Alrtoimmnnit~.1992, Vol.
13, pp. 27-34 Reprints available directly from the publisher Photocopying permitted by license only
0 1992 Harwood Academic Publishers GmbH Printed in the United Kingdom
CYTOKINES AND IMMUNE REGULATION IN THYROID AUTOIMMUNITY SHJGENOBLJ NAGATAKI and KATSUMI EGUCHI First Department oj'littri.na1 Medic,ine, Nagasaki University School of Medic,ine, 7-1 Sakamoto-muchi. Na,gasaki X.52. Japan
Autoimmunity Downloaded from informahealthcare.com by McMaster University on 11/04/14 For personal use only.
(Rewived March 24. 1992)
We studied the role of cytokines and immune regulation in thyroid tissue from patients with Graves' disease. lmmunohistochemistry showed that the thyroid glands are characterized by an aberrant expression of HLA class I1 antigens on thyrorytes, generation of new blood vessels and infiltration of mononuclear cells. We demonstrated that CD4' memory cells were more frequent in thyroid glands from Graves' patients than were CD4' naive cells. The intrathyroidal T cells demonstrated an enhanced expression of the adhesion molecules LFA-I, CD2, VLA-4 and VLA-5, and vascular endothelial cells of capillaries and thyrocytes in thyroid glands reacted with anti-ICAM-l monoclonal antibody. The adhesion molecules and HLA antigens on both vascular endothelial cells and thyrocytes were regulated by inflammatory cytokines. These results suggest that circulating lymphocytes migrate into thyroid tissues and that memory T cells are retained in the thyroid tissues by cellular interactions with thyrocytes or with extracellular matrix.
INTRODUCTION
In normal thyroid tissue, thyroid follicular cells (thyrocytes) express low levels of HLA class I (HLAA, B, C) and no HLA class I1 antigens (HLA-DR, DP, DQ)'. Thyrocytes from patients with autoimmune thyroid disorders (Graves' disease and Hashimoto's thyroiditis) have been shown to express an increased density of class I antigens and to inappropriately express class I1 antigens"'. Immunohistological study shows that the inflammation in Graves' disease and Hashimoto's thyroiditis tend to occur as large lymphoid aggregates, often showing germinal center formation.'.'. Expression of HLA antigens is known to be regulated by cytokines. Table I lists the effects of cytokines on HLA class I and class I1 antigens on the surface of thyrocytes. Interferon-T (IFN-T) is an important modulator of the expression of class 1 and class I1 antigens on classical antigen-presenting cells and non-lymphoid cells such as melanoma cell line, fibroblasts and vascular endothelial cells",". We and other investigators demonstrated that the effect of IFN-T on the expression of HLA antigens in thyrocytes is dependent on concentration and time, and that at least 3 days are required to achieve maximal e f f e ~ t " " ~In . the presence of IFN-l-, class 11 antigen expression persists; when IFN-l- is removed, there is loss of antigen expression. This is a direct effect of IFN-T, because enhancement is abolished by incubating the cells with IFN-T in the presence of a neutralizing antibodyI5. Lymphocytes secreting immunoreactive IFN-T are found in human autoimmune thyroid diseases, and abetrant HLA class I1 expression on thyrocytes is localized adjacent to IFN-T-expressing lymphocytes". These findings support the view that in
There is increasing evidence that autoimmune processes may have an important pathogenetic role in autoimmune thyroid diseases','. The thyroid gland is thought to be the major site of the production of autoantibodies such as anti-microsomal, anti-thyroglobulin and anti-TSH receptor antibodie~',~. Thyroid gland is characterized by generation of new blood vessels and by a variety of mononuclear cells in aggregates or diffusely distributed between and within the thyroid It seems likely that the infiltrated mononuclear cells migrate from peripheral blood into thyroid tissue where they are retained by interaction with other cells including thyroid epithelial cells (thyrocytes). Recently, it became apparent that cytokines and adhesion molecules play important roles in this migration of mononuclear cells and the cellular interactions of chronic inflammatory processes. The present study has focused on the role of cytokines and adhesion monelules in thyroid glands from patients with autoimmune thyroid disorders.
EXPRESSION O F HLA CLASS I AND HLA CLASS I1 ANTIGENS ON THYROID EPITHELIAL CELLS Substantial evidence exists to show that the major histocompatibility antigen (HLA in humans) has an essential role in the regulation of immune responses. Correspondence to: Shigenobu Nagataki, M.D., First Department of- Internal Medicine, Nagasaki University School of Medicine, 7- 1 Sakamoto-machi, Nagasaki 852, Japan.
27
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28
S. NAGATAKI AND K. EGUCHI
autoimmune thyroid diseases expression of HLA class I1 on thyrocytes is the result of local release of IFN-T. Tumor necrosis factor a (TNF-a), which is principally produced by monocytes, has a direct cytotoxic/cytostatic effect in vitro and an anti-tumor activity in vivo. We have shown that TNF-a alone enhances HLA class I antigen expression on cultured thyrocytes, but has no effect on the class I1 antigen expression". TNF-a acts synergistically with JFN-T on both HLA class I and HLA class I1 antigen expression on thyrocytes. Interleukin 1 (IL-1) has many of the same biological effects as TNF-a, and a broad range of inflammatory and immunoregulatory actions. IL- 1 is necessary for the optimal induction of antigen-specific T cell proliferation and is shown to be produced by endothelial cells" and thyrocytes, as well as monocytes in the autoimmune thyroid Previously we reported that IL- lp stimulates 3H-thymidine incorporation into human thyrocytes, and increases the proportion of thyrocytes in the S phase of the cell cycle". IL-1 alone has little effect on HLA class I and class I1 antigen expression on thyrocytes, although IL- lp in combination with IFN-T enhanced the expression of HLA class I and class I1 antigens beyond that produced by IFN-T alone17.These results support the idea that both IL-I and TNF act synergistically with IFN-T on HLA class I and class I1 antigen expression of thyrocytes in vitro. Interleukin 6 (IL-6) is a biologically active B cell stimulatory factor. Both IL-6 mRNA and protein are found in thyrocytes as well as infiltrating cells in thyroid glands from patients with Graves' disease and Hashimoto's thyroiditis". In contrast to IL-1, TNF and IFN-T, neither class I nor class I1 expression is induced by IL-6 alone or in combination with IFN-T". PHENOTYPIC AND FUNCTIONAL CHARACTERISTICS OF INTRATHYROIDAL MONONUCLEAR CELLS IN PATIENTS WITH GRAVES' DISEASE Many investigators have studied the phenotypic and functional characteristics of intrathyroidal mononuclear cells from patients with autoimmune thyroid diseases, but the results have been conflicting22-2s. Immunohistochemical staining shows that the distribution of CD4' and CD8' cells differs in different regions of Graves' thyroid tissue. CD4' cells (helper/inducer T cells) are mainly found in lymphoid aggregates, whereas CD8' cells (suppressor/cytotoxic T cells) are scattered among the thyroid follicle^^.^. Intrathyroidal mononuclear cells were separated from thyroid glands by mechanical aggregation and enzymatic digestion. Dual immunofluorescence staining was used to compare the expression of surface
markers associated with activation of T cells from the thyroid gland and from the corresponding peripheral blood of Graves' patients. Thyroid tissue from treated patients contained greater percentages of HLA-DR' T cells and CD25' (IL-2 receptor a') T cells than the peripheral bloodz9 (Table 2). These results show that intrathyroidal mononuclear cells from patients with Graves' disease comprise an increased proportion of activated T cells. We have also reported that the thyroid gland contains increased numbers of CD4TD29' cells, which results in decreased numbers of the reciprocal CD4'CD45RA' cells. CD4'CD29' cells amplify the production of immunoglobulins after stimulation with
Table 1 Effects of cytokines on antigen expression on thyroid epithelial cells Cytokine
HLA class I
IL- 1 IL-2 IL-6 IFN-a IFN-P IFN- y TNF-a IFN-ytIL-lP IFN-pTNF-a IFN- ytIFN- a
HLA class I1
+* + t + 7
t t tt
77
+
ICAM- 1
t
+
-+ + -3 +
NT** NT NT NT
t 7 tt tt
7
-3
tt tt 1
NT
*+=no change; f=increased; J=decreased. **NT=not tested (or not known).
Table 2 Summary of phenotypic characteristics of intrathyroidal mononuclear cells from patients with Graves' disease. T cells Activated and memory T cells HLA-DR+T cells CD2S' (IL-2Ra+) T cells CD26' (Tali) T cells
t t* tt tt
Helper and memory T cells CD4' CD29' cells
tt
Suppressor-inducer and naive T cells CD4' CD45RA+ cells
11
Natural killer cells Dull CD8' CD1 I b+ cells
1
Adhesion molecule expression LFA- 1 CD2 VLA-4 VLA-S
tt tf tt tt
B cells Activated and differentiated B cells CD20+ CD21- cells IgM'IgD- cells CD20' transferrin receptor+cells PCA-I'cells
tt tt tt tl'
*T=increase& J=decreaaed.
Autoimmunity Downloaded from informahealthcare.com by McMaster University on 11/04/14 For personal use only.
IMMUNE REGULATION IN THYROID AUTOIMMUNITY
pokeweed mitogen (PWM) by inducing a "helperinducer" subpopulation. Conversely, CD4'CD45RA+ cells induce suppressor cells which downregulate immunoglobulin production after stimulation by PWM29,30.A different interpretation is that the presence of CD29 and CD45RA antigens indicates a different maturational status. According to this notion, CD45RA' cells are naive cells that do not respond to recall antigens, whereas CD29' cells are memory cells that have been activated by prior exposure to antigens33. We have shown that the proportion of CD26' cells in the thyroid gland was similar to that in the peripheral blood, but markedly increased in comparison to the value for normal subjects. We also demonstrated that the proliferative responses to thyroglobulin and microsomal antigens of a CD26' cellenriched population isolated from the peripheral blood of patients with Graves' disease were greater than for a CD26' cell-depleted p ~ p u l a t i o nThese ~ ~ . results suggest that the CD26' cells in the thyroid gland from patients with Graves' disease may include antigentriggered memory cells that react with thyroid specific antigens. The percentages of CD8' cells and CD8'CDl Ibcells (cytotoxic T cells) in thyroid glands of Graves' patients are significantly greater than those in their peripheral blood and the peripheral blood from normal subjects. The CD8'CDl Ib' cells are subdivided into two subpopulations on the basis of CD8 antigen density. The percentage of dull CD8'CDllb' cells (natural killer cells) is lower than that in peripheral blood. There is no significant difference in bright CD8+CDl1b+ cells (suppressor-effector T cells) when comparing thyroid glands and peripheral Next, the functional characteristics of intrathyroidal mononuclear cells from patients with Graves' disease were We used specific monoclonal antibodies to separate specific T cell subsets from one another, so that their functions could be individually examined by cell-mixing techniques. By these means, abnormalities of suppression are found in intrathyroidal mononuclear cells from Graves' thyroid glands. These abnormalities are heterogenous in nature, involving decreased suppressor-inducer activity in CD4' cells, decreased suppressor-effector activity in CD8' cells, or both. These results support the possibility that the impairment of suppressor-inducer T cell function may be attributed to reduced numbers of intrathyroidal CD4TD45RA' cells. Thyroid-infiltrating B cells from Graves' patients were also investigated in regard to their phenotypic profiles and spontaneous production of immunoglobulin G (IgG) and anti-thyroidal autoantibodie~'~.The thyroid glands from Graves' patients had greater percentages of IgM'IgD- cells, CD20' transferrin receptor' cells, CD20TD21- cells, and PCA-1' cells compared to the peripheral blood of the same patients
29
and normal subjects. Furthermore, B cells from thyroid glands were able to synthesize IgG, anti-thyroglobulin and anti-microsomal antibodies in the culture supernatants. These findings suggest that the population of phenotypically different B cells that characterize the proliferative and differentiative phases of B cell maturation is expanded in the thyroid of patients with Graves' disease. On the basis of the observations presented in these studies, we suggest that the combination of an increase in the number of activated B cells and impaired function of suppressor T cells may lead to excessive production of immunoglobulins and antithyroidal autoantibodies in thyroid glands from patients with Graves' disease. NEOVASCULARIZATION AND EMIGRATION OF MONONUCLEAR CELLS FROM PERIPHERAL BLOOD TO THYROID TISSUES Thyroid tissues from patients with Graves' disease were stained with anti-factor VIII monoclonal antibody. As shown in Figure 1, the generation of new blood vessels (neovascularization) was observed in the lymphoid follicles. The mononuclear cells which infiltrated around the capillaries contained preferentially CD4' cells. High endothelial venules (HEV) derive their name from their morphological appearance and consist of cuboidal endothelial cells. HEVs are found in T-cell areas of a wide variety of lymphoid tissues. The function of the HEVs is to attract lymphocytes to lymphoid tissues, and they are thus of importance in lym-
Figure 1 Frozen section of Graves' thyroid disease immunostained with anti-factor VIII monoclonal antibody (ABC method). Endothelial cells of blood vessels in the lymphoid follicles are stained positively for anti-factor VIII monoclonal antibody (Original magnification ~ 1 0 0 ) .
Autoimmunity Downloaded from informahealthcare.com by McMaster University on 11/04/14 For personal use only.
30
S. NAGATAKI AND K. EGUCHI
phocyte recirculationTx.Using a monoclonal antibody which recognizes an epitope on human HEVs and immunohistochemical techniques, HEVs are found in lymphoid cell accumulation in thyroid glands from patients with Graves’ disease and Hashimoto’s thyroiditis”. Recently, the migration of lymphocytes has been shown to be controlled by the expression of adhesion molecules on the lymphocytes and of ligands on the vascular endothelial cells. T cells become eligible for migration if they express sufficient levels of adhesion molecules and come into contact with endothelial cells which express sufficient levels of ligands. Peripheral blood T cells bind to human umbilical vein endothelial cells (HUVEC) in vitro. As shown in Figure 2, the percentage of ConA-stimulated T cells adhering to HUVEC was significantly greater than that of unstimulated T cells. Furthermore, the percentage T cells adhering to IFN-T-stimulated HUVEC was increased compared with that to unstimulated HUVEC4’. Thus, the expression of adhesion molecules on T cells is enhanced by antigenic or mitogenic stimulation, and the expression of ligands on the endothelial cells is enhanced by cytokines. The adhesion of T cells to endothelial cells is mediated by at least two adhesion molecules, namely lymphocyte function-associated antigen 1 (LFA- I , C D 1 1 a/ CDI 8)4’, which reacts with intercellular adhesion molecule 1 (ICAM-1)43 and with ICAM-244 and the so-called “very late antigen 4” (VLA-4), which binds to vascular cell adhesion molecule 1 (VCAMThe expressions of ICAM- 1 and VCAM- 1 are upregulated by IFN-T, IL-I and TNF47-4’. In the thyroid tissues from patients with autoimmune thyroid diseases, the mononuclear cells which surround the capillaries
Figure 2 Adherence of T cells to IFNy-stimulated human umbilical vein endothelial cells (HUVEC). T cells were separatcd from blood and incubated for 72 hr with or without ConA and PMA. HUVEC were cultured with various amounts of IFN-y for 24 hr. After incubation, the percentage T-cell adhesion was determined by ”Cr adhesion assay. Open column depicts the untreated T cells. Closed column depicts the stimulated T cells. *P
13, pp. 27-34 Reprints available directly from the publisher Photocopying permitted by license only
0 1992 Harwood Academic Publishers GmbH Printed in the United Kingdom
CYTOKINES AND IMMUNE REGULATION IN THYROID AUTOIMMUNITY SHJGENOBLJ NAGATAKI and KATSUMI EGUCHI First Department oj'littri.na1 Medic,ine, Nagasaki University School of Medic,ine, 7-1 Sakamoto-muchi. Na,gasaki X.52. Japan
Autoimmunity Downloaded from informahealthcare.com by McMaster University on 11/04/14 For personal use only.
(Rewived March 24. 1992)
We studied the role of cytokines and immune regulation in thyroid tissue from patients with Graves' disease. lmmunohistochemistry showed that the thyroid glands are characterized by an aberrant expression of HLA class I1 antigens on thyrorytes, generation of new blood vessels and infiltration of mononuclear cells. We demonstrated that CD4' memory cells were more frequent in thyroid glands from Graves' patients than were CD4' naive cells. The intrathyroidal T cells demonstrated an enhanced expression of the adhesion molecules LFA-I, CD2, VLA-4 and VLA-5, and vascular endothelial cells of capillaries and thyrocytes in thyroid glands reacted with anti-ICAM-l monoclonal antibody. The adhesion molecules and HLA antigens on both vascular endothelial cells and thyrocytes were regulated by inflammatory cytokines. These results suggest that circulating lymphocytes migrate into thyroid tissues and that memory T cells are retained in the thyroid tissues by cellular interactions with thyrocytes or with extracellular matrix.
INTRODUCTION
In normal thyroid tissue, thyroid follicular cells (thyrocytes) express low levels of HLA class I (HLAA, B, C) and no HLA class I1 antigens (HLA-DR, DP, DQ)'. Thyrocytes from patients with autoimmune thyroid disorders (Graves' disease and Hashimoto's thyroiditis) have been shown to express an increased density of class I antigens and to inappropriately express class I1 antigens"'. Immunohistological study shows that the inflammation in Graves' disease and Hashimoto's thyroiditis tend to occur as large lymphoid aggregates, often showing germinal center formation.'.'. Expression of HLA antigens is known to be regulated by cytokines. Table I lists the effects of cytokines on HLA class I and class I1 antigens on the surface of thyrocytes. Interferon-T (IFN-T) is an important modulator of the expression of class 1 and class I1 antigens on classical antigen-presenting cells and non-lymphoid cells such as melanoma cell line, fibroblasts and vascular endothelial cells",". We and other investigators demonstrated that the effect of IFN-T on the expression of HLA antigens in thyrocytes is dependent on concentration and time, and that at least 3 days are required to achieve maximal e f f e ~ t " " ~In . the presence of IFN-l-, class 11 antigen expression persists; when IFN-l- is removed, there is loss of antigen expression. This is a direct effect of IFN-T, because enhancement is abolished by incubating the cells with IFN-T in the presence of a neutralizing antibodyI5. Lymphocytes secreting immunoreactive IFN-T are found in human autoimmune thyroid diseases, and abetrant HLA class I1 expression on thyrocytes is localized adjacent to IFN-T-expressing lymphocytes". These findings support the view that in
There is increasing evidence that autoimmune processes may have an important pathogenetic role in autoimmune thyroid diseases','. The thyroid gland is thought to be the major site of the production of autoantibodies such as anti-microsomal, anti-thyroglobulin and anti-TSH receptor antibodie~',~. Thyroid gland is characterized by generation of new blood vessels and by a variety of mononuclear cells in aggregates or diffusely distributed between and within the thyroid It seems likely that the infiltrated mononuclear cells migrate from peripheral blood into thyroid tissue where they are retained by interaction with other cells including thyroid epithelial cells (thyrocytes). Recently, it became apparent that cytokines and adhesion molecules play important roles in this migration of mononuclear cells and the cellular interactions of chronic inflammatory processes. The present study has focused on the role of cytokines and adhesion monelules in thyroid glands from patients with autoimmune thyroid disorders.
EXPRESSION O F HLA CLASS I AND HLA CLASS I1 ANTIGENS ON THYROID EPITHELIAL CELLS Substantial evidence exists to show that the major histocompatibility antigen (HLA in humans) has an essential role in the regulation of immune responses. Correspondence to: Shigenobu Nagataki, M.D., First Department of- Internal Medicine, Nagasaki University School of Medicine, 7- 1 Sakamoto-machi, Nagasaki 852, Japan.
27
Autoimmunity Downloaded from informahealthcare.com by McMaster University on 11/04/14 For personal use only.
28
S. NAGATAKI AND K. EGUCHI
autoimmune thyroid diseases expression of HLA class I1 on thyrocytes is the result of local release of IFN-T. Tumor necrosis factor a (TNF-a), which is principally produced by monocytes, has a direct cytotoxic/cytostatic effect in vitro and an anti-tumor activity in vivo. We have shown that TNF-a alone enhances HLA class I antigen expression on cultured thyrocytes, but has no effect on the class I1 antigen expression". TNF-a acts synergistically with JFN-T on both HLA class I and HLA class I1 antigen expression on thyrocytes. Interleukin 1 (IL-1) has many of the same biological effects as TNF-a, and a broad range of inflammatory and immunoregulatory actions. IL- 1 is necessary for the optimal induction of antigen-specific T cell proliferation and is shown to be produced by endothelial cells" and thyrocytes, as well as monocytes in the autoimmune thyroid Previously we reported that IL- lp stimulates 3H-thymidine incorporation into human thyrocytes, and increases the proportion of thyrocytes in the S phase of the cell cycle". IL-1 alone has little effect on HLA class I and class I1 antigen expression on thyrocytes, although IL- lp in combination with IFN-T enhanced the expression of HLA class I and class I1 antigens beyond that produced by IFN-T alone17.These results support the idea that both IL-I and TNF act synergistically with IFN-T on HLA class I and class I1 antigen expression of thyrocytes in vitro. Interleukin 6 (IL-6) is a biologically active B cell stimulatory factor. Both IL-6 mRNA and protein are found in thyrocytes as well as infiltrating cells in thyroid glands from patients with Graves' disease and Hashimoto's thyroiditis". In contrast to IL-1, TNF and IFN-T, neither class I nor class I1 expression is induced by IL-6 alone or in combination with IFN-T". PHENOTYPIC AND FUNCTIONAL CHARACTERISTICS OF INTRATHYROIDAL MONONUCLEAR CELLS IN PATIENTS WITH GRAVES' DISEASE Many investigators have studied the phenotypic and functional characteristics of intrathyroidal mononuclear cells from patients with autoimmune thyroid diseases, but the results have been conflicting22-2s. Immunohistochemical staining shows that the distribution of CD4' and CD8' cells differs in different regions of Graves' thyroid tissue. CD4' cells (helper/inducer T cells) are mainly found in lymphoid aggregates, whereas CD8' cells (suppressor/cytotoxic T cells) are scattered among the thyroid follicle^^.^. Intrathyroidal mononuclear cells were separated from thyroid glands by mechanical aggregation and enzymatic digestion. Dual immunofluorescence staining was used to compare the expression of surface
markers associated with activation of T cells from the thyroid gland and from the corresponding peripheral blood of Graves' patients. Thyroid tissue from treated patients contained greater percentages of HLA-DR' T cells and CD25' (IL-2 receptor a') T cells than the peripheral bloodz9 (Table 2). These results show that intrathyroidal mononuclear cells from patients with Graves' disease comprise an increased proportion of activated T cells. We have also reported that the thyroid gland contains increased numbers of CD4TD29' cells, which results in decreased numbers of the reciprocal CD4'CD45RA' cells. CD4'CD29' cells amplify the production of immunoglobulins after stimulation with
Table 1 Effects of cytokines on antigen expression on thyroid epithelial cells Cytokine
HLA class I
IL- 1 IL-2 IL-6 IFN-a IFN-P IFN- y TNF-a IFN-ytIL-lP IFN-pTNF-a IFN- ytIFN- a
HLA class I1
+* + t + 7
t t tt
77
+
ICAM- 1
t
+
-+ + -3 +
NT** NT NT NT
t 7 tt tt
7
-3
tt tt 1
NT
*+=no change; f=increased; J=decreased. **NT=not tested (or not known).
Table 2 Summary of phenotypic characteristics of intrathyroidal mononuclear cells from patients with Graves' disease. T cells Activated and memory T cells HLA-DR+T cells CD2S' (IL-2Ra+) T cells CD26' (Tali) T cells
t t* tt tt
Helper and memory T cells CD4' CD29' cells
tt
Suppressor-inducer and naive T cells CD4' CD45RA+ cells
11
Natural killer cells Dull CD8' CD1 I b+ cells
1
Adhesion molecule expression LFA- 1 CD2 VLA-4 VLA-S
tt tf tt tt
B cells Activated and differentiated B cells CD20+ CD21- cells IgM'IgD- cells CD20' transferrin receptor+cells PCA-I'cells
tt tt tt tl'
*T=increase& J=decreaaed.
Autoimmunity Downloaded from informahealthcare.com by McMaster University on 11/04/14 For personal use only.
IMMUNE REGULATION IN THYROID AUTOIMMUNITY
pokeweed mitogen (PWM) by inducing a "helperinducer" subpopulation. Conversely, CD4'CD45RA+ cells induce suppressor cells which downregulate immunoglobulin production after stimulation by PWM29,30.A different interpretation is that the presence of CD29 and CD45RA antigens indicates a different maturational status. According to this notion, CD45RA' cells are naive cells that do not respond to recall antigens, whereas CD29' cells are memory cells that have been activated by prior exposure to antigens33. We have shown that the proportion of CD26' cells in the thyroid gland was similar to that in the peripheral blood, but markedly increased in comparison to the value for normal subjects. We also demonstrated that the proliferative responses to thyroglobulin and microsomal antigens of a CD26' cellenriched population isolated from the peripheral blood of patients with Graves' disease were greater than for a CD26' cell-depleted p ~ p u l a t i o nThese ~ ~ . results suggest that the CD26' cells in the thyroid gland from patients with Graves' disease may include antigentriggered memory cells that react with thyroid specific antigens. The percentages of CD8' cells and CD8'CDl Ibcells (cytotoxic T cells) in thyroid glands of Graves' patients are significantly greater than those in their peripheral blood and the peripheral blood from normal subjects. The CD8'CDl Ib' cells are subdivided into two subpopulations on the basis of CD8 antigen density. The percentage of dull CD8'CDllb' cells (natural killer cells) is lower than that in peripheral blood. There is no significant difference in bright CD8+CDl1b+ cells (suppressor-effector T cells) when comparing thyroid glands and peripheral Next, the functional characteristics of intrathyroidal mononuclear cells from patients with Graves' disease were We used specific monoclonal antibodies to separate specific T cell subsets from one another, so that their functions could be individually examined by cell-mixing techniques. By these means, abnormalities of suppression are found in intrathyroidal mononuclear cells from Graves' thyroid glands. These abnormalities are heterogenous in nature, involving decreased suppressor-inducer activity in CD4' cells, decreased suppressor-effector activity in CD8' cells, or both. These results support the possibility that the impairment of suppressor-inducer T cell function may be attributed to reduced numbers of intrathyroidal CD4TD45RA' cells. Thyroid-infiltrating B cells from Graves' patients were also investigated in regard to their phenotypic profiles and spontaneous production of immunoglobulin G (IgG) and anti-thyroidal autoantibodie~'~.The thyroid glands from Graves' patients had greater percentages of IgM'IgD- cells, CD20' transferrin receptor' cells, CD20TD21- cells, and PCA-1' cells compared to the peripheral blood of the same patients
29
and normal subjects. Furthermore, B cells from thyroid glands were able to synthesize IgG, anti-thyroglobulin and anti-microsomal antibodies in the culture supernatants. These findings suggest that the population of phenotypically different B cells that characterize the proliferative and differentiative phases of B cell maturation is expanded in the thyroid of patients with Graves' disease. On the basis of the observations presented in these studies, we suggest that the combination of an increase in the number of activated B cells and impaired function of suppressor T cells may lead to excessive production of immunoglobulins and antithyroidal autoantibodies in thyroid glands from patients with Graves' disease. NEOVASCULARIZATION AND EMIGRATION OF MONONUCLEAR CELLS FROM PERIPHERAL BLOOD TO THYROID TISSUES Thyroid tissues from patients with Graves' disease were stained with anti-factor VIII monoclonal antibody. As shown in Figure 1, the generation of new blood vessels (neovascularization) was observed in the lymphoid follicles. The mononuclear cells which infiltrated around the capillaries contained preferentially CD4' cells. High endothelial venules (HEV) derive their name from their morphological appearance and consist of cuboidal endothelial cells. HEVs are found in T-cell areas of a wide variety of lymphoid tissues. The function of the HEVs is to attract lymphocytes to lymphoid tissues, and they are thus of importance in lym-
Figure 1 Frozen section of Graves' thyroid disease immunostained with anti-factor VIII monoclonal antibody (ABC method). Endothelial cells of blood vessels in the lymphoid follicles are stained positively for anti-factor VIII monoclonal antibody (Original magnification ~ 1 0 0 ) .
Autoimmunity Downloaded from informahealthcare.com by McMaster University on 11/04/14 For personal use only.
30
S. NAGATAKI AND K. EGUCHI
phocyte recirculationTx.Using a monoclonal antibody which recognizes an epitope on human HEVs and immunohistochemical techniques, HEVs are found in lymphoid cell accumulation in thyroid glands from patients with Graves’ disease and Hashimoto’s thyroiditis”. Recently, the migration of lymphocytes has been shown to be controlled by the expression of adhesion molecules on the lymphocytes and of ligands on the vascular endothelial cells. T cells become eligible for migration if they express sufficient levels of adhesion molecules and come into contact with endothelial cells which express sufficient levels of ligands. Peripheral blood T cells bind to human umbilical vein endothelial cells (HUVEC) in vitro. As shown in Figure 2, the percentage of ConA-stimulated T cells adhering to HUVEC was significantly greater than that of unstimulated T cells. Furthermore, the percentage T cells adhering to IFN-T-stimulated HUVEC was increased compared with that to unstimulated HUVEC4’. Thus, the expression of adhesion molecules on T cells is enhanced by antigenic or mitogenic stimulation, and the expression of ligands on the endothelial cells is enhanced by cytokines. The adhesion of T cells to endothelial cells is mediated by at least two adhesion molecules, namely lymphocyte function-associated antigen 1 (LFA- I , C D 1 1 a/ CDI 8)4’, which reacts with intercellular adhesion molecule 1 (ICAM-1)43 and with ICAM-244 and the so-called “very late antigen 4” (VLA-4), which binds to vascular cell adhesion molecule 1 (VCAMThe expressions of ICAM- 1 and VCAM- 1 are upregulated by IFN-T, IL-I and TNF47-4’. In the thyroid tissues from patients with autoimmune thyroid diseases, the mononuclear cells which surround the capillaries
Figure 2 Adherence of T cells to IFNy-stimulated human umbilical vein endothelial cells (HUVEC). T cells were separatcd from blood and incubated for 72 hr with or without ConA and PMA. HUVEC were cultured with various amounts of IFN-y for 24 hr. After incubation, the percentage T-cell adhesion was determined by ”Cr adhesion assay. Open column depicts the untreated T cells. Closed column depicts the stimulated T cells. *P
