHIKO KOTANI* Department of Anatomy, Kumamoto University School of Medicine, Honjo, Kumamato and +Kyoto College. of Medical Technology, Imakita, Koyama-Higashi-Machi, Sonobe-Cho, Kyoto, Japan
Autoimmunity Downloaded from informahealthcare.com by UB Kiel on 12/25/14 For personal use only.
(Received May 5.1992; infinal form September 28,1992)
We examined the possible relationship between thymic mast cells and increased vascular permeability in the thymus of autoimmune N Z B W F, mice. Light-microscopic observation of tissue sections showed that nonautoimmune BDF, mast cells increased with age. In contrast, autoimmune N Z B W F, mast cells did not increase in the thymic parenchyma at the age of 9 weeks. However, N Z B W F, mast cells resumed the ageassociated increase from the age of 12 weeks and exceeded the number of BDF, mast cells at the age of 30 weeks. Blood histamine levels of 9-week-old N Z B W FImice were higher than those of BDF, mice of comparable age. Furthermore, peritoneal mast cells of NZBW F, mice were more sensitive to compound 48/80 than those of BDF, mice. Increased blood histamine levels of N Z B N F,mice seem to be due to the enhanced histamine release from mast cells. These results suggest a possible correlation between the high histamine levels by degranulation of mast cells and increased vascular permeability in the thymus of NZBW F, mice.
KEY WORDS: Autoimmune N Z B M F,, thymus. vascular permeability, mast cells, histamine.
INTRODUCTION We previously reported increased vascular permeability in the thymus of autoimmune NZB/W FI mice'.'. However, we have not yet elucidated the mechanism of the increased vascular permeability. Many investigators have reported various substances that increase vascular permeability, such as histamine, serotonin, bradykinin, kallikrein'. Such substances satisfactorily explain various states of increased vascular permeability. B ~ r n e t ~first ' ~ ' ~reported that thymic mast cells increased with age in autoimmune mice. Since then there have been no reports concerning mast cells in the thymus of autoimmune mice. Mast cells release histamine and serotonin via IgE and IgG, receptors'**or by other substances includin neuropeptides9*lo*l',lymphokines, bacterial lectins", and complement peptided3. Kotani et al."*'' reported the generation of germinal centers in the rat thymus after injection of histamine or serotonin, together with antigens. The germinal centers were induced by antigens that easily pass through the blood vessels of thymus in which vascular permeability increased with histamine or serotonin. In the present study we examined whether histamine and other vascular perCorrespondence to: Dr Jun Ohmori. Department of Anatomy. Kumamoio University School of Medicine, 2-2-1 Honjo, Kumamoto 860.Japan.
meability factors released from mast cells could be responsible for the induction of increased vascular permeability in the thymus of NZBW F,mice.
MATERIALS AND METHODS Mice
Female autoimmune (NZBxNZW) F,(NZB/W FI) and non-autoimmune (C57BL/6xDBA/2) FI (BDF,) mice were purchased from Shizuoka Agricultural Cooperative for Laboratory Animals (Shizuoka, Japan). We used only one normal mouse strain, which shared the same H-2d haplotype. Histological analysis
Mice were killed by cervical dislocation. The thymuses were fixed in Carnoy's fluid or buffered neutral formalin solution, and embedded in paraffin wax. Serial sections cut at 6 p m thickness were stained with methyl green and pyronin (MGP) and/or toluidine blue (Merck, Darmstadt, Germany). Serial tissue sections (30) from each individual mouse were counted by light microscopy and results expressed per 100 mm'. The mast cell counts were MGP stained cells. In addition, to examine whether mast cells are connective tissue type mast cells (CTMCs) or mucosal
137
I38
J. OHMORI AND M. KOTANI
The histamine content in the plasma was determined spectrofluorometrically according to the method of Shore, Burkhalter and Cohn”. A standard curve was prepared using histamine diphosphate (Nacalai Tesque, Inc., Kyoto, Japan).
0.1% gelatin). The cell suspensions were centrifuged at 15Og for 5 min, and then resuspended in the same solution. The cell suspensions were incubated with 100 and/or 10pg/ml of compound 48/80 (Sigma Chemical Co., St. Louis, MO) for 10 min at 37°C. The supernatant was harvested by centrifugation at 150 g for 5 min. The histamine content of the supernatants and cell fractions was determined by the method described above. Histamine release was expressed as a percentage of the total cellular content of histamine after corrections for the spontaneous release found in controls.
Measurement of histamine release from peritoneal mast cells
Statistical analysis
Peritoneal mast cells were collected from 4 and 30week-old mice by peritoneal lavage with Tyrode solution (pH 7.4) (124mM NaCl, 4 mM KCI, 0.64mM NaH2P04, 1.6mM CaC12, 1 mM MgCl?, 10mM NaHCO,, 5.5 mM glucose, 10mM Hepes, 5 mM 4-morpholinoethanesulfonic acid (MES), and
The numbers of mast cells are given as the geometric mean. The histamine levels and % histamine release are given as the arithmetric mean. Statistical significance was determined using Mann-Whitney test for mast cell count and histamine levels, and Student’s t-test for % histamine release.
type mast cells, formalin-fixed sections16were stained with alcian blue (pH 0.3) and safranin-0, and frozen sections were stained with berberine sulfate according to the method of Enerback”.
Autoimmunity Downloaded from informahealthcare.com by UB Kiel on 12/25/14 For personal use only.
Measurement of blood histamine
Figure 1 (a) Thymus of a 9-week-old N Z B N F, mouse. Mast cells are seen in the cortex and medulla. Magnification: x188. (b) Thymus of a 9-week-old BDF, mouse. Mast cells are observed in the cortex and capsule. Magnification: x 188. (c) Thymus of a 30-week-old NZBW FI mouse. Large numbers of mast cells are seen in the cortex. Some are located in the medulla close to the germinal center. Magnification: X93. (d) Thymus of a 30-week-old BDF, mouse. Mast cells are seen in the cortex. cortico-medullary junction and capsule. Magnification: X93. Mast cells are identified by metachromatic granules after staining with toluidine blue. All mast cells are indicated by arrow heads.
VASCULAR PERMEABILITY AND MAST CELLS IN THE THYMUS
139
9 weeks. They resumed the age-associated increase from the age of 12 weeks and then exceeded the number of BDF, mast cells at the age of 30 weeks. Histological analysis We also counted mast cells in the mesenteric lymph We examined the number of mast cells in the thy- node (MLN) and spleen. The numbers of mast cells muses of N Z B N F, and BDF, mice at 4 , 9 , 12 and 30 changed in the MLN and spleen in a pattern similar to weeks of age by light microscopy. Mast cells counted that seen in the thymus. The number of mast cells was within 30 serial tissue sections were expressed as the lower in the 9-week-old N Z B N F I MLN than that in number of mast cells per 100 mm2 after staining with 4-week-old N Z B N FI MLN (Figure 3). However, the MGP or toluidine blue. Figure I shows representative change of the number of mast cells was moderate in tissue sections of both strains at 9, and 30 weeks of the spleen. There was no significant difference age. Mast cells are seen in the cortex, medulla, and between 4 and 9 weeks of age in either NZB/W F, or capsule at the a g e o f 9 weeks (Figures l a and Ib). In BDFl spleens (Figure 4). These mast cells in the parboth strains aged 30 weeks, the concentrations of mast enchyma of thymus, MLN, and spleen were conneccells containing metachromatic granules was tive tissue type mast cells (CTMCs), the cytoplasm of increased (Figures Ic and Id). As shown in Figure 2, which exhibited intense fluorescence by berberine BDF, mast cells increased with age. The number of sulfate staining. In addition, they were stained with NZBW FI mast cells was more than that of compar- alcian blue and safranin-o after the fixation with forable BDF, mast cells at the age of 4 weeks. However, malin solution (data not shown). N Z B W FI mast cells were not increased at the age of
Autoimmunity Downloaded from informahealthcare.com by UB Kiel on 12/25/14 For personal use only.
RESULTS
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Figure 2 Numbers of mast cells per 100 mm2 of thymus. Mast cells in the thymic parenchyma were counted after tissue sections had been stained with MGP. Bars indicate geometric mean. Statistical significance was tested using Mann-Whitney test comparisons between 2 groups linked by line: (*) Pc0.05; (**) P
Autoimmunity. 1992, Vol. 14, pp. 137-142 Reprints available directly from the publisher Photocopying permitted by license only
A STUDY OF MAST CELLS IN AUTOIMMUNE NZB/W F1 MICE: POSSIBLE RELATIONSHIP BETWEEN MAST CELLS AND INCREASED VASCULAR PERMEABILITY IN THE THYMUS OF NZB/W F1 MICE JUN OHMORI and MASAHIKO KOTANI* Department of Anatomy, Kumamoto University School of Medicine, Honjo, Kumamato and +Kyoto College. of Medical Technology, Imakita, Koyama-Higashi-Machi, Sonobe-Cho, Kyoto, Japan
Autoimmunity Downloaded from informahealthcare.com by UB Kiel on 12/25/14 For personal use only.
(Received May 5.1992; infinal form September 28,1992)
We examined the possible relationship between thymic mast cells and increased vascular permeability in the thymus of autoimmune N Z B W F, mice. Light-microscopic observation of tissue sections showed that nonautoimmune BDF, mast cells increased with age. In contrast, autoimmune N Z B W F, mast cells did not increase in the thymic parenchyma at the age of 9 weeks. However, N Z B W F, mast cells resumed the ageassociated increase from the age of 12 weeks and exceeded the number of BDF, mast cells at the age of 30 weeks. Blood histamine levels of 9-week-old N Z B W FImice were higher than those of BDF, mice of comparable age. Furthermore, peritoneal mast cells of NZBW F, mice were more sensitive to compound 48/80 than those of BDF, mice. Increased blood histamine levels of N Z B N F,mice seem to be due to the enhanced histamine release from mast cells. These results suggest a possible correlation between the high histamine levels by degranulation of mast cells and increased vascular permeability in the thymus of NZBW F, mice.
KEY WORDS: Autoimmune N Z B M F,, thymus. vascular permeability, mast cells, histamine.
INTRODUCTION We previously reported increased vascular permeability in the thymus of autoimmune NZB/W FI mice'.'. However, we have not yet elucidated the mechanism of the increased vascular permeability. Many investigators have reported various substances that increase vascular permeability, such as histamine, serotonin, bradykinin, kallikrein'. Such substances satisfactorily explain various states of increased vascular permeability. B ~ r n e t ~first ' ~ ' ~reported that thymic mast cells increased with age in autoimmune mice. Since then there have been no reports concerning mast cells in the thymus of autoimmune mice. Mast cells release histamine and serotonin via IgE and IgG, receptors'**or by other substances includin neuropeptides9*lo*l',lymphokines, bacterial lectins", and complement peptided3. Kotani et al."*'' reported the generation of germinal centers in the rat thymus after injection of histamine or serotonin, together with antigens. The germinal centers were induced by antigens that easily pass through the blood vessels of thymus in which vascular permeability increased with histamine or serotonin. In the present study we examined whether histamine and other vascular perCorrespondence to: Dr Jun Ohmori. Department of Anatomy. Kumamoio University School of Medicine, 2-2-1 Honjo, Kumamoto 860.Japan.
meability factors released from mast cells could be responsible for the induction of increased vascular permeability in the thymus of NZBW F,mice.
MATERIALS AND METHODS Mice
Female autoimmune (NZBxNZW) F,(NZB/W FI) and non-autoimmune (C57BL/6xDBA/2) FI (BDF,) mice were purchased from Shizuoka Agricultural Cooperative for Laboratory Animals (Shizuoka, Japan). We used only one normal mouse strain, which shared the same H-2d haplotype. Histological analysis
Mice were killed by cervical dislocation. The thymuses were fixed in Carnoy's fluid or buffered neutral formalin solution, and embedded in paraffin wax. Serial sections cut at 6 p m thickness were stained with methyl green and pyronin (MGP) and/or toluidine blue (Merck, Darmstadt, Germany). Serial tissue sections (30) from each individual mouse were counted by light microscopy and results expressed per 100 mm'. The mast cell counts were MGP stained cells. In addition, to examine whether mast cells are connective tissue type mast cells (CTMCs) or mucosal
137
I38
J. OHMORI AND M. KOTANI
The histamine content in the plasma was determined spectrofluorometrically according to the method of Shore, Burkhalter and Cohn”. A standard curve was prepared using histamine diphosphate (Nacalai Tesque, Inc., Kyoto, Japan).
0.1% gelatin). The cell suspensions were centrifuged at 15Og for 5 min, and then resuspended in the same solution. The cell suspensions were incubated with 100 and/or 10pg/ml of compound 48/80 (Sigma Chemical Co., St. Louis, MO) for 10 min at 37°C. The supernatant was harvested by centrifugation at 150 g for 5 min. The histamine content of the supernatants and cell fractions was determined by the method described above. Histamine release was expressed as a percentage of the total cellular content of histamine after corrections for the spontaneous release found in controls.
Measurement of histamine release from peritoneal mast cells
Statistical analysis
Peritoneal mast cells were collected from 4 and 30week-old mice by peritoneal lavage with Tyrode solution (pH 7.4) (124mM NaCl, 4 mM KCI, 0.64mM NaH2P04, 1.6mM CaC12, 1 mM MgCl?, 10mM NaHCO,, 5.5 mM glucose, 10mM Hepes, 5 mM 4-morpholinoethanesulfonic acid (MES), and
The numbers of mast cells are given as the geometric mean. The histamine levels and % histamine release are given as the arithmetric mean. Statistical significance was determined using Mann-Whitney test for mast cell count and histamine levels, and Student’s t-test for % histamine release.
type mast cells, formalin-fixed sections16were stained with alcian blue (pH 0.3) and safranin-0, and frozen sections were stained with berberine sulfate according to the method of Enerback”.
Autoimmunity Downloaded from informahealthcare.com by UB Kiel on 12/25/14 For personal use only.
Measurement of blood histamine
Figure 1 (a) Thymus of a 9-week-old N Z B N F, mouse. Mast cells are seen in the cortex and medulla. Magnification: x188. (b) Thymus of a 9-week-old BDF, mouse. Mast cells are observed in the cortex and capsule. Magnification: x 188. (c) Thymus of a 30-week-old NZBW FI mouse. Large numbers of mast cells are seen in the cortex. Some are located in the medulla close to the germinal center. Magnification: X93. (d) Thymus of a 30-week-old BDF, mouse. Mast cells are seen in the cortex. cortico-medullary junction and capsule. Magnification: X93. Mast cells are identified by metachromatic granules after staining with toluidine blue. All mast cells are indicated by arrow heads.
VASCULAR PERMEABILITY AND MAST CELLS IN THE THYMUS
139
9 weeks. They resumed the age-associated increase from the age of 12 weeks and then exceeded the number of BDF, mast cells at the age of 30 weeks. Histological analysis We also counted mast cells in the mesenteric lymph We examined the number of mast cells in the thy- node (MLN) and spleen. The numbers of mast cells muses of N Z B N F, and BDF, mice at 4 , 9 , 12 and 30 changed in the MLN and spleen in a pattern similar to weeks of age by light microscopy. Mast cells counted that seen in the thymus. The number of mast cells was within 30 serial tissue sections were expressed as the lower in the 9-week-old N Z B N F I MLN than that in number of mast cells per 100 mm2 after staining with 4-week-old N Z B N FI MLN (Figure 3). However, the MGP or toluidine blue. Figure I shows representative change of the number of mast cells was moderate in tissue sections of both strains at 9, and 30 weeks of the spleen. There was no significant difference age. Mast cells are seen in the cortex, medulla, and between 4 and 9 weeks of age in either NZB/W F, or capsule at the a g e o f 9 weeks (Figures l a and Ib). In BDFl spleens (Figure 4). These mast cells in the parboth strains aged 30 weeks, the concentrations of mast enchyma of thymus, MLN, and spleen were conneccells containing metachromatic granules was tive tissue type mast cells (CTMCs), the cytoplasm of increased (Figures Ic and Id). As shown in Figure 2, which exhibited intense fluorescence by berberine BDF, mast cells increased with age. The number of sulfate staining. In addition, they were stained with NZBW FI mast cells was more than that of compar- alcian blue and safranin-o after the fixation with forable BDF, mast cells at the age of 4 weeks. However, malin solution (data not shown). N Z B W FI mast cells were not increased at the age of
Autoimmunity Downloaded from informahealthcare.com by UB Kiel on 12/25/14 For personal use only.
RESULTS
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Figure 2 Numbers of mast cells per 100 mm2 of thymus. Mast cells in the thymic parenchyma were counted after tissue sections had been stained with MGP. Bars indicate geometric mean. Statistical significance was tested using Mann-Whitney test comparisons between 2 groups linked by line: (*) Pc0.05; (**) P
