0021-972X/90/7105-1230$02.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1990 by The Endocrine Society
Vol. 71, No. 5 Printed in U.S.A.
ANNIE W. C. KUNG, BRIAN M. JONES, AND C. L. LAI Departments of Medicine and Pathology (B.M.J.), University of Hong Kong, Queen Mary Hospital, Hong Kong
ABSTRACT. Treatment with human leucocyte
lymphocyte subpopulations. None of the patients treated with
interferon
rhIFNY developed thyroglobulin or thyroid microsomal antibodies. However, four patients developed antinuclear and smooth muscle autoantibodies during or after rhIFNY treatment. Treatment with rhIFN7 resulted in a significant increase in circulating T-lymphocytes and HLA-DR-positive T-cells (P < 0.05), with equal proportions of circulating CD4+ and CD8+ Tcells becoming DR positive. The percentage of HLA-DR-positive T-cells remained elevated after discontinuation of rhIFNY. Also, rhIFN7 treatment led to a decrease in the number of circulating granulocytes and interleukin-2 receptor-positive cells. In conclusion, we did not observe any thyroid dysfunction or thyroid autoimmunity in our patients treated with the studied dose of rhIFN7, but induction of other autoantibodies was observed. (J Clin Endocrinol Metab 7 1 : 1230-1234, 1990)
(IFNa) has been associated with the development of thyroid autoimmunity and hypothyroidism, but it is not certain whether this phenomenon was a result of contamination with IFN7, which induced HLA class II antigen expression on T-lymphocytes. We prospectively studied 11 subjects (5 females and 6 males; mean age, 26.8 yr; range, 18-36) with chronic active hepatitis B who were randomized to receive recombinant human IFN7 (rhIFN?; 106 U/m2-day, im, 3 times/week) for 16 weeks. Goiter was not present, and no patient had a history or family history of autoimmune diseases. Serial thyroid functions, including serum T4, T3, free thyroid hormone index, free T4 and TSH before, during, and on subsequent follow-up for a period of 1 yr were all normal. Eight patients had adequate serial samples for study of serological and
T
HE ABILITY of interferons (IFNs) to induce and modulate the expression of class I and II HLA antigen and, hence, regulate the immune response is well recognized. The presence of class II antigens on antigenpresenting accessory cells such as macrophages allows appropriate recognition of antigenic peptides by T-helper cells and induction of immunity. IFN7 can increase the expression of class II antigens, while a- and /?-interferons have little or no such activity (1, 2). Normal epithelial cells express class I, but not class II, HLA antigen on the surface. However, treatment with recombinant human IFN7 (rhIFN7) in vitro can induce aberrant HLA class II antigen expression by normal thyrocytes (3, 4). The clinical use of IFN7 had been associated with the development of primary hypothyroidism with an elevation of thyroid antibodies (5, 6). It was speculated that the development of autoimmune thyroid diseases during long term treatment with IFNa was a result of contaminating IFN7, which induced expression of class II antigens on thyrocytes.
The aim of the present study was to investigate whether Chinese patients treated for 16 weeks with rhIFN7 for chronic type B hepatitis would develop features of thyroid autoimmunity or any of the autoantibodies that typically appear in chronic liver diseases.
Subjects and Methods Nineteen patients with biopsy-proven hepatitis B surface antigen-positive (HBsAg-positive) chronic active hepatitis were randomized to received either rhIFN7 or placebo. All individuals had HBsAg, hepatitis B e antigen, and hepatitis B virus DNA in their serum at the beginning of the study. Sera were drawn before, during, and after IFN therapy and stored at -20 C. The study was approved by the Ethics Committee of the University of Hong Kong, and informed written consent was obtained from all patients. Goiter was not found, and thyroid function tests were all normal before therapy. No patient had a history or family history of autoimmune diseases. Treatment
Received March 8, 1990. Address all correspondence and requests for reprints to: Annie W. C. Kung, Department of Medicine, University of Hong Kong, Queen Mary Hospital, Hong Kong.
The subjects randomized to treatment received rhIFN7 (SA, 1 X 107 U/mg; Boehringer Ingelheim, Germany) at a dose of 1 x 106 U/m2-day, im, three times per week for 16 weeks. 1230
Downloaded from https://academic.oup.com/jcem/article-abstract/71/5/1230/2652715 by Leiden University / LUMC user on 17 January 2019
Effects of Interferon-7 Therapy on Thyroid Function, T Lymphocyte Subpopulations and Induction of Autoantibodies
AUTOIMMUNITY AND rhIFN 7 THERAPY Thyroid function
Serological tests
Immunoglobin G (IgG), IgA, and IgM levels were measured by turbidimetry using antisera and calibrator from Atlantic (Stillwater, MN) with the Cobas Bio fast centrifugal analyzer (Roche, Basel, Switzerland). Antibodies to thyroglobulin (TGA) and thyroid microsomal antigen (TMA) were assessed using Thymune-T and Thymune-M hemagglutinatioin kits (Wellcome, Dartford, United Kingdom). Antinuclear antibodies (ANA), anti-DNA antibodies, antismooth muscle antibodies (SMA), and antimitochondrial antibodies (AMA) were evaluated by indirect immunofluorescence, using as substrates HEp2 cells, Crithidia luciliae kinetoplast, rat stomach, and rat kidney, respectively. Thyroid-stimulating antibodies were determined by measuring cAMP released from cultured human thyrocytes in monolayers (7). Antibodies The monoclonal antibodies (mabs) employed in this study were 0KT3 (anti-CD3, mouse IgG2a), 0KT4 (anti-CD4, IgG2b), 0KT8 (anti-CD8, IgG2), and OKIal (anti-HLA-DR framework, IgG2) purchased from Ortho (High Wycombe, United Kingdom); Leu lib (anti-CD16, IgM) from Becton-Dickinson (Mountain View, CA); and anti-Tac (anti-CD25, IgG2a) from Serotec (Bicester, United Kingdom). The polyclonal antibodies used were goat antihuman Ig conjugated to fluorescein isothiocyanate (FITC), F(ab') 2 fragment, from Ortho, FITC-conjugated sheep antimouse (SAM) IgG from Silenus (Hawthorn, Victoria, Australia), and FITC goat antimouse (GAM) IgM from Cappell (Cochranville, PA). Lymphocyte subpopulation analysis Peripheral blood was taken into 10 U/mL preservative-free heparin and mononuclear cells (MNC) separated over a cushion of Ficoll-Hypaque (specific gravity, 1.077). Monocytes were labeled by allowing them to ingest 1-fim diameter latex particles for 30 min at 37 C and 30 min at room temperature, after which aliquots of 8 x 105 MNC were stained with 50 fiL 1:20 diluted FITC goat antihuman Ig (for surface Ig-bearing B-lymphocytes) or with 100 fiL of the mabs listed above (OKT3 and OKT8 diluted 1:160; OKT4, OKIal, and Leu l i b diluted 1:20; anti-Tac diluted 1:200). In each case staining was performed at 4 C for 30 min and then the cells were washed in 0.01 M phosphate-buffered saline (PBS); pH 7.2, containing 1% BSA. Binding of mab was revealed by a second 30-min incubation at 4 C with 50 /xL 1:10 diluted FITC SAM IgG or FITC GAM IgM as appropriate. After further washings in PBS plus 1% BSA, cells were resuspended in 1 drop of mounting fluid, placed onto
a slide, and examined under oil immersion using the Laborlux D UV microscope with a 3-X Ploemopak filter block 12. At least 200 cells were scanned for each marker. Background staining was measured by incubating MNC with FITC SAM IgG or FITC GAM IgM without prior mab treatment and was subtracted from the result for mab-stained MNC. Isolation of T-cells
MNC were suspended in PBS containing 30% fetal calf serum and 1.2% sheep erythrocytes (ShE), the latter having been previously treated with 2-aminoethyl isothiouronium bromide. After incubating at 37 C for 5 min, the cells were pelleted and incubated at 0 C for 60 min. They were then resuspended, layered over Ficoll-Hypaque, and centrifuged at 1000 X g for 20 min. The pelleted ShE plus rosetted T-cells were treated with 0.84% NH4C1 in 0.01 M Tris to lyse the ShE. T-Cells prepared in this way were at least 95% CD3+ by indirect immunofluorescence. Depletion of CD4+ and CD8+ T-cells
To determine whether HLA-DR had been induced preferentially on CD4+ or CD8+ cells, T-lymphocytes prepared by rosetting, which were then stained with OKIal followed by FITC SAM IgG, were depleted of CD4+ or CD8+ cells using immunomagnetic particles coated with anti-CD4 or anti-CD8 mabs (Dynabeads, Dynal, Oslo, Norway). The T-cells were mixed with Dynabeads at a ratio of 10 beads/T-cell in 3 mL PBS plus 0.1% BSA and mixed gently using the roller-mixer for 30 min. The tubes were then placed on a samarium cobalt magnet, which removed Dynabeads plus cells that had bound to Dynabeads. The residual cells were washed and reexamined by UV microscopy for numbers of HLA-DR"1" cells. In preliminary studies using T-cells that had not been stained with OKIal, removal of CD4+ cells or CD8+ cells resulted in populations that were more than 90% CD8+ or more than 90% CD4+, respectively. Statistics Results were analyzed using one-way analysis of variance. Values were expressed as the mean ± SE, and P < 0.05 was considered statistically significant.
Results Thyroid function There was no significant change in thyroid function in any of the subjects over a mean follow-up period of 54.6 weeks (range, 44-64 weeks). Serum total T4, T3, free thyroid hormone index, free T4, and TSH remained normal before, during, and after rhIFN7 therapy (Fig. 1). Serological tests
Only 8 patients in the rhIFN7 treatment group had adequate serial samples for serological and lymphocyte
Downloaded from https://academic.oup.com/jcem/article-abstract/71/5/1230/2652715 by Leiden University / LUMC user on 17 January 2019
Thyroid functions were estimated with routine biochemical methods. Serum T4 and T4 uptake tests were measured using fluorescent polarization immunoassay (Abbott, Chicago, IL), serum free T4 and T 3 by RIA [Amersham (United Kingdom) and Diagnostic Product Corp. (Los Angeles, CA), respectively], and TSH by microparticle enzyme immunoassay (Abbott, North Chicago, IL).
1231
1232
JCE & M • 1990 Vol 71 • No 5
RUNG, JONES, AND LAI
Jo.o 10
16
32
48
Time (weeks)
subpopulation studies. None of the patients treated with rhIFN7 developed TGA or TMA during the study period. Tests for TGA and TMA were all less than 1:10 and 1:100, respectively (normal for TGA,
Vol. 71, No. 5 Printed in U.S.A.
ANNIE W. C. KUNG, BRIAN M. JONES, AND C. L. LAI Departments of Medicine and Pathology (B.M.J.), University of Hong Kong, Queen Mary Hospital, Hong Kong
ABSTRACT. Treatment with human leucocyte
lymphocyte subpopulations. None of the patients treated with
interferon
rhIFNY developed thyroglobulin or thyroid microsomal antibodies. However, four patients developed antinuclear and smooth muscle autoantibodies during or after rhIFNY treatment. Treatment with rhIFN7 resulted in a significant increase in circulating T-lymphocytes and HLA-DR-positive T-cells (P < 0.05), with equal proportions of circulating CD4+ and CD8+ Tcells becoming DR positive. The percentage of HLA-DR-positive T-cells remained elevated after discontinuation of rhIFNY. Also, rhIFN7 treatment led to a decrease in the number of circulating granulocytes and interleukin-2 receptor-positive cells. In conclusion, we did not observe any thyroid dysfunction or thyroid autoimmunity in our patients treated with the studied dose of rhIFN7, but induction of other autoantibodies was observed. (J Clin Endocrinol Metab 7 1 : 1230-1234, 1990)
(IFNa) has been associated with the development of thyroid autoimmunity and hypothyroidism, but it is not certain whether this phenomenon was a result of contamination with IFN7, which induced HLA class II antigen expression on T-lymphocytes. We prospectively studied 11 subjects (5 females and 6 males; mean age, 26.8 yr; range, 18-36) with chronic active hepatitis B who were randomized to receive recombinant human IFN7 (rhIFN?; 106 U/m2-day, im, 3 times/week) for 16 weeks. Goiter was not present, and no patient had a history or family history of autoimmune diseases. Serial thyroid functions, including serum T4, T3, free thyroid hormone index, free T4 and TSH before, during, and on subsequent follow-up for a period of 1 yr were all normal. Eight patients had adequate serial samples for study of serological and
T
HE ABILITY of interferons (IFNs) to induce and modulate the expression of class I and II HLA antigen and, hence, regulate the immune response is well recognized. The presence of class II antigens on antigenpresenting accessory cells such as macrophages allows appropriate recognition of antigenic peptides by T-helper cells and induction of immunity. IFN7 can increase the expression of class II antigens, while a- and /?-interferons have little or no such activity (1, 2). Normal epithelial cells express class I, but not class II, HLA antigen on the surface. However, treatment with recombinant human IFN7 (rhIFN7) in vitro can induce aberrant HLA class II antigen expression by normal thyrocytes (3, 4). The clinical use of IFN7 had been associated with the development of primary hypothyroidism with an elevation of thyroid antibodies (5, 6). It was speculated that the development of autoimmune thyroid diseases during long term treatment with IFNa was a result of contaminating IFN7, which induced expression of class II antigens on thyrocytes.
The aim of the present study was to investigate whether Chinese patients treated for 16 weeks with rhIFN7 for chronic type B hepatitis would develop features of thyroid autoimmunity or any of the autoantibodies that typically appear in chronic liver diseases.
Subjects and Methods Nineteen patients with biopsy-proven hepatitis B surface antigen-positive (HBsAg-positive) chronic active hepatitis were randomized to received either rhIFN7 or placebo. All individuals had HBsAg, hepatitis B e antigen, and hepatitis B virus DNA in their serum at the beginning of the study. Sera were drawn before, during, and after IFN therapy and stored at -20 C. The study was approved by the Ethics Committee of the University of Hong Kong, and informed written consent was obtained from all patients. Goiter was not found, and thyroid function tests were all normal before therapy. No patient had a history or family history of autoimmune diseases. Treatment
Received March 8, 1990. Address all correspondence and requests for reprints to: Annie W. C. Kung, Department of Medicine, University of Hong Kong, Queen Mary Hospital, Hong Kong.
The subjects randomized to treatment received rhIFN7 (SA, 1 X 107 U/mg; Boehringer Ingelheim, Germany) at a dose of 1 x 106 U/m2-day, im, three times per week for 16 weeks. 1230
Downloaded from https://academic.oup.com/jcem/article-abstract/71/5/1230/2652715 by Leiden University / LUMC user on 17 January 2019
Effects of Interferon-7 Therapy on Thyroid Function, T Lymphocyte Subpopulations and Induction of Autoantibodies
AUTOIMMUNITY AND rhIFN 7 THERAPY Thyroid function
Serological tests
Immunoglobin G (IgG), IgA, and IgM levels were measured by turbidimetry using antisera and calibrator from Atlantic (Stillwater, MN) with the Cobas Bio fast centrifugal analyzer (Roche, Basel, Switzerland). Antibodies to thyroglobulin (TGA) and thyroid microsomal antigen (TMA) were assessed using Thymune-T and Thymune-M hemagglutinatioin kits (Wellcome, Dartford, United Kingdom). Antinuclear antibodies (ANA), anti-DNA antibodies, antismooth muscle antibodies (SMA), and antimitochondrial antibodies (AMA) were evaluated by indirect immunofluorescence, using as substrates HEp2 cells, Crithidia luciliae kinetoplast, rat stomach, and rat kidney, respectively. Thyroid-stimulating antibodies were determined by measuring cAMP released from cultured human thyrocytes in monolayers (7). Antibodies The monoclonal antibodies (mabs) employed in this study were 0KT3 (anti-CD3, mouse IgG2a), 0KT4 (anti-CD4, IgG2b), 0KT8 (anti-CD8, IgG2), and OKIal (anti-HLA-DR framework, IgG2) purchased from Ortho (High Wycombe, United Kingdom); Leu lib (anti-CD16, IgM) from Becton-Dickinson (Mountain View, CA); and anti-Tac (anti-CD25, IgG2a) from Serotec (Bicester, United Kingdom). The polyclonal antibodies used were goat antihuman Ig conjugated to fluorescein isothiocyanate (FITC), F(ab') 2 fragment, from Ortho, FITC-conjugated sheep antimouse (SAM) IgG from Silenus (Hawthorn, Victoria, Australia), and FITC goat antimouse (GAM) IgM from Cappell (Cochranville, PA). Lymphocyte subpopulation analysis Peripheral blood was taken into 10 U/mL preservative-free heparin and mononuclear cells (MNC) separated over a cushion of Ficoll-Hypaque (specific gravity, 1.077). Monocytes were labeled by allowing them to ingest 1-fim diameter latex particles for 30 min at 37 C and 30 min at room temperature, after which aliquots of 8 x 105 MNC were stained with 50 fiL 1:20 diluted FITC goat antihuman Ig (for surface Ig-bearing B-lymphocytes) or with 100 fiL of the mabs listed above (OKT3 and OKT8 diluted 1:160; OKT4, OKIal, and Leu l i b diluted 1:20; anti-Tac diluted 1:200). In each case staining was performed at 4 C for 30 min and then the cells were washed in 0.01 M phosphate-buffered saline (PBS); pH 7.2, containing 1% BSA. Binding of mab was revealed by a second 30-min incubation at 4 C with 50 /xL 1:10 diluted FITC SAM IgG or FITC GAM IgM as appropriate. After further washings in PBS plus 1% BSA, cells were resuspended in 1 drop of mounting fluid, placed onto
a slide, and examined under oil immersion using the Laborlux D UV microscope with a 3-X Ploemopak filter block 12. At least 200 cells were scanned for each marker. Background staining was measured by incubating MNC with FITC SAM IgG or FITC GAM IgM without prior mab treatment and was subtracted from the result for mab-stained MNC. Isolation of T-cells
MNC were suspended in PBS containing 30% fetal calf serum and 1.2% sheep erythrocytes (ShE), the latter having been previously treated with 2-aminoethyl isothiouronium bromide. After incubating at 37 C for 5 min, the cells were pelleted and incubated at 0 C for 60 min. They were then resuspended, layered over Ficoll-Hypaque, and centrifuged at 1000 X g for 20 min. The pelleted ShE plus rosetted T-cells were treated with 0.84% NH4C1 in 0.01 M Tris to lyse the ShE. T-Cells prepared in this way were at least 95% CD3+ by indirect immunofluorescence. Depletion of CD4+ and CD8+ T-cells
To determine whether HLA-DR had been induced preferentially on CD4+ or CD8+ cells, T-lymphocytes prepared by rosetting, which were then stained with OKIal followed by FITC SAM IgG, were depleted of CD4+ or CD8+ cells using immunomagnetic particles coated with anti-CD4 or anti-CD8 mabs (Dynabeads, Dynal, Oslo, Norway). The T-cells were mixed with Dynabeads at a ratio of 10 beads/T-cell in 3 mL PBS plus 0.1% BSA and mixed gently using the roller-mixer for 30 min. The tubes were then placed on a samarium cobalt magnet, which removed Dynabeads plus cells that had bound to Dynabeads. The residual cells were washed and reexamined by UV microscopy for numbers of HLA-DR"1" cells. In preliminary studies using T-cells that had not been stained with OKIal, removal of CD4+ cells or CD8+ cells resulted in populations that were more than 90% CD8+ or more than 90% CD4+, respectively. Statistics Results were analyzed using one-way analysis of variance. Values were expressed as the mean ± SE, and P < 0.05 was considered statistically significant.
Results Thyroid function There was no significant change in thyroid function in any of the subjects over a mean follow-up period of 54.6 weeks (range, 44-64 weeks). Serum total T4, T3, free thyroid hormone index, free T4, and TSH remained normal before, during, and after rhIFN7 therapy (Fig. 1). Serological tests
Only 8 patients in the rhIFN7 treatment group had adequate serial samples for serological and lymphocyte
Downloaded from https://academic.oup.com/jcem/article-abstract/71/5/1230/2652715 by Leiden University / LUMC user on 17 January 2019
Thyroid functions were estimated with routine biochemical methods. Serum T4 and T4 uptake tests were measured using fluorescent polarization immunoassay (Abbott, Chicago, IL), serum free T4 and T 3 by RIA [Amersham (United Kingdom) and Diagnostic Product Corp. (Los Angeles, CA), respectively], and TSH by microparticle enzyme immunoassay (Abbott, North Chicago, IL).
1231
1232
JCE & M • 1990 Vol 71 • No 5
RUNG, JONES, AND LAI
Jo.o 10
16
32
48
Time (weeks)
subpopulation studies. None of the patients treated with rhIFN7 developed TGA or TMA during the study period. Tests for TGA and TMA were all less than 1:10 and 1:100, respectively (normal for TGA,
