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Demonstration and characterization of thyroglobulin-binding peripheral blood cells in Hashimoto patients by fluoroimmunocytoadherence
Demonstration and Characterization of Thyroglobulin-Binding Peripheral Blood Cells in Hashimoto Patients by Fluoroimmunocytoadherence E. RICHTER,.*
G. WICK,? N. ZAMBELIS,$ AND G. SCHERNTHANER~
H. Lunwrc;,#
*Em -Now - Thront -Clinic, *fn.stitute fiw Grttercrl trnd Exprrinrentctl Prrtholo,qy, and Slmtitute for NucIe~~r Medicine. i7nir.ersit.v of InnshrucX. InnshrucX. rrnd Wecond Depctrtmettt of Intemnl Mvdicitw. UGlvr.sity of Virnnrr. VicJntrtr. A~striu
Received March 15, 1978 The number of thyroglobulin-binding peripheral blood cells (TG-BL) was determined in 24 patients with Hashimoto thyroiditis: I2 non-Hashimoto. non-Graves disease goiter patients; and IO unrelated controls using the fluoroimmunocytoadherence (FICA) technique. Hashimoto patients showed significantly elevated numbers of TG-BL as compared to members of the two other groups. For further characterization of TG-BL, inhibition experiments were performed by preincubation of peripheral blood lymphocytes with TG, normal human serum, rabbit-y-globulin, or bovine serum albumin. The results of these experiments indicate that the total number of TG-BL in Hashimoto patients consists of a majority of cells actively synthesizing their TG receptors and a minority adhering to TG-coated beads due to passive adsorption of TG autoantibody onto their surface or in a nonspecific fashion. No correlation between the total number of TG-BL and the presence and/or titer of autoantibodies to TG. the second colloid antigen, or microsomal antigens was found. Evidence is presented that the determination of TG-BL provides a specific tool for the diagnosis of Hashimoto thyroiditis superior to conventional thyroid autoantibody analysis.
INTRODUCTION
The Obese strain (OS)’ of chickens is a well-defined animal model for human Hashimoto thyroiditis and some essential data for understanding of the human counterpart derive from studies of this strain (1). In an earlier chronological investigation on the occurrence of thyroglobulin (TG)-binding cells in peripheral lymphoid organs of OS chickens, the number of TG rosette-forming cells (RFC) was shown to reach peak values at 2-4 weeks of age, i.e. clearly before maximum severity of spontaneous autoimmune thyroiditis and frequency of circulating TG autoantibodies (AAB) were attained (2). The B-cell nature of TG-RFC actively synthesizing their TG-receptor Ig molecules was confirmed by inhibition experiments using B- and T-cell-specific turkey antisera. Though circulating IgM and IgG anti-TG autoantibodies can be found in OS chickens, only anti-p sera significantly inhibited TG-rosette formation, indicating that antigen receptors I Abbreviations used: TG, thyroglobulin: TG-BL, thyroglobulin-binding lymphocytes: FICA. fluoroimmunocytoadherence; CA, and CA,, first and second colloid antigens; OS, Obese strain; RFC, rosette-forming cells: AAB, autoantibodies: BSA, bovine serum albumin: PBS, phosphatebuffered saline; IIF, indirect immunofluorescence; CRBC. chicken red blood cells: PBL. peripheral blood lymphocytes; NHS, normal human serum. 178 OOYO-1229/78/0112-0178$01.00/O Copynght All
rights
0 of
1978 reproductmn
by
Academic in any
Press.
Inc.
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THYROGLOBULIN-BINDING
BLOOD
CELLS
179
of active TG-RFC are IgM molecules. The existence of passive TG-RFC in peripheral lymphoid organs of OS chickens was suggested by the competitive inhibitory effect of normal chicken serum on the total number of TG-RFC. Only anti-T-cell serum-sensitive RFC were shown to adsorb passively TG antibodies of the IgM class (3), in agreement with the previous results of Webb and Cooper in sheep red blood cell-immunized normal chickens (4). The rosette test, using antigen-coated erythrocytes as indicator particles, is time consuming and exact quantification of the degree of antigen coating is difficult. Therefore, a new, generally applicable method-fluoroimmunocytoadherence (FICA)-was developed. Beads of cross-linked dextran (Sephadex G-25) were chosen as indicator particles and the antigen in question was coupled to their surface via a spacer chain. The main advantages and performance characteristics of this method have been described in detail (5). Essentially, such spacer-armed beads can be stored ready for use in the coupling with any protein ligand. Also after coupling the antigen-coated beads are stable for many months. Slight modifications introduced for the present purposes will be reported under Materials and Methods. The main purpose of the present investigation was the enumeration of TGbinding blood lymphoid cells by the FICA method and the clarification of their eventual significance for the diagnosis of Hashimoto thyroiditis. To answer these questions the individual number of TG-binding cells in Hashimoto patients was compared to the serological findings and the cytological appearance of needle biopsies in selected cases. The mean numbers of TG-binding lymphocytes of Hashimoto patients; non-Hashimoto, non-Graves disease goiter patients; non thyroid disease patients; and healthy individuals were determined and compared statistically. Finally the following control experiments were performed: (i) specificity testing of TG-binding cells in FICA; (ii) production of passive TGbinding cells; (iii) inhibition of nonspecific antigen-binding cells. MATERIALS AND METHODS Human Thyroglobulin Human rhyroglobulin was prepared using a modification by Shulman ef al. (6). Human thyroid glands obtained
of a method described at surgery were sliced and extracted in 0.15 M saline solution for 16 hr at 4°C and the extract was subjected to precipitation with saturated (NH&SO,. The reconstituted precipitate was centrifuged at 30,OOOg for 30 min and the supernatant was dialyzed against saline. After passage through a Sephadex G-200 column (Pharmacia, Uppsala, Sweden) two peaks were collected. The first peak containing TG, but contaminated with y-globulin, was further purified by absorption with glutaraldehydeimmobilized rabbit anti-human y-globulin serum. The purity of the TG was assessed by immunoelectrophoresis, and the specificity, by its inhibitory capacity in passive hemagglutination tests.
Fluoroimmunocytoadherencr
(FICA)
This method has already been described in detail elsewhere (5). In short, fluorochrome-labeled dextran beads are coated with antigen. These beads are then incubated with the lymphoid cells under study, which, in case they carry antigen-specific receptors, will adhere to the beads coated with the respective
180
KI(:H 1EK Et A/.
antigen. Beads carrying a different antigen and labeled with another fluorochrome (or left unstained) are added as a control for specificity testing. ((1) Acti\wtiott of hrcrtls. Cross-linked dextran beads (Sephadex G-25 Superfine, Lot No. 0998, Pharmacia, Uppsala, Sweden) were selected due to their size characteristics, their aptitude for lyophilization, and their advantages for the possible fluorometric evaluation of the degree of antigen coating (5). Briefly. beads swollen in distilled water were sieved to obtain particles of a mean diameter of 2627 pm. These beads were then activated with CNBr and armed with a spacer consisting of 3,3’-diaminodipropylamine. succinic anhydride, and N-hydroxysuccinimide. The spacer-armed beads can be stored in the dark in dioxane (water free) for many months and can be used immediately without further manipulation for the coupling of any ligand containing primary amino groups. (h) Coupling with antigens. Human TG or bovine serum albumin (BSA. Berhringwerke AG, Marburg, FRG) was coupled onto the spacer-armed beads as described in detail (5). (c) Deacti\wtion. The procedure used for deactivation of coated ligand beads differed slightly from that recently described (5). Conventional deactivation. appropriate for affinity chromatography, does not suffice for FICA. Thus. during prolonged (weeks) storage of coupled and 1 M glycine-deactivated beads, small amounts of protein are lost and free active sites are reexposed, leading to unspecific adherence of lymphoid cells. This problem can be overcome by admixture of an appropriate (not cross-reacting with the specific antigen under investigation) normal serum or purified serum protein to the antigen-coated beads, thus blocking residual active sites. For this purpose rabbit fluorochromelabeled or unlabeled y-globulin (Pentex. Miles Laboratories, Kankakee, Ill.) preparations in phosphate-buffered saline (PBS, pH 7.2) were used in the present study. In experiments with only two different antigen-coated bead preparations only one charge was treated with fluorescein isothiocynate-conjugated rabbit y-globulin, the second charge was deactivated with unlabeled rabbit y-globulin. After storage of coated and deactivated preparations in suspension, unlabeled rabbit y-globulin (1%) was again added and the beads were vertically rotated for 60 min at 4”C, followed by three washings in PBS just before use in an FICA test. (d) Specificity of coating. This was assessed in indirect immunofluorescence (IIF) tests using a semiautomated micromethod described elsewhere (7). (P) Pevfkmcrnce of the test. PBS was used as a medium throughout. Fivetenths of a milliliter of a suspension of acridine orange-stained cells (4 x lo6 cells/ml) was added to 0.5 ml of a mixture of BSA- and TG-coated beads [both types of beads as 5% (v/v) suspensions] in 13 x 75ml plastic tubes, the ratio of lymphoid cells to beads equaling 1: 10. When analyzed, mixtures of unstained beads and lymphocytes were observed under a conventional light microscope. Simultaneous observation of labeled and unstained beads was done under a Reichert Immunopan fluorescence microscope. Double readings were performed after incubation for 1 and 18 hr at 4°C and vigorous resuspension with Pasteur pipets just before mounting a drop of the suspension on the slide. Only beads with two or more adherent cells were evaluated.
THYROGL.OBULIN-BINDING
BLOOD
CELLS
181
Lqmphocytes PBS (pH 7.2) was used as a medium throughout. Ten milliliters of defibrinated venous blood was collected and lymphocytes were isolated by the method of Bqiyum (8). Through collaboration with (and some financial motivation of) express train personnel, blood collected from 22 patients in Vienna could also be tested late on the same day in Innsbruck. Mononuclear cells were removed by incubation and passage, through nylon wool (Leukopak, Hyland Division of Travenol Laboratories)-packed syringes. Viability, as assessed by trypan blue exclusion, was always above 90%. Finally, the vital dye acridine orange used for staining of the final preparation also permitted a morphological classification of cells (less than 1% monocytes) in suspension and those adhering to beads. Specificity Tests FICA irlhibition experiments. Before admixture to the beads the blood lymphocytes were incubated for 45 min at room temperature with human TG, rabbit y-globulin, BSA, PBS only, or aggregated rabbit y-globulin prepared according to Dickler and Kunkel (9). Corwsrztional rosette rests. Chicken red blood cells (CRBC) were coated with human TG, BSA, or rabbit y-globulin by means of a modified chromium chloride method ( 10) and the rosette tests were performed as described earlier (2). Pnssive FICA. (a) Lymphocytes of a healthy donor were incubated for 1 hr at 37°C with sera (all sera undiluted and heat inactivated at 56°C for 30 min) from Hashimoto patients with different serological reaction patterns (i.e., high or low titers of microsomal and/or TG autoantibodies). This procedure was followed by three washings and readjustments to 4 x lo6 cells/ml prior to FICA testing. (b) Inhibition of passive FICA: After incubation of normal lymphoid cells with a patients’ sera containing TG antibodies (titer of 2500 in passive hemagglutination) and three washings, lymphocytes were resuspended for a second incubation in undiluted normal human serum for 30 min at 37°C before further washings and readjustment for FICA testing wuth supplementation of 10% normal human serum to the medium. Serology Autoantibodies to human TG were detected by tanned red cell hemagglutination (Thyroglobulin Test Kit, Wellcome Reagents, Ltd., Beckenham, England) and in IIF tests on methanol-fixed cryostat sections of colloid goiter. Microsomal autoantibodies were detected by tanned red cell hemagglutination (Microsome Test Kit, Fujizoki, Pharmaceutical Co., Ltd., Tokyo, Japan) and in IIF tests on unfixed cryostat sections of thyrotoxic goiter. Antibodies to the second colloid antigen (CA,) were demonstrated by IIF on methanol-fixed colloid goiter sections after absorption of antisera with TG-coated sephadex G-25 beads. One-tenth of a milliliter of packed TG beads was added to 0.1 ml of undiluted serum followed by incubation overnight at 4°C. The procedure was repeated until no TG autoantibodies were detectable in the tanned red cell hemagglutination test (1 1- 13).
182
Rl(:H
I-ER
E7 Al
Sera and defibrinated blood were collected from 10 individuals consisting mainly of laboratory personnel, patients hospitalized for reasons other than autoimmune or thyroid disease, 12 patients with various thyroid diseases except Hashimoto and Graves diseases and 24 patients with Hashimoto thyroiditis. The diagnosis of Hashimoto thyroiditis was established by the following criteria: presence of goiters of typical consistency, results of thyroid function tests, high titers of TG and/or microsomal autoantibodies, and cytological appearance of thyroid tissue obtained by needle biopsies. Biopsies were only performed in questionable instances (the age and sex distribution of patients and controls is evident from Table 1). RESULTS
Active TG-Binding Cells The serological results and the mean numbers of TG-binding lymphocytes (TG-BL) for the three groups tested are shown in Table 1. In all 24 Hashimoto patients tested, TG-binding lymphocytes were significantly elevated. Of 12 patients with thyroid diseases other than Hashimoto or Graves disease, two had high counts (19 and 13/103 cells, respectively) of TG-BL. One of these proved to be serologically negative, the other showed a low titer of TG antibodies in passive hemagglutination. The controls from the third group showed only background values of TG-BL corresponding to those observed in the case of BSA-BL. No correlation emerged between the number of TG-BL and individual autoantibody titers to TG, CA.), or microsomal antigens in passive hemagglutination or IIF tests (Table 2). The mean number of TG-BL in the Hashimoto group (25.2 + 7.8) was significantly higher than those of non-Hashimoto, non-Graves goiter patients (6.6 & 4.9) and normal controls (3.9 ? 1.7). The mean values for BSA-BL were in the same range for all three groups. The frequencies of TG and microsomal antibodies in passive hemagglutination (HA) and IIF tests and of antibodies to the second colloid antigen in IIF tests are also given in Table 1. Five Hashimoto patients were serologically negative. Only one patient of the non-Hashimoto, non-Graves goiter group had a low TG-autoantibody titer of I:40 in passive HA. The specificity of TG-BL was assessed by inhibition studies. Table 3 shows the results of two representative experiments. Preincubation of lymphocytes of Hashimoto patients for 4.5 min at room temperature with 2% human TG significantly reduced the number of TG-BL to background values, while 2% rabbit y-globulin or 1% aggregated rabbit y-globulin only had a partial inhibitory effect. Preincubation with 2% TG + 2% rabbit y-globulin resulted in more pronounced reduction, but not complete inhibition. No reduction of TG-BL or lymphoid cells adhering to BSA beads occurred after preincubation with 2% BSA as an unrelated antigen (not shown in Table 3). Incubation with rabbit y-globulin or aggregated rabbit y-globulin, but not with TG alone, reduced the number of background values in both Hashimoto patients and controls. From these data it was concluded that the actual number of lymphoid cells of Hashimoto patients adhering to TG beads consists of a majority of TG-BL
N.D.
0112
18/24 N.D.
14124
BSA 5.0 It 1.4
TG 25.2 + 7.8
W
L f:
812
32 (21-45)
o/10
N.D.
N.D.
o/10
N.D.
8 E: F: in
x F
N.D.I
2124
IIF”
n Titer >20 in tanned cell hemagglutination test. b Titer > 10 in IIF on methanol-fixed, frozen sections of a human colloid goiter. c Same as in Footnote b; sera absorbed with human thyroglobulin. ‘I Titer > 10 in IIF on unfixed, frozen sections of human thyrotoxic goiter. (1Mean numbers of lymphocytes / lo3 PBL adhering to antigen-coated beads, only two or more cells adhering to a single bead counted as positive. f Not done. y Statistical significant difference to Hashimoto group (p < 0.005). h Statistical significant difference to Hashimoto group (p i 0.001).
l/l2
17124
CA,’
Microsome-AAB
Antigen-binding cells (FICA)/103 PBL’
g
45 (19-64)
17124
IIF (CA,)*
IIF
PATIENTS
Passive HA”
IN HASHIMW~O
Serology
CELLS
3.8 t 1.5
10
Normal + other controls
913
57 (35-78)
Passive HA”
TG-AAB
1
3.9 +- 1.7*
12
Non-Hashimoto, non-Graves disease goiter
2212
TABLE .~ND ANTIGEN-BINDING
4.2 i 1.3
24
Hashimoto thyroiditis
(range)
Mean age
SEROLOGY
6.6 + 4.9O
N
Patients
Sex (F/M)
THYROID
Antigen-binding lymphocytes/IO’ PBL
Serd0gy _____--.-.--.TG-AAB Patient W.M. F.A. C.R.’ P.A. SM.' K.J. ” ’ ’ ” “ ’
Passive HA” 65 69 50 60 63 65
F F F F F F
250,000 40,960 40 250,000 <20 <20
IIF (CA,)” 80 40 40 80
IIF (CA,)’ (10
Microsome-AAB Passive HA” IIF” 20,480 40,960 320 80,000
20
TG
BSA
18 31 20 22 39 28
6 3 5 5 4 1
Titer in tanned cell hemagglutination test. Titer in IIF on methanol-fixed, frozen sections of a human colloid goiter Same as in Footnote b; sera absorbed with human thyroglobulin. Titer in IIF on unfixed, frozen sections of human thyrotoxic goiter. Number of lymphocytes adhering to beads per IO3 PBL. Needle biopsy.
and a minority of cells attached to the beads via an Fc receptor (to the rabbit y-globulin used for bead deactivation) or in an unspecific fashion. Passive TG-Binding Cells A series of control experiments was performed in order to assess further (a) the proportion of TG-BL in Hashimoto patients, which had acquired their antigenbinding capacity by passive adsorption of TG-autoantibodies; and (b) the possibility of producing TG-BL by preincubation of normal lymphoid cells with TG antibody-containing serum. For elucidation of the first question peripheral blood lymphocytes (PBL) from patients with Hashimoto thyroiditis were isolated and washed as usual and then incubated with heat-inactivated, undiluted normal human serum (NHS) assuming a possible displacement of passively adsorbed TG autoantibodies. The PBS used for subsequent washing and performance of FICA was supplemented with 10% NHS. The results of this experiment are presented in Table 4. A clear-cut inhibition of a portion of TG-BL was brought about by this treatment, the remaining values, however, still were significant. To answer the second question normal blood lymphocytes were exposed to different heat-inactivated Hashimoto sera, normal serum, and PBS for 60 min at 37°C (Table 5). Antigen-binding capacity was only acquired by lymphocytes preincubated with sera containing autoantibodies to TG. Hashimoto sera without circulating antibody detectable in passive HA or IIF tests and normal human serum produced only background values in the range of the value obtained by preincubation with PBS only. Table 6 shows that the phenomenon of passive TG binding is reversible. A Hashimoto serum containing a high titer of TG autoantibodies in passive HA was used for the first incubation step, for the second incubation, after three washings, the lymphocytes were resuspended in undiluted normal human serum, resulting in a reduction of passive TG-BL.
Expt 2
cells/IO3 PBL
30
TG 26 7
BSA 8
PBS
8
TG 8 7
BSA 9
2% TG
23
TG 20 3
BSA 4
2% rabbit y-globulin TG 25 N.D.
BSA 7
0.1% TG 18 N.D.
BSA 3
0.5% TG 18
Aggregated rabbit y-globulin
TABLE 3 OF ANTIGEN-BINDING CELLS IN FICA
” Forty-five minutes at room temperature followed by addition of antigen-coated beads. b Not done.
Expt 1
Preincubation of lymphocytes” Antigen coating Number of antigen-binding
INHIBITION
N.D.
1% BSA 3
4
TG N.D.D
2
BSA N.D.
2%TG + 2% rabbit y-globulin
2; 5 : 5 ? z E z n F 8 u E FVT
Number of antigen-binding PBL/lOZ lymphocytes in FIC.4 after preincubation with” -___ PBS NHS Patient
Diagnosis
TG
BSA
H” H N“ N
26 20 5 6
5 7 6 6
J.M. S.M. H.M. F.J.
TG __-__ 17 14 3 3
BSA 3 3 3 3
’ Preincubation was for I5 min at 37°C in medium only or in medium + 10% NHS, followed by three washings and readjustment. b Hashimoto disease. c Normal control.
DISCUSSION
The FICA technique was developed in this laboratory as a new method for the demonstration, enumeration, characterization, and separation (5, 14) of antigenbinding cells. The antigen in question is coupled via a spacer onto the surface of Sephadex G-25 beads. Adherence of lymphocytes results in formation of
TABLE PASSIVE
ANTIGEN
5
BINDING BY PREINCUBATION OF NORMAL WITH DIFFERENT HASHIMOTO SERA
Characterization
BLOOD
of serum donors
AAB titer in passive HA testb Serum used for incubation”
TGAAB
PBS (medium only) NHS U.J. S.E. F.A. J.M.
110 <20 <20 40,960 2,500
MicrosomeAAB
L~MPHOWTES
Passive antigen-binding cells (FICA)/lO’ PBL” CA, (IIF)’ N.D.’
FICA TGbinding cells
TG
BSA
N.D. 3 34 31 16
4 3 5 3 I3 12
3 3 4 4 4 5
n Undiluted serum was used for 60 min at 37°C followed by three washings and readjustment to 4 x lo6 PBL/ml before addition of TG or BSA beads. ’ Titer in tanned cell hemagglutination test. c Titer in IIF on methanol-fixed, frozen sections of human colloid goiter; sera absorbed with human thyroglobulin. ’ Number of lymphocytes adhering to beads per 10’ PBL. ’ Not done.
THYROGLOBULIN-BINDING
PASSIVE TG-BINDING
NORMAL
BLOOD
TABLE 6 PBL AND INHIBITION
187
CELLS
WITH NORMAL
HUMAN
SERUM”
Passive antigen-binding cells (FICA) / lo3 PBLC Incubationb
TG
BSA
NHS, 30 min, 37°C PBS, 30 min, 37°C
6 13
6 6
n Normal PBL were preincubated with undiluted Hashimoto patient serum (J.M. of Table 6) for 30 min at 37”C, followed by three washings before addition of NHS (undiluted) or PBS. b Incubation with NHS or PBS (medium only) followed by three washings and readjustment to 4 x 106 PBUml before addition of beads. r Number of lymphocytes adhering to beads per 10) PBL, mean of two measurements after 90 min and 18 hr.
reversed rosettes, i.e., the beads in the center are surrounded by clusters of lymphocytes. The main advantages of this technique are (a) the constant availability of spacer-armed beads for coating with any protein ligand, (b) the stability of ready to use antigen-coated beads for many months, and (c) the possibility to incorporate beads coated with a control antigen into the same preparation which can be evaluated simultaneously under a fluorescence microscope. In the present study TG and BSA-BL were enumerated by means of this technique in 24 hashimoto and Graves disease and 10 control persons consisting of healthy laboratory personnel and nonthyroid disease patients. The specificity of TG binding was confirmed by a number of control experiments. In the peripheral blood of all Hashimoto patients the observed numbers of TG-BL were significantly elevated in comparison to those of the control groups. The only exception were two patients in the non-Hashimoto thyroid group which showed elevated numbers of TG-BL. Unfortunately, no needle biopsies were available from these patients to complement the clinical diagnosis. The values of all other individuals were in the range of the background counts attained with BSA-coated beads. These data are in agreement with those in earlier publications of other authors (15- 17) on TG-BL in Hashimoto patients and results obtained in the Obese strain (OS) of chickens, an animal model for human Hashimoto thyroiditis (l-3). Khalid et al. (18) have studied the occurrence of peripheral blood lymphocytes binding microsomal membranes in patients with different thyroid diseases, including six Hashimoto patients, and observed a significant increase in the number of antigen-BL in patients with circulating antibodies to thyroid cytoplasmic antibodies, while the number of antigen-BL in patients without autoantibodies was within the normal range. They concluded that the occurrence of antigen-BL may be a response to an excess of antigen in the circulation. The binding capacity of peripheral blood lymphocytes for thyroid microsomal antigens was not evaluated in the present study. However, in regard to the binding of TG, no correlation was found between the presence and titer of autoantibodies to this antigen and the absolute numbers of TG-BL. This fact again parallels the findings in OS chickens, but is in contrast to observations published by others (19).
188
KICH’I‘EK
ET Al
Our studies on passive TG rosette-forming cells do, however, lend support to the notion that a certain, although minor, portion of TG-BL in Hashimoto patients consists of cells which have passively adsorbed TG autoantibodies onto their surface. This phenomenon could be competitively inhibited by incubation with normal human serum. Similar findings have been reported for passive TG-BL in OS chickens, in which these cells had been identified as T cells adsorbing IgM anti-TG (3), and TG-BL actively synthesizing their receptors were shown to be B cells (2). That passive adsorption of autoantibodies may contribute to the total number of antigen-binding cells in humans was also shown by the effect of an incubation of normal peripheral blood lymphocytes from Hashimoto patients. The characterization of active and passive TG-BL and the elucidation of the class of antibodies responsible for the formation of the latter will be the subjects of further studies. Incubation of Hashimoto lymphocytes with rabbit y-globulin and aggregated rabbit y-globulin at lower concentrations slightly reduced the number of antigen-BL. No reduction resulted by incubation with BSA and no complete inhibition of antigen BL could be obtained by preincubation of lymphocytes with 2% TG + 2% rabbit y-globulin. As rabbit y-globulin was used in addition to glycine for deactivation of all bead preparations, small amounts of this protein are bound by residual active sites. This occurs in a random fashion, i.e., the Fc portion being exposed only in some of the rabbit Ig molecules and thus being accessible to cells with Fc receptors. This explains the relatively small number of cells adhering to the beads via an Fc receptor and inhibitable by rabbit -y-globulin. In conclusion the present study demonstrates a significantly increased number of TG-BL in patients with autoimmune thyroiditis. The total number of TG-BL in the peripheral blood of Hashimoto patients is composed of: (a) a majority of lymphoid cells binding TG via their own membrane-associated receptors (active TG-BL) and (b) a minor portion consisting of three types of cells: lymphoid cells which possess passively acquired receptors for TG; cells with Fc receptors binding to the rabbit y-globulin used for bead deactivation, and cells adhering nonspecifically to the surface of beads. The proportion of passive TG-BL may vary in different patients and test systems, thus accounting for the discrepancies in respect to a possible correlation between the autoantibody titers and the number of TG-BL reported by different authors. As increased counts of TG-BL were shown to be a constant finding in Hashimoto’s thyroiditis, the FICA technique might prove an ideal test system for monitoring ongoing autoimmune thyroid disease and perhaps other autoimmune conditions as well. ACKNOWLEDGMENTS This work was supported by the Austrian Research Council (Project No. 31201.
REFERENCES I. 2. 3. 4. 5. 6. 7. 8.
Wick, G., Sundick, R. S., and Albini, B., C/i/r. Ir,rntuno/. Ir,,n~lr/,opntho/. 3. 272, 1974. Richter, E., and Wick, G., J. I~r~nu~ol. 114, 757. 1975. Richter, E., Wick, G., and Schauenstein. K.. El/r. J. I/u~n~rrol. 5, 554. 1975. Webb, S. R., and Cooper. M. D.. J. f/rr~nr/to/. 111, 275, 1973. Richter, E., and Wick. G.. 2. I/,l/,lrcllirtrfr.~~~~.~~,/~,152, 351, 1977. Shulman, S., Mates, G., and Bronson. P.. Biwhim. Biophys. AC/U 147, 208, 1967. Schauenstein, K., Wick. G.. and Kink. H., .I. f~rr~trc/ro/. Mctlto~/.t 10, 173, 1976. Boyum, A., Scu&. J. t/i/r. I.&. I,r~r.~r. 21 (Suppl. 97). 1968.
THYROGLOBULIN-BINDING
9. IO. Il. 12. 13. 14. 15. 16. 17. 18. 19.
BLOOD
CELLS
Dickler, H. B., and Kunkel, H. G., J. E.rp. Med. 136, 191, 1972. Kofler, R., and Wick, G., J. Immunol. Methods 16, 201. 1977. Roitt, I. M., and Doniach, D., Lancer 1, 1027, 1958. Beutner, E. H., Thyroid 5, 152, 1964. Balfour, B., Doniach, D., Roitt, I. M., and Couchman, K. G.. &it. J. Exp. Parhol. 307, 1961. Gattringer. C., Wolf, H., and Wick, G., Folia Biol. (Prague), 23, 432, 1977. Perrudet-Badoux, A., and Frei, P. C., C/in. Exp. Immunol. 5, 117, 1969. Roberts, Isabel M., Wittingham, Senga, and Mackay, I. R., Lance? 1, 936, 1973. Urbaniak, S. J., Torrigiani, G., and Allison, A. C., C/in. Exp. fmmunol. 15, 345, 1973. Kahlid, 8. A. K., Hamilton, N. T., and Cauchi, M. N., C/in. Exp. Immunol. 23, 28, 1976. Bankhurst. A. D.. Tonigiani, G.. and Allison, A. C., trrncet 1, 226, 1973.
189
42,
G. WICK,? N. ZAMBELIS,$ AND G. SCHERNTHANER~
H. Lunwrc;,#
*Em -Now - Thront -Clinic, *fn.stitute fiw Grttercrl trnd Exprrinrentctl Prrtholo,qy, and Slmtitute for NucIe~~r Medicine. i7nir.ersit.v of InnshrucX. InnshrucX. rrnd Wecond Depctrtmettt of Intemnl Mvdicitw. UGlvr.sity of Virnnrr. VicJntrtr. A~striu
Received March 15, 1978 The number of thyroglobulin-binding peripheral blood cells (TG-BL) was determined in 24 patients with Hashimoto thyroiditis: I2 non-Hashimoto. non-Graves disease goiter patients; and IO unrelated controls using the fluoroimmunocytoadherence (FICA) technique. Hashimoto patients showed significantly elevated numbers of TG-BL as compared to members of the two other groups. For further characterization of TG-BL, inhibition experiments were performed by preincubation of peripheral blood lymphocytes with TG, normal human serum, rabbit-y-globulin, or bovine serum albumin. The results of these experiments indicate that the total number of TG-BL in Hashimoto patients consists of a majority of cells actively synthesizing their TG receptors and a minority adhering to TG-coated beads due to passive adsorption of TG autoantibody onto their surface or in a nonspecific fashion. No correlation between the total number of TG-BL and the presence and/or titer of autoantibodies to TG. the second colloid antigen, or microsomal antigens was found. Evidence is presented that the determination of TG-BL provides a specific tool for the diagnosis of Hashimoto thyroiditis superior to conventional thyroid autoantibody analysis.
INTRODUCTION
The Obese strain (OS)’ of chickens is a well-defined animal model for human Hashimoto thyroiditis and some essential data for understanding of the human counterpart derive from studies of this strain (1). In an earlier chronological investigation on the occurrence of thyroglobulin (TG)-binding cells in peripheral lymphoid organs of OS chickens, the number of TG rosette-forming cells (RFC) was shown to reach peak values at 2-4 weeks of age, i.e. clearly before maximum severity of spontaneous autoimmune thyroiditis and frequency of circulating TG autoantibodies (AAB) were attained (2). The B-cell nature of TG-RFC actively synthesizing their TG-receptor Ig molecules was confirmed by inhibition experiments using B- and T-cell-specific turkey antisera. Though circulating IgM and IgG anti-TG autoantibodies can be found in OS chickens, only anti-p sera significantly inhibited TG-rosette formation, indicating that antigen receptors I Abbreviations used: TG, thyroglobulin: TG-BL, thyroglobulin-binding lymphocytes: FICA. fluoroimmunocytoadherence; CA, and CA,, first and second colloid antigens; OS, Obese strain; RFC, rosette-forming cells: AAB, autoantibodies: BSA, bovine serum albumin: PBS, phosphatebuffered saline; IIF, indirect immunofluorescence; CRBC. chicken red blood cells: PBL. peripheral blood lymphocytes; NHS, normal human serum. 178 OOYO-1229/78/0112-0178$01.00/O Copynght All
rights
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1978 reproductmn
by
Academic in any
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Inc.
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reserved.
THYROGLOBULIN-BINDING
BLOOD
CELLS
179
of active TG-RFC are IgM molecules. The existence of passive TG-RFC in peripheral lymphoid organs of OS chickens was suggested by the competitive inhibitory effect of normal chicken serum on the total number of TG-RFC. Only anti-T-cell serum-sensitive RFC were shown to adsorb passively TG antibodies of the IgM class (3), in agreement with the previous results of Webb and Cooper in sheep red blood cell-immunized normal chickens (4). The rosette test, using antigen-coated erythrocytes as indicator particles, is time consuming and exact quantification of the degree of antigen coating is difficult. Therefore, a new, generally applicable method-fluoroimmunocytoadherence (FICA)-was developed. Beads of cross-linked dextran (Sephadex G-25) were chosen as indicator particles and the antigen in question was coupled to their surface via a spacer chain. The main advantages and performance characteristics of this method have been described in detail (5). Essentially, such spacer-armed beads can be stored ready for use in the coupling with any protein ligand. Also after coupling the antigen-coated beads are stable for many months. Slight modifications introduced for the present purposes will be reported under Materials and Methods. The main purpose of the present investigation was the enumeration of TGbinding blood lymphoid cells by the FICA method and the clarification of their eventual significance for the diagnosis of Hashimoto thyroiditis. To answer these questions the individual number of TG-binding cells in Hashimoto patients was compared to the serological findings and the cytological appearance of needle biopsies in selected cases. The mean numbers of TG-binding lymphocytes of Hashimoto patients; non-Hashimoto, non-Graves disease goiter patients; non thyroid disease patients; and healthy individuals were determined and compared statistically. Finally the following control experiments were performed: (i) specificity testing of TG-binding cells in FICA; (ii) production of passive TGbinding cells; (iii) inhibition of nonspecific antigen-binding cells. MATERIALS AND METHODS Human Thyroglobulin Human rhyroglobulin was prepared using a modification by Shulman ef al. (6). Human thyroid glands obtained
of a method described at surgery were sliced and extracted in 0.15 M saline solution for 16 hr at 4°C and the extract was subjected to precipitation with saturated (NH&SO,. The reconstituted precipitate was centrifuged at 30,OOOg for 30 min and the supernatant was dialyzed against saline. After passage through a Sephadex G-200 column (Pharmacia, Uppsala, Sweden) two peaks were collected. The first peak containing TG, but contaminated with y-globulin, was further purified by absorption with glutaraldehydeimmobilized rabbit anti-human y-globulin serum. The purity of the TG was assessed by immunoelectrophoresis, and the specificity, by its inhibitory capacity in passive hemagglutination tests.
Fluoroimmunocytoadherencr
(FICA)
This method has already been described in detail elsewhere (5). In short, fluorochrome-labeled dextran beads are coated with antigen. These beads are then incubated with the lymphoid cells under study, which, in case they carry antigen-specific receptors, will adhere to the beads coated with the respective
180
KI(:H 1EK Et A/.
antigen. Beads carrying a different antigen and labeled with another fluorochrome (or left unstained) are added as a control for specificity testing. ((1) Acti\wtiott of hrcrtls. Cross-linked dextran beads (Sephadex G-25 Superfine, Lot No. 0998, Pharmacia, Uppsala, Sweden) were selected due to their size characteristics, their aptitude for lyophilization, and their advantages for the possible fluorometric evaluation of the degree of antigen coating (5). Briefly. beads swollen in distilled water were sieved to obtain particles of a mean diameter of 2627 pm. These beads were then activated with CNBr and armed with a spacer consisting of 3,3’-diaminodipropylamine. succinic anhydride, and N-hydroxysuccinimide. The spacer-armed beads can be stored in the dark in dioxane (water free) for many months and can be used immediately without further manipulation for the coupling of any ligand containing primary amino groups. (h) Coupling with antigens. Human TG or bovine serum albumin (BSA. Berhringwerke AG, Marburg, FRG) was coupled onto the spacer-armed beads as described in detail (5). (c) Deacti\wtion. The procedure used for deactivation of coated ligand beads differed slightly from that recently described (5). Conventional deactivation. appropriate for affinity chromatography, does not suffice for FICA. Thus. during prolonged (weeks) storage of coupled and 1 M glycine-deactivated beads, small amounts of protein are lost and free active sites are reexposed, leading to unspecific adherence of lymphoid cells. This problem can be overcome by admixture of an appropriate (not cross-reacting with the specific antigen under investigation) normal serum or purified serum protein to the antigen-coated beads, thus blocking residual active sites. For this purpose rabbit fluorochromelabeled or unlabeled y-globulin (Pentex. Miles Laboratories, Kankakee, Ill.) preparations in phosphate-buffered saline (PBS, pH 7.2) were used in the present study. In experiments with only two different antigen-coated bead preparations only one charge was treated with fluorescein isothiocynate-conjugated rabbit y-globulin, the second charge was deactivated with unlabeled rabbit y-globulin. After storage of coated and deactivated preparations in suspension, unlabeled rabbit y-globulin (1%) was again added and the beads were vertically rotated for 60 min at 4”C, followed by three washings in PBS just before use in an FICA test. (d) Specificity of coating. This was assessed in indirect immunofluorescence (IIF) tests using a semiautomated micromethod described elsewhere (7). (P) Pevfkmcrnce of the test. PBS was used as a medium throughout. Fivetenths of a milliliter of a suspension of acridine orange-stained cells (4 x lo6 cells/ml) was added to 0.5 ml of a mixture of BSA- and TG-coated beads [both types of beads as 5% (v/v) suspensions] in 13 x 75ml plastic tubes, the ratio of lymphoid cells to beads equaling 1: 10. When analyzed, mixtures of unstained beads and lymphocytes were observed under a conventional light microscope. Simultaneous observation of labeled and unstained beads was done under a Reichert Immunopan fluorescence microscope. Double readings were performed after incubation for 1 and 18 hr at 4°C and vigorous resuspension with Pasteur pipets just before mounting a drop of the suspension on the slide. Only beads with two or more adherent cells were evaluated.
THYROGL.OBULIN-BINDING
BLOOD
CELLS
181
Lqmphocytes PBS (pH 7.2) was used as a medium throughout. Ten milliliters of defibrinated venous blood was collected and lymphocytes were isolated by the method of Bqiyum (8). Through collaboration with (and some financial motivation of) express train personnel, blood collected from 22 patients in Vienna could also be tested late on the same day in Innsbruck. Mononuclear cells were removed by incubation and passage, through nylon wool (Leukopak, Hyland Division of Travenol Laboratories)-packed syringes. Viability, as assessed by trypan blue exclusion, was always above 90%. Finally, the vital dye acridine orange used for staining of the final preparation also permitted a morphological classification of cells (less than 1% monocytes) in suspension and those adhering to beads. Specificity Tests FICA irlhibition experiments. Before admixture to the beads the blood lymphocytes were incubated for 45 min at room temperature with human TG, rabbit y-globulin, BSA, PBS only, or aggregated rabbit y-globulin prepared according to Dickler and Kunkel (9). Corwsrztional rosette rests. Chicken red blood cells (CRBC) were coated with human TG, BSA, or rabbit y-globulin by means of a modified chromium chloride method ( 10) and the rosette tests were performed as described earlier (2). Pnssive FICA. (a) Lymphocytes of a healthy donor were incubated for 1 hr at 37°C with sera (all sera undiluted and heat inactivated at 56°C for 30 min) from Hashimoto patients with different serological reaction patterns (i.e., high or low titers of microsomal and/or TG autoantibodies). This procedure was followed by three washings and readjustments to 4 x lo6 cells/ml prior to FICA testing. (b) Inhibition of passive FICA: After incubation of normal lymphoid cells with a patients’ sera containing TG antibodies (titer of 2500 in passive hemagglutination) and three washings, lymphocytes were resuspended for a second incubation in undiluted normal human serum for 30 min at 37°C before further washings and readjustment for FICA testing wuth supplementation of 10% normal human serum to the medium. Serology Autoantibodies to human TG were detected by tanned red cell hemagglutination (Thyroglobulin Test Kit, Wellcome Reagents, Ltd., Beckenham, England) and in IIF tests on methanol-fixed cryostat sections of colloid goiter. Microsomal autoantibodies were detected by tanned red cell hemagglutination (Microsome Test Kit, Fujizoki, Pharmaceutical Co., Ltd., Tokyo, Japan) and in IIF tests on unfixed cryostat sections of thyrotoxic goiter. Antibodies to the second colloid antigen (CA,) were demonstrated by IIF on methanol-fixed colloid goiter sections after absorption of antisera with TG-coated sephadex G-25 beads. One-tenth of a milliliter of packed TG beads was added to 0.1 ml of undiluted serum followed by incubation overnight at 4°C. The procedure was repeated until no TG autoantibodies were detectable in the tanned red cell hemagglutination test (1 1- 13).
182
Rl(:H
I-ER
E7 Al
Sera and defibrinated blood were collected from 10 individuals consisting mainly of laboratory personnel, patients hospitalized for reasons other than autoimmune or thyroid disease, 12 patients with various thyroid diseases except Hashimoto and Graves diseases and 24 patients with Hashimoto thyroiditis. The diagnosis of Hashimoto thyroiditis was established by the following criteria: presence of goiters of typical consistency, results of thyroid function tests, high titers of TG and/or microsomal autoantibodies, and cytological appearance of thyroid tissue obtained by needle biopsies. Biopsies were only performed in questionable instances (the age and sex distribution of patients and controls is evident from Table 1). RESULTS
Active TG-Binding Cells The serological results and the mean numbers of TG-binding lymphocytes (TG-BL) for the three groups tested are shown in Table 1. In all 24 Hashimoto patients tested, TG-binding lymphocytes were significantly elevated. Of 12 patients with thyroid diseases other than Hashimoto or Graves disease, two had high counts (19 and 13/103 cells, respectively) of TG-BL. One of these proved to be serologically negative, the other showed a low titer of TG antibodies in passive hemagglutination. The controls from the third group showed only background values of TG-BL corresponding to those observed in the case of BSA-BL. No correlation emerged between the number of TG-BL and individual autoantibody titers to TG, CA.), or microsomal antigens in passive hemagglutination or IIF tests (Table 2). The mean number of TG-BL in the Hashimoto group (25.2 + 7.8) was significantly higher than those of non-Hashimoto, non-Graves goiter patients (6.6 & 4.9) and normal controls (3.9 ? 1.7). The mean values for BSA-BL were in the same range for all three groups. The frequencies of TG and microsomal antibodies in passive hemagglutination (HA) and IIF tests and of antibodies to the second colloid antigen in IIF tests are also given in Table 1. Five Hashimoto patients were serologically negative. Only one patient of the non-Hashimoto, non-Graves goiter group had a low TG-autoantibody titer of I:40 in passive HA. The specificity of TG-BL was assessed by inhibition studies. Table 3 shows the results of two representative experiments. Preincubation of lymphocytes of Hashimoto patients for 4.5 min at room temperature with 2% human TG significantly reduced the number of TG-BL to background values, while 2% rabbit y-globulin or 1% aggregated rabbit y-globulin only had a partial inhibitory effect. Preincubation with 2% TG + 2% rabbit y-globulin resulted in more pronounced reduction, but not complete inhibition. No reduction of TG-BL or lymphoid cells adhering to BSA beads occurred after preincubation with 2% BSA as an unrelated antigen (not shown in Table 3). Incubation with rabbit y-globulin or aggregated rabbit y-globulin, but not with TG alone, reduced the number of background values in both Hashimoto patients and controls. From these data it was concluded that the actual number of lymphoid cells of Hashimoto patients adhering to TG beads consists of a majority of TG-BL
N.D.
0112
18/24 N.D.
14124
BSA 5.0 It 1.4
TG 25.2 + 7.8
W
L f:
812
32 (21-45)
o/10
N.D.
N.D.
o/10
N.D.
8 E: F: in
x F
N.D.I
2124
IIF”
n Titer >20 in tanned cell hemagglutination test. b Titer > 10 in IIF on methanol-fixed, frozen sections of a human colloid goiter. c Same as in Footnote b; sera absorbed with human thyroglobulin. ‘I Titer > 10 in IIF on unfixed, frozen sections of human thyrotoxic goiter. (1Mean numbers of lymphocytes / lo3 PBL adhering to antigen-coated beads, only two or more cells adhering to a single bead counted as positive. f Not done. y Statistical significant difference to Hashimoto group (p < 0.005). h Statistical significant difference to Hashimoto group (p i 0.001).
l/l2
17124
CA,’
Microsome-AAB
Antigen-binding cells (FICA)/103 PBL’
g
45 (19-64)
17124
IIF (CA,)*
IIF
PATIENTS
Passive HA”
IN HASHIMW~O
Serology
CELLS
3.8 t 1.5
10
Normal + other controls
913
57 (35-78)
Passive HA”
TG-AAB
1
3.9 +- 1.7*
12
Non-Hashimoto, non-Graves disease goiter
2212
TABLE .~ND ANTIGEN-BINDING
4.2 i 1.3
24
Hashimoto thyroiditis
(range)
Mean age
SEROLOGY
6.6 + 4.9O
N
Patients
Sex (F/M)
THYROID
Antigen-binding lymphocytes/IO’ PBL
Serd0gy _____--.-.--.TG-AAB Patient W.M. F.A. C.R.’ P.A. SM.' K.J. ” ’ ’ ” “ ’
Passive HA” 65 69 50 60 63 65
F F F F F F
250,000 40,960 40 250,000 <20 <20
IIF (CA,)” 80 40 40 80
IIF (CA,)’ (10
Microsome-AAB Passive HA” IIF” 20,480 40,960 320 80,000
20
TG
BSA
18 31 20 22 39 28
6 3 5 5 4 1
Titer in tanned cell hemagglutination test. Titer in IIF on methanol-fixed, frozen sections of a human colloid goiter Same as in Footnote b; sera absorbed with human thyroglobulin. Titer in IIF on unfixed, frozen sections of human thyrotoxic goiter. Number of lymphocytes adhering to beads per IO3 PBL. Needle biopsy.
and a minority of cells attached to the beads via an Fc receptor (to the rabbit y-globulin used for bead deactivation) or in an unspecific fashion. Passive TG-Binding Cells A series of control experiments was performed in order to assess further (a) the proportion of TG-BL in Hashimoto patients, which had acquired their antigenbinding capacity by passive adsorption of TG-autoantibodies; and (b) the possibility of producing TG-BL by preincubation of normal lymphoid cells with TG antibody-containing serum. For elucidation of the first question peripheral blood lymphocytes (PBL) from patients with Hashimoto thyroiditis were isolated and washed as usual and then incubated with heat-inactivated, undiluted normal human serum (NHS) assuming a possible displacement of passively adsorbed TG autoantibodies. The PBS used for subsequent washing and performance of FICA was supplemented with 10% NHS. The results of this experiment are presented in Table 4. A clear-cut inhibition of a portion of TG-BL was brought about by this treatment, the remaining values, however, still were significant. To answer the second question normal blood lymphocytes were exposed to different heat-inactivated Hashimoto sera, normal serum, and PBS for 60 min at 37°C (Table 5). Antigen-binding capacity was only acquired by lymphocytes preincubated with sera containing autoantibodies to TG. Hashimoto sera without circulating antibody detectable in passive HA or IIF tests and normal human serum produced only background values in the range of the value obtained by preincubation with PBS only. Table 6 shows that the phenomenon of passive TG binding is reversible. A Hashimoto serum containing a high titer of TG autoantibodies in passive HA was used for the first incubation step, for the second incubation, after three washings, the lymphocytes were resuspended in undiluted normal human serum, resulting in a reduction of passive TG-BL.
Expt 2
cells/IO3 PBL
30
TG 26 7
BSA 8
PBS
8
TG 8 7
BSA 9
2% TG
23
TG 20 3
BSA 4
2% rabbit y-globulin TG 25 N.D.
BSA 7
0.1% TG 18 N.D.
BSA 3
0.5% TG 18
Aggregated rabbit y-globulin
TABLE 3 OF ANTIGEN-BINDING CELLS IN FICA
” Forty-five minutes at room temperature followed by addition of antigen-coated beads. b Not done.
Expt 1
Preincubation of lymphocytes” Antigen coating Number of antigen-binding
INHIBITION
N.D.
1% BSA 3
4
TG N.D.D
2
BSA N.D.
2%TG + 2% rabbit y-globulin
2; 5 : 5 ? z E z n F 8 u E FVT
Number of antigen-binding PBL/lOZ lymphocytes in FIC.4 after preincubation with” -___ PBS NHS Patient
Diagnosis
TG
BSA
H” H N“ N
26 20 5 6
5 7 6 6
J.M. S.M. H.M. F.J.
TG __-__ 17 14 3 3
BSA 3 3 3 3
’ Preincubation was for I5 min at 37°C in medium only or in medium + 10% NHS, followed by three washings and readjustment. b Hashimoto disease. c Normal control.
DISCUSSION
The FICA technique was developed in this laboratory as a new method for the demonstration, enumeration, characterization, and separation (5, 14) of antigenbinding cells. The antigen in question is coupled via a spacer onto the surface of Sephadex G-25 beads. Adherence of lymphocytes results in formation of
TABLE PASSIVE
ANTIGEN
5
BINDING BY PREINCUBATION OF NORMAL WITH DIFFERENT HASHIMOTO SERA
Characterization
BLOOD
of serum donors
AAB titer in passive HA testb Serum used for incubation”
TGAAB
PBS (medium only) NHS U.J. S.E. F.A. J.M.
110 <20 <20 40,960 2,500
MicrosomeAAB
L~MPHOWTES
Passive antigen-binding cells (FICA)/lO’ PBL” CA, (IIF)’ N.D.’
FICA TGbinding cells
TG
BSA
N.D. 3 34 31 16
4 3 5 3 I3 12
3 3 4 4 4 5
n Undiluted serum was used for 60 min at 37°C followed by three washings and readjustment to 4 x lo6 PBL/ml before addition of TG or BSA beads. ’ Titer in tanned cell hemagglutination test. c Titer in IIF on methanol-fixed, frozen sections of human colloid goiter; sera absorbed with human thyroglobulin. ’ Number of lymphocytes adhering to beads per 10’ PBL. ’ Not done.
THYROGLOBULIN-BINDING
PASSIVE TG-BINDING
NORMAL
BLOOD
TABLE 6 PBL AND INHIBITION
187
CELLS
WITH NORMAL
HUMAN
SERUM”
Passive antigen-binding cells (FICA) / lo3 PBLC Incubationb
TG
BSA
NHS, 30 min, 37°C PBS, 30 min, 37°C
6 13
6 6
n Normal PBL were preincubated with undiluted Hashimoto patient serum (J.M. of Table 6) for 30 min at 37”C, followed by three washings before addition of NHS (undiluted) or PBS. b Incubation with NHS or PBS (medium only) followed by three washings and readjustment to 4 x 106 PBUml before addition of beads. r Number of lymphocytes adhering to beads per 10) PBL, mean of two measurements after 90 min and 18 hr.
reversed rosettes, i.e., the beads in the center are surrounded by clusters of lymphocytes. The main advantages of this technique are (a) the constant availability of spacer-armed beads for coating with any protein ligand, (b) the stability of ready to use antigen-coated beads for many months, and (c) the possibility to incorporate beads coated with a control antigen into the same preparation which can be evaluated simultaneously under a fluorescence microscope. In the present study TG and BSA-BL were enumerated by means of this technique in 24 hashimoto and Graves disease and 10 control persons consisting of healthy laboratory personnel and nonthyroid disease patients. The specificity of TG binding was confirmed by a number of control experiments. In the peripheral blood of all Hashimoto patients the observed numbers of TG-BL were significantly elevated in comparison to those of the control groups. The only exception were two patients in the non-Hashimoto thyroid group which showed elevated numbers of TG-BL. Unfortunately, no needle biopsies were available from these patients to complement the clinical diagnosis. The values of all other individuals were in the range of the background counts attained with BSA-coated beads. These data are in agreement with those in earlier publications of other authors (15- 17) on TG-BL in Hashimoto patients and results obtained in the Obese strain (OS) of chickens, an animal model for human Hashimoto thyroiditis (l-3). Khalid et al. (18) have studied the occurrence of peripheral blood lymphocytes binding microsomal membranes in patients with different thyroid diseases, including six Hashimoto patients, and observed a significant increase in the number of antigen-BL in patients with circulating antibodies to thyroid cytoplasmic antibodies, while the number of antigen-BL in patients without autoantibodies was within the normal range. They concluded that the occurrence of antigen-BL may be a response to an excess of antigen in the circulation. The binding capacity of peripheral blood lymphocytes for thyroid microsomal antigens was not evaluated in the present study. However, in regard to the binding of TG, no correlation was found between the presence and titer of autoantibodies to this antigen and the absolute numbers of TG-BL. This fact again parallels the findings in OS chickens, but is in contrast to observations published by others (19).
188
KICH’I‘EK
ET Al
Our studies on passive TG rosette-forming cells do, however, lend support to the notion that a certain, although minor, portion of TG-BL in Hashimoto patients consists of cells which have passively adsorbed TG autoantibodies onto their surface. This phenomenon could be competitively inhibited by incubation with normal human serum. Similar findings have been reported for passive TG-BL in OS chickens, in which these cells had been identified as T cells adsorbing IgM anti-TG (3), and TG-BL actively synthesizing their receptors were shown to be B cells (2). That passive adsorption of autoantibodies may contribute to the total number of antigen-binding cells in humans was also shown by the effect of an incubation of normal peripheral blood lymphocytes from Hashimoto patients. The characterization of active and passive TG-BL and the elucidation of the class of antibodies responsible for the formation of the latter will be the subjects of further studies. Incubation of Hashimoto lymphocytes with rabbit y-globulin and aggregated rabbit y-globulin at lower concentrations slightly reduced the number of antigen-BL. No reduction resulted by incubation with BSA and no complete inhibition of antigen BL could be obtained by preincubation of lymphocytes with 2% TG + 2% rabbit y-globulin. As rabbit y-globulin was used in addition to glycine for deactivation of all bead preparations, small amounts of this protein are bound by residual active sites. This occurs in a random fashion, i.e., the Fc portion being exposed only in some of the rabbit Ig molecules and thus being accessible to cells with Fc receptors. This explains the relatively small number of cells adhering to the beads via an Fc receptor and inhibitable by rabbit -y-globulin. In conclusion the present study demonstrates a significantly increased number of TG-BL in patients with autoimmune thyroiditis. The total number of TG-BL in the peripheral blood of Hashimoto patients is composed of: (a) a majority of lymphoid cells binding TG via their own membrane-associated receptors (active TG-BL) and (b) a minor portion consisting of three types of cells: lymphoid cells which possess passively acquired receptors for TG; cells with Fc receptors binding to the rabbit y-globulin used for bead deactivation, and cells adhering nonspecifically to the surface of beads. The proportion of passive TG-BL may vary in different patients and test systems, thus accounting for the discrepancies in respect to a possible correlation between the autoantibody titers and the number of TG-BL reported by different authors. As increased counts of TG-BL were shown to be a constant finding in Hashimoto’s thyroiditis, the FICA technique might prove an ideal test system for monitoring ongoing autoimmune thyroid disease and perhaps other autoimmune conditions as well. ACKNOWLEDGMENTS This work was supported by the Austrian Research Council (Project No. 31201.
REFERENCES I. 2. 3. 4. 5. 6. 7. 8.
Wick, G., Sundick, R. S., and Albini, B., C/i/r. Ir,rntuno/. Ir,,n~lr/,opntho/. 3. 272, 1974. Richter, E., and Wick, G., J. I~r~nu~ol. 114, 757. 1975. Richter, E., Wick, G., and Schauenstein. K.. El/r. J. I/u~n~rrol. 5, 554. 1975. Webb, S. R., and Cooper. M. D.. J. f/rr~nr/to/. 111, 275, 1973. Richter, E., and Wick. G.. 2. I/,l/,lrcllirtrfr.~~~~.~~,/~,152, 351, 1977. Shulman, S., Mates, G., and Bronson. P.. Biwhim. Biophys. AC/U 147, 208, 1967. Schauenstein, K., Wick. G.. and Kink. H., .I. f~rr~trc/ro/. Mctlto~/.t 10, 173, 1976. Boyum, A., Scu&. J. t/i/r. I.&. I,r~r.~r. 21 (Suppl. 97). 1968.
THYROGLOBULIN-BINDING
9. IO. Il. 12. 13. 14. 15. 16. 17. 18. 19.
BLOOD
CELLS
Dickler, H. B., and Kunkel, H. G., J. E.rp. Med. 136, 191, 1972. Kofler, R., and Wick, G., J. Immunol. Methods 16, 201. 1977. Roitt, I. M., and Doniach, D., Lancer 1, 1027, 1958. Beutner, E. H., Thyroid 5, 152, 1964. Balfour, B., Doniach, D., Roitt, I. M., and Couchman, K. G.. &it. J. Exp. Parhol. 307, 1961. Gattringer. C., Wolf, H., and Wick, G., Folia Biol. (Prague), 23, 432, 1977. Perrudet-Badoux, A., and Frei, P. C., C/in. Exp. Immunol. 5, 117, 1969. Roberts, Isabel M., Wittingham, Senga, and Mackay, I. R., Lance? 1, 936, 1973. Urbaniak, S. J., Torrigiani, G., and Allison, A. C., C/in. Exp. fmmunol. 15, 345, 1973. Kahlid, 8. A. K., Hamilton, N. T., and Cauchi, M. N., C/in. Exp. Immunol. 23, 28, 1976. Bankhurst. A. D.. Tonigiani, G.. and Allison, A. C., trrncet 1, 226, 1973.
189
42,