Clin. exp. Immunol. (1976) 23, 429-435.
Indirect immunofluorescence staining of human thyroid by antibodies occurring in Yersimna enterocolitica infections K. LIDMAN, ULLA ERIKSSON,* RENEE NORBERG & ASTRIDFAGRAEUSDepartment of Immunology and *Department of Bacteriology, National Bacteriological Laboratory, S-105 21 Stockholm, Sweden
(Received 21 July 1975)
SUMMARY
In the diagnostic routine for tissue antibodies, using indirect immunofluorescence on cryostat sections of human thyrotoxic thyroid, rat stomach and kidney, ninety-six out of 48,388 sera showed a marginal staining of the membrane region of thyroid epithelial cells but no other reaction. Twenty-six of these sera were from patients with acute Yersinia enterocolitica serotype 3 infection but without signs of thyroid disease. Fifty of sixty-three sera with agglutinins against Y. enterocolitica serotype 3 and three out of four sera with agglutinins against Y. enterocolitica serotype 9 also showed this reaction on thyroid sections. It was due to antibodies, mostly of the IgG class, occurring in low titre, which react with intracytoplasmic antigens in thyroid, as staining of live thyroid epithelial cells was negative. The pattern of immunofluorescence on thyroid sections caused by these antibodies could not be distinguished from that caused by smooth muscle antibodies by appearance only. However, smooth muscle antibodies react also on other tissue sections and extracts of contractile proteins which absorb out these, did not change the reaction on thyroid of Y. enterocolitica sera. Absorption with sonicated Y. enterocolitica 3 and 9 antigens, but not with heat-killed whole bacteria, extinguished the reaction on thyroid. This indicates the presence of a cross-reactivity between antigens in these bacteria, different from the 0 antigen, and antigens in thyroid epithelial cells. Knowledge of this pattern of immunofluorescence on thyroid sections can be of diagnostic significance.
INTRODUCTION Smooth muscle antibodies (SMA) appear predominantly in sera from patients with active chronic hepatitis and are demonstrable by indirect IFL on cryostat sections of rat stomach (Johnson, Holborow & Glynn, 1965). SMA also react with other tissues, e.g. renal glomeruli (Whittingham, Mackay & Irwin, 1966) and the epithelial cells in cryostat sections of human thyroid (Biberfeld, Fagraeus & Lenkei, 1974; Sutton et al., 1974). In the routine examination for autoantibodies we have observed a number of sera which gave only a reaction with the membrane region of thyroid epithelial cells (Biberfeld et al., 1974) and not with smooth muscle fibres or rat renal glomeruli. Checking of the clinical diagnosis of thirteen patients whose sera gave this isolated reaction with thyroid, revealed that five of them had a serologically verified infection with Yersinia enterocolitica (Y. enterocolitica) and further preliminary investigation still indicated this connection (Lidman et al., 1974). Y. enterocolitica is not uncommon in the Scandinavian countries. It causes infection in Correspondence: Dr Knut Lidman, Department of Immunology, National Bacteriological Laboratory, 105 21 Stockholm, Sweden.
429
430
K. Lidman et al.
the gastrointestinal tract, sometimes with symptoms of acute terminal ileitis (Winblad, Nilehn & Sternby, 1966; Persson et al., 1974). Acute arthritis (Ahvonen, Sievers & Aho, 1969) and erythema nodosum (Winblad, 1969; Hannuksela & Ahvonen, 1969) may occur during the course of Y. enterocolitica infection. Diagnosis is based on the finding of agglutinating antibodies against 0 antigens from Y. enterocolitica or on the isolation of the pathogen. Y. enterocolitica serotype 3 (Y. enterocolitica 3) dominates as the cause of human cases in Europe including Sweden (Winblad, 1973). Cases of Y. enterocolitica serotype 9 (Y. enterocolitica 9) are rare in Sweden but fairly common in Finland, Belgium and the Netherlands (Winblad, 1973). The occurrence in Y. enterocolitica infections of antibodies that in IFL gives a marginal staining of the thyroid epithelial cells has been investigated. Evidence is presented that these antibodies are separate from SMA and are directed against related antigens in Y. enterocolitica bacteria and thyroid cells. MATERIALS AND METHODS Sera. Most sera used in this study were sent in from various hospitals to the National Bacteriological Laboratory for routine testing for tissue antibodies or estimation of agglutinins against Y. enterocolitica or Salmonella antigens. Sera from patients with acute terminal ileitis caused by Y. enterocolitica 3 and from patients with Crohn's disease were kindly provided by Dr Dan Danielsson, Orebro. Sera from patients with Y. enterocolitica 9 were kindly provided by Dr Lars Burman, Umeat. Immunofluorescence (IFL). Cryostat sections of human thyrotoxic thyroid (acetone-fixed sections), rat stomach (unfixed) and rat kidney (unfixed) were used as antigens in all routine testing for tissue antibodies by indirect IFL. Selected sera were also tested on cryostat sections of the following organs: atoxic human, monkey and rat thyroid, mouse thymus, rat liver, pig stomach, human foetal and adult ileum. Tests were also performed on smears of lymphoblastoid cells and human fibroblasts grown on glass as described by Norberg, Lidman & Fagraeus (1975). Thyroid monolayer cultures were prepared from surgically resected glands and after various time of culture (0-3-5-7 days) the cells were dispersed with 0-25% trypsin solution. Cyto-centrifuged preparations of the cells were then made and stained by indirect IFL as described by Biberfeld et al. (1974). Staining of live suspended thyroid cells from the cultures was made by mixing 106 cells with 0 2 ml of test serum dilution. After 30 min at + 4°C the cells were washed and 0 2 ml of the appropriately diluted FITC-conjugate was added. The patients' sera were heated at 56°C for 30 min and examined in a dilution of 1/10, if not otherwise stated. The following fluorescein isothiocyanate (FITC) conjugates were employed: (1) a sheep anti-human immunoglobulin (SBL 480040); (2) Wellcome anti-IgG lot number K 8351; (3) sheep anti-IgM (SBL 76101); (4) sheep anti-IgA (SBL 0904"). The preparations were examined in a Zeiss fluorescence microscope equipped with a HBO mercury lamp and a dry dark-field condenser using primary filter BG 3 and secondary filters 44 or 47. Bacterial strains. Y. enterocolitica 3 (482/IIL), Y. enterocolitica 9 (486/IX), E. coli 945, E. coli 014: K7: H-, Staphylococcus aureus 209, Streptococcus pyogenes type 12 group A (Squibb 1300) were received from strain collections at the National Bacteriological Laboratory. Y. enterocolitica 3 patients strains from different geographical regions were isolated in the diagnostic routine and some were also kindly provided by Dr P. Toivanen and Dr L. Olkkonen, Turku, Finland. Agglutination technique. Tube agglutination was performed by the conventional Widal technique for 0 antigens using heat-killed Y. enterocolitica 3 and 9 bacteria. A titre of 160 or more was regarded diagnostic for an acute Y. enterocolitica infection (Arvaston, Damgaard & Winblad, 1971; Ahvonen, 1972; Toivanen et al., 1973). All sera found in the routine with the typical IFL-pattern on thyroid were tested for agglutinins against Y. enterocolitica 3 and 9. Absorption of sera. (A) Bacteria were cultured on agar plates (Oxoid blood agar base CM 55 (Oxoid Ltd, London)+ 1%Y glucose) for 20 hr at 37°C. Cells were harvested in PBS, pH 7-4 and washed twice. For absorption four different antigen preparations were made. (1) Treatment at + 100°C for 60 min followed by two washings in PBS. (2) Formaldehyde was added to a final concentration of 1 .. After 20 hr at + 4°C the antigen was washed twice in PBS. (3) Tetracycline-HCl lot number VN-238-304 (Pfizer, Brussels) was added to a final concentration of 2 pg/ml in order to obtain bacteriostatic condition.
Antibodies reacting with human thyroid
431
(4) Treatment of bacteria (1 mg dry weight/ml) in a Braunsonic 300 S (B. Braun, Melsungen, W. Germany) at 70%. for 5 min. The supernatant from centrifugation at 10,000 g for 30 min was used. Absorption with whole bacterial antigens (1-3) was performed by resuspending 1010 packed bacteria in 0 9 ml PBS (with tetracycline included for antigen 3) and adding 0 1 ml serum. The mixture was incubated for 4 hr at + 40C and then centrifuged at 3000 g for 15 min. The supernatant was repeatedly absorbed with the same amount of packed bacteria at + 40C overnight. After centrifugation the supernatant was examined for complete absorption by tube agglutination and then tested by IFL. Four volumes of soluble antigen (4) was mixed with one volume of serum and incubated at + 40C for 4 hr. After centrifugation (35,000 g for 60 min) repeated absorption of serum was made with equal amounts of antigen and serum dilution at + 40C over night. The supernatant after centrifugation was then tested by Widal agglutination and by IFL. (B) Antigens containing contractile proteins were prepared from the muscularis of pig stomach (smooth muscle antigen) and from lymphoblastoid cells and used for absorption as previously described (Biberfeld et al., 1974; Fagraeus, Lidman & Biberfeld, 1974).
RESULTS
During the period January 1973 to June 1975, 48,388 patients sera were routinely tested by IFL for tissue antibodies. Ninety-six of these sera (0.2%) (Table 1) gave a typical marginal staining of thyroid cells as shown in Fig. 1. In contrast to SMA-positive sera, there was no reaction of these sera on sections of rat stomach, kidney, liver or on human foetal and adult ileum. Smears of lymphoblastoid cells did not reveal any IFL-staining of microvilli and human fibroblasts grown on glass were negative. The only positive tissue reaction, except with thyroid, that sometimes could be seen with these sera was a 'streaky' staining of sections of mouse thymus.
FIG. 1. Indirect inmmunofluorescence reaction on cryostat sections of human thyrotoxic thyroid with serum from a patient with Yersinia enterocolitica serotype 3 infection. Note the staining of the membrane region of the epithelial cells surrounding a follicle in the gland. (Magnification x 600.)
Twenty-six (270 ) of the ninety-six sera, which gave this marginal staining on thyroid, patients with coexisting antibodies to Y. enterocolitica 3 in titre . 160 (Table 1). Clinically fourteen of them had reported diarrhoea, twenty had postinfectious arthritis affecting one or several joints and three had erythema nodosum but in no case were there any apparent symptoms of thyroid disease. The cause of the disease symptoms was previously unknown in twelve of these cases and was revealed by the finding of thyroid reaction and then Y. enterocolitica 3 agglutinins. came from different
c
K. Lidman
432
et
al.
TABLE 1. Sera reacting only with the membrane region of thyroid epithelial cells found in routine-IFL
Total number of sera 1973 1974 1975
Sera with reaction on thyroid and Sera reacting Y. enterocolitica 3 agglutinins in on thyroid in dilution . 160 dilution . 10
17109 20040 11239
13 56 27
5 13 8
48388
96
26
(1/130/6)
Sera reacted with the thyroid epithelial cells usually in low titres, 10-20, occasionally in titres of 80-160. Antibodies that gave a marginal staining on thyroid usually disappeared earlier than the agglutinating antibodies in convalescent sera. The reason for reactivity of the 70 Yersinia-negative sera (730%) was not revealed and preliminary checking did not show any apparent disease in common in these cases. None of the ninety-six sera found at the examination by IFL had agglutinins against Y. enterocolitica 9. This might depend upon the rare occurrence of infections with Y. enterocolitica 9, as three out of four sera with agglutinins against Y. enterocolitica 9 showed a faint staining of thyroid epithelial cells. Forty-seven sera sent in primarily for Widal agglutination and found with a Y. enterocolitica 3 titre 2 160, were also tested in IFL. Thirty-seven (84%) of them gave the typical pattern and ten were negative. Thirteen of sixteen sera from cases of acute terminal ileitis caused by Y. enterocolitica 3 were positive on thyroid sections. However, nineteen sera from patients with Crohn's disease and ten Salmonella agglutinin-positive sera were negative. Testing with monospecific conjugates showed that the majority of the thyroid reacting antibodies were of IgG-class (Table 2) but antibodies of IgM and IgA class occurred. The IFL reaction of Yersinia-positive sera was best seen on thyrotoxic epithelial cells. Tests on sections of six atoxic glands showed no or only a faint reaction in the membrane region of the cells. Staining of thyroid from monkey and rat were negative. Sera that were positive on thyroid sections and for Y. enterocolitica 3 agglutinins did not react with live cells from thyrotoxic or atoxic glands. Staining of cytocentrifuged thyrotoxic epithelial cells before culture, however, revealed fluorescent irregular 'rings' in the cytoplasm of the cells (Fig. 2). On day 3 of culture almost all 'rings' had disappeared in the cytocentrifuged specimens. Cytocentrifuged cell preparations from atoxic glands were never stained. Absorption of agglutinin and 'thyroid-positive' Y. enterocolitica 3 sera with heat-killed Y. enterocolitica 3 bacteria resulted in the disappearance of the 0 agglutinins but the IFLTABLE 2. Ig-class of Ab reacting in IFL with thyroid in 33 Y. enterocolitica serotype 3 (titre . 160) sera, from acute phase of the disease revealed by monospecific conjugates
IgG IgM IgG and IgM IgG and IgA IgG, IgM and IgA
16 1 7 3 6
Antibodies reacting with human thyroid
ia
433
1~j
D FIG. 2. Indirect immunofluorescence reaction with serum from a patient with Yersinia enterocolitica serotype 3 infection on cytocentrifuged preparations of thyrotoxic thyroid cells before culture. Fluorescent 'rings' appear (a) in one single cell; (b) in several cells in an aggregate. (Magnification x 700.)
reaction on thyroid was unchanged. The effect of absorption with formaldehyde or tetracycline-treated bacteria was a decrease in titre of the thyroid-reacting antibodies and disappearance of the 0 agglutinins. Total absorption of both antibody reactions was, however, achieved when sonicated Y. enterocolitica 3 or 9 bacteria were used (Table 3). Absorption with Staphylococcus aureus 209, Streptococcus pyogenes type 12 group A and E. coli 014 and 945 did not influence the antibody titres. The titre of sera positive for thyroid cytoplasmic antibodies or mitochondrial antibodies, ANF and SMA were not changed after absorption with sonicated Y. enterocolitica 3 antigen. The reactivity on thyroid by the Y. enterocolitica 3 positive sera was unchanged after absorption with extracts of pig stomach or lymphoblastoid cells. TABLE 3. Absorption of antibodies in Y. enterocolitica 3-positive sera reacting in IFL only with the membrane region of thyroid
epithelial cells
Sonicated bacterial antigens used Yersinia enterocolitica 3 (five strains)
Indirect immunofluorescence staining of human thyroid by antibodies occurring in Yersimna enterocolitica infections K. LIDMAN, ULLA ERIKSSON,* RENEE NORBERG & ASTRIDFAGRAEUSDepartment of Immunology and *Department of Bacteriology, National Bacteriological Laboratory, S-105 21 Stockholm, Sweden
(Received 21 July 1975)
SUMMARY
In the diagnostic routine for tissue antibodies, using indirect immunofluorescence on cryostat sections of human thyrotoxic thyroid, rat stomach and kidney, ninety-six out of 48,388 sera showed a marginal staining of the membrane region of thyroid epithelial cells but no other reaction. Twenty-six of these sera were from patients with acute Yersinia enterocolitica serotype 3 infection but without signs of thyroid disease. Fifty of sixty-three sera with agglutinins against Y. enterocolitica serotype 3 and three out of four sera with agglutinins against Y. enterocolitica serotype 9 also showed this reaction on thyroid sections. It was due to antibodies, mostly of the IgG class, occurring in low titre, which react with intracytoplasmic antigens in thyroid, as staining of live thyroid epithelial cells was negative. The pattern of immunofluorescence on thyroid sections caused by these antibodies could not be distinguished from that caused by smooth muscle antibodies by appearance only. However, smooth muscle antibodies react also on other tissue sections and extracts of contractile proteins which absorb out these, did not change the reaction on thyroid of Y. enterocolitica sera. Absorption with sonicated Y. enterocolitica 3 and 9 antigens, but not with heat-killed whole bacteria, extinguished the reaction on thyroid. This indicates the presence of a cross-reactivity between antigens in these bacteria, different from the 0 antigen, and antigens in thyroid epithelial cells. Knowledge of this pattern of immunofluorescence on thyroid sections can be of diagnostic significance.
INTRODUCTION Smooth muscle antibodies (SMA) appear predominantly in sera from patients with active chronic hepatitis and are demonstrable by indirect IFL on cryostat sections of rat stomach (Johnson, Holborow & Glynn, 1965). SMA also react with other tissues, e.g. renal glomeruli (Whittingham, Mackay & Irwin, 1966) and the epithelial cells in cryostat sections of human thyroid (Biberfeld, Fagraeus & Lenkei, 1974; Sutton et al., 1974). In the routine examination for autoantibodies we have observed a number of sera which gave only a reaction with the membrane region of thyroid epithelial cells (Biberfeld et al., 1974) and not with smooth muscle fibres or rat renal glomeruli. Checking of the clinical diagnosis of thirteen patients whose sera gave this isolated reaction with thyroid, revealed that five of them had a serologically verified infection with Yersinia enterocolitica (Y. enterocolitica) and further preliminary investigation still indicated this connection (Lidman et al., 1974). Y. enterocolitica is not uncommon in the Scandinavian countries. It causes infection in Correspondence: Dr Knut Lidman, Department of Immunology, National Bacteriological Laboratory, 105 21 Stockholm, Sweden.
429
430
K. Lidman et al.
the gastrointestinal tract, sometimes with symptoms of acute terminal ileitis (Winblad, Nilehn & Sternby, 1966; Persson et al., 1974). Acute arthritis (Ahvonen, Sievers & Aho, 1969) and erythema nodosum (Winblad, 1969; Hannuksela & Ahvonen, 1969) may occur during the course of Y. enterocolitica infection. Diagnosis is based on the finding of agglutinating antibodies against 0 antigens from Y. enterocolitica or on the isolation of the pathogen. Y. enterocolitica serotype 3 (Y. enterocolitica 3) dominates as the cause of human cases in Europe including Sweden (Winblad, 1973). Cases of Y. enterocolitica serotype 9 (Y. enterocolitica 9) are rare in Sweden but fairly common in Finland, Belgium and the Netherlands (Winblad, 1973). The occurrence in Y. enterocolitica infections of antibodies that in IFL gives a marginal staining of the thyroid epithelial cells has been investigated. Evidence is presented that these antibodies are separate from SMA and are directed against related antigens in Y. enterocolitica bacteria and thyroid cells. MATERIALS AND METHODS Sera. Most sera used in this study were sent in from various hospitals to the National Bacteriological Laboratory for routine testing for tissue antibodies or estimation of agglutinins against Y. enterocolitica or Salmonella antigens. Sera from patients with acute terminal ileitis caused by Y. enterocolitica 3 and from patients with Crohn's disease were kindly provided by Dr Dan Danielsson, Orebro. Sera from patients with Y. enterocolitica 9 were kindly provided by Dr Lars Burman, Umeat. Immunofluorescence (IFL). Cryostat sections of human thyrotoxic thyroid (acetone-fixed sections), rat stomach (unfixed) and rat kidney (unfixed) were used as antigens in all routine testing for tissue antibodies by indirect IFL. Selected sera were also tested on cryostat sections of the following organs: atoxic human, monkey and rat thyroid, mouse thymus, rat liver, pig stomach, human foetal and adult ileum. Tests were also performed on smears of lymphoblastoid cells and human fibroblasts grown on glass as described by Norberg, Lidman & Fagraeus (1975). Thyroid monolayer cultures were prepared from surgically resected glands and after various time of culture (0-3-5-7 days) the cells were dispersed with 0-25% trypsin solution. Cyto-centrifuged preparations of the cells were then made and stained by indirect IFL as described by Biberfeld et al. (1974). Staining of live suspended thyroid cells from the cultures was made by mixing 106 cells with 0 2 ml of test serum dilution. After 30 min at + 4°C the cells were washed and 0 2 ml of the appropriately diluted FITC-conjugate was added. The patients' sera were heated at 56°C for 30 min and examined in a dilution of 1/10, if not otherwise stated. The following fluorescein isothiocyanate (FITC) conjugates were employed: (1) a sheep anti-human immunoglobulin (SBL 480040); (2) Wellcome anti-IgG lot number K 8351; (3) sheep anti-IgM (SBL 76101); (4) sheep anti-IgA (SBL 0904"). The preparations were examined in a Zeiss fluorescence microscope equipped with a HBO mercury lamp and a dry dark-field condenser using primary filter BG 3 and secondary filters 44 or 47. Bacterial strains. Y. enterocolitica 3 (482/IIL), Y. enterocolitica 9 (486/IX), E. coli 945, E. coli 014: K7: H-, Staphylococcus aureus 209, Streptococcus pyogenes type 12 group A (Squibb 1300) were received from strain collections at the National Bacteriological Laboratory. Y. enterocolitica 3 patients strains from different geographical regions were isolated in the diagnostic routine and some were also kindly provided by Dr P. Toivanen and Dr L. Olkkonen, Turku, Finland. Agglutination technique. Tube agglutination was performed by the conventional Widal technique for 0 antigens using heat-killed Y. enterocolitica 3 and 9 bacteria. A titre of 160 or more was regarded diagnostic for an acute Y. enterocolitica infection (Arvaston, Damgaard & Winblad, 1971; Ahvonen, 1972; Toivanen et al., 1973). All sera found in the routine with the typical IFL-pattern on thyroid were tested for agglutinins against Y. enterocolitica 3 and 9. Absorption of sera. (A) Bacteria were cultured on agar plates (Oxoid blood agar base CM 55 (Oxoid Ltd, London)+ 1%Y glucose) for 20 hr at 37°C. Cells were harvested in PBS, pH 7-4 and washed twice. For absorption four different antigen preparations were made. (1) Treatment at + 100°C for 60 min followed by two washings in PBS. (2) Formaldehyde was added to a final concentration of 1 .. After 20 hr at + 4°C the antigen was washed twice in PBS. (3) Tetracycline-HCl lot number VN-238-304 (Pfizer, Brussels) was added to a final concentration of 2 pg/ml in order to obtain bacteriostatic condition.
Antibodies reacting with human thyroid
431
(4) Treatment of bacteria (1 mg dry weight/ml) in a Braunsonic 300 S (B. Braun, Melsungen, W. Germany) at 70%. for 5 min. The supernatant from centrifugation at 10,000 g for 30 min was used. Absorption with whole bacterial antigens (1-3) was performed by resuspending 1010 packed bacteria in 0 9 ml PBS (with tetracycline included for antigen 3) and adding 0 1 ml serum. The mixture was incubated for 4 hr at + 40C and then centrifuged at 3000 g for 15 min. The supernatant was repeatedly absorbed with the same amount of packed bacteria at + 40C overnight. After centrifugation the supernatant was examined for complete absorption by tube agglutination and then tested by IFL. Four volumes of soluble antigen (4) was mixed with one volume of serum and incubated at + 40C for 4 hr. After centrifugation (35,000 g for 60 min) repeated absorption of serum was made with equal amounts of antigen and serum dilution at + 40C over night. The supernatant after centrifugation was then tested by Widal agglutination and by IFL. (B) Antigens containing contractile proteins were prepared from the muscularis of pig stomach (smooth muscle antigen) and from lymphoblastoid cells and used for absorption as previously described (Biberfeld et al., 1974; Fagraeus, Lidman & Biberfeld, 1974).
RESULTS
During the period January 1973 to June 1975, 48,388 patients sera were routinely tested by IFL for tissue antibodies. Ninety-six of these sera (0.2%) (Table 1) gave a typical marginal staining of thyroid cells as shown in Fig. 1. In contrast to SMA-positive sera, there was no reaction of these sera on sections of rat stomach, kidney, liver or on human foetal and adult ileum. Smears of lymphoblastoid cells did not reveal any IFL-staining of microvilli and human fibroblasts grown on glass were negative. The only positive tissue reaction, except with thyroid, that sometimes could be seen with these sera was a 'streaky' staining of sections of mouse thymus.
FIG. 1. Indirect inmmunofluorescence reaction on cryostat sections of human thyrotoxic thyroid with serum from a patient with Yersinia enterocolitica serotype 3 infection. Note the staining of the membrane region of the epithelial cells surrounding a follicle in the gland. (Magnification x 600.)
Twenty-six (270 ) of the ninety-six sera, which gave this marginal staining on thyroid, patients with coexisting antibodies to Y. enterocolitica 3 in titre . 160 (Table 1). Clinically fourteen of them had reported diarrhoea, twenty had postinfectious arthritis affecting one or several joints and three had erythema nodosum but in no case were there any apparent symptoms of thyroid disease. The cause of the disease symptoms was previously unknown in twelve of these cases and was revealed by the finding of thyroid reaction and then Y. enterocolitica 3 agglutinins. came from different
c
K. Lidman
432
et
al.
TABLE 1. Sera reacting only with the membrane region of thyroid epithelial cells found in routine-IFL
Total number of sera 1973 1974 1975
Sera with reaction on thyroid and Sera reacting Y. enterocolitica 3 agglutinins in on thyroid in dilution . 160 dilution . 10
17109 20040 11239
13 56 27
5 13 8
48388
96
26
(1/130/6)
Sera reacted with the thyroid epithelial cells usually in low titres, 10-20, occasionally in titres of 80-160. Antibodies that gave a marginal staining on thyroid usually disappeared earlier than the agglutinating antibodies in convalescent sera. The reason for reactivity of the 70 Yersinia-negative sera (730%) was not revealed and preliminary checking did not show any apparent disease in common in these cases. None of the ninety-six sera found at the examination by IFL had agglutinins against Y. enterocolitica 9. This might depend upon the rare occurrence of infections with Y. enterocolitica 9, as three out of four sera with agglutinins against Y. enterocolitica 9 showed a faint staining of thyroid epithelial cells. Forty-seven sera sent in primarily for Widal agglutination and found with a Y. enterocolitica 3 titre 2 160, were also tested in IFL. Thirty-seven (84%) of them gave the typical pattern and ten were negative. Thirteen of sixteen sera from cases of acute terminal ileitis caused by Y. enterocolitica 3 were positive on thyroid sections. However, nineteen sera from patients with Crohn's disease and ten Salmonella agglutinin-positive sera were negative. Testing with monospecific conjugates showed that the majority of the thyroid reacting antibodies were of IgG-class (Table 2) but antibodies of IgM and IgA class occurred. The IFL reaction of Yersinia-positive sera was best seen on thyrotoxic epithelial cells. Tests on sections of six atoxic glands showed no or only a faint reaction in the membrane region of the cells. Staining of thyroid from monkey and rat were negative. Sera that were positive on thyroid sections and for Y. enterocolitica 3 agglutinins did not react with live cells from thyrotoxic or atoxic glands. Staining of cytocentrifuged thyrotoxic epithelial cells before culture, however, revealed fluorescent irregular 'rings' in the cytoplasm of the cells (Fig. 2). On day 3 of culture almost all 'rings' had disappeared in the cytocentrifuged specimens. Cytocentrifuged cell preparations from atoxic glands were never stained. Absorption of agglutinin and 'thyroid-positive' Y. enterocolitica 3 sera with heat-killed Y. enterocolitica 3 bacteria resulted in the disappearance of the 0 agglutinins but the IFLTABLE 2. Ig-class of Ab reacting in IFL with thyroid in 33 Y. enterocolitica serotype 3 (titre . 160) sera, from acute phase of the disease revealed by monospecific conjugates
IgG IgM IgG and IgM IgG and IgA IgG, IgM and IgA
16 1 7 3 6
Antibodies reacting with human thyroid
ia
433
1~j
D FIG. 2. Indirect immunofluorescence reaction with serum from a patient with Yersinia enterocolitica serotype 3 infection on cytocentrifuged preparations of thyrotoxic thyroid cells before culture. Fluorescent 'rings' appear (a) in one single cell; (b) in several cells in an aggregate. (Magnification x 700.)
reaction on thyroid was unchanged. The effect of absorption with formaldehyde or tetracycline-treated bacteria was a decrease in titre of the thyroid-reacting antibodies and disappearance of the 0 agglutinins. Total absorption of both antibody reactions was, however, achieved when sonicated Y. enterocolitica 3 or 9 bacteria were used (Table 3). Absorption with Staphylococcus aureus 209, Streptococcus pyogenes type 12 group A and E. coli 014 and 945 did not influence the antibody titres. The titre of sera positive for thyroid cytoplasmic antibodies or mitochondrial antibodies, ANF and SMA were not changed after absorption with sonicated Y. enterocolitica 3 antigen. The reactivity on thyroid by the Y. enterocolitica 3 positive sera was unchanged after absorption with extracts of pig stomach or lymphoblastoid cells. TABLE 3. Absorption of antibodies in Y. enterocolitica 3-positive sera reacting in IFL only with the membrane region of thyroid
epithelial cells
Sonicated bacterial antigens used Yersinia enterocolitica 3 (five strains)
