British Journal of Haematology, 1979, 43, 223-233.
Ferritin, a Sensitizing Substance in the Leucocyte Migration Inhibition Test in Patients with Malignant Lymphoma B. W. HANCOCK, LESLEY BRUCE,K. MAYA N D J. RICHMOND University of S h d j e l d Department $Medicine, Hallamshire Hospital, Shejjeld (Received 28 February 1978; acceptedfor publication 3 0 February 1979) SUMMARY. The nature of the factor in Hodgkin’s disease involved spleen to which many patients with malignant lymphoma react in the leucocyte migration inhibition test has been investigated. Our results suggest that ferritin from Hodgkin’s disease involved spleens is antigenically different to that prepared from normal spleen. Isoelectric focusing shows the presence of more acidic ‘isoferritins’in ferritin prepared from Hodgkin’s disease involved spleen than in that prepared from normal spleen. Further observations using the leucocyte migration inhibition test suggest that sensitization to the abnormal ferritin, acting as an onco-fetal tumour associated substance, may be responsible for the reaction of patients with malignant lymphoma in this test. The immunological derangements found in malignant lymphoma and the varying prognosis seen in relationship to the proportion of lymphocytes in tissue biopsies in Hodgkin’s disease suggest the possibility of an immunological response in the host against the tumour. Recent reports indicate that there may be tumour associated ‘antigens’ in the spleen in Hodgkin’s disease (Order et al, 1972; Long et al, 1973). Elevated levels of carcinoembryonic antigen have also been found in spleens from such patients (Khoo et al, 1973); in addition elevated serum levels of other onco-fetal substances have been recorded in malignant lymphoma (Lawrence & Neville, 1972). Using the leucocyte migration inhibition test we have demonstrated sensitization to a factor present in Hodgkin’s disease involved spleen obtained at necropsy in approximately 50% of patients with malignant lymphoma tested (Hancock et al, 1976a) and it appears that follow-up assessment of sensitization may be of prognostic value (Hancock et al, 1976b). The nature of the Hodgkin’s disease tissue culture ‘antigen’ ofLong et a1 (1973, 1974a, b) has not been established. Eshaar et al(1974) have concluded that the antigen that they detected with fast electrophoretic activity (F antigen) in non-cultured spleen is normal tissue ferritin. High serum ferritin levels have been found in patients with leukaemia and with Hodgkin’s disease (Jones et al, 1973) but may often be related to the non-specific anaemia of neoplastic disease with its associated increase in the reticulo-endothelial iron stores and decreased serum Correspondence: Dr B. W. Hancock, Department of Medicine, Floor M, Hallamshire Hospital, Sheffield S10 2JF. 0007-1048/79/1ooCr0223$02.00 01979 Blackwekl Scientific Publications
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iron concentrations (Jacobs& Worwood, 1975).Increased ferritin synthesisby neoplastic tissue has been shown in acute leukaemia (White et al, 1974) but may reflect an overall increase in protein synthesis rather than tumour cell over-production of a specific ferritin. Increased ferritin synthesis and release by peripheral blood lymphocytes has also been demonstrated in Hodgkin’s disease (Sarcione et al, 1977) and ferritin has also been implicated as a possible blocking substance in Hodgkin’s disease (Moroz t f al, 1977). There have been reports of abnormal isoferritin profiles in malignancy (Drysdale & Alpert, 1975; Halliday et al, 1976)and differences in the quantitative immunological reactions of various isoferritins have also been documented (Worwood et al, 1976; Hazard et al, 1977a, b). It has been suggested that abnormal tissue isoferritins are onco-fetal substances. It was decided to determine whether ferritin was concerned in the sensitization which we had demonstrated to Hodgkin’s disease involved spleen in the leucocyte migration inhibition test. We also investigated the difference between ferritins from normal and Hodgkin’s disease involved spleens. METHODS Preparation offerritin. Ferritin was prepared from normal and Hodgkin’s disease involved human spleens obtained at necropsy by the method of Worwood et a1 (1975). Briefly, the homogenate was heated to 70°C, cooled, acidified, centrifuged and the supernatant collected. Protein was then precipitated by adding solid ammonium sulphate to 50% saturation. The precipitate containing ferritin was dissolved in phosphate buffer, dialysed and subjected to gel filtration, on Sepharose 6B (Pharmacia GI3 Ltd) the gel filtration then being repeated. Ferritins were similarly prepared from necropsy specimens of Hodgkin’s disease involved lymph nodes, of adenocarcinoma involved liver and of fetal livers. Raising ofantisera. Antisera to Hodgkin’s disease spleen ferritin were raised by injecting New Zealand white rabbits with 1 mg of ferritin together with Freund’s complete adjuvant. Injection of ferritin was repeated three times at 2-3 week intervals and 2 weeks after the final injection the rabbits were bled and the antisera stored at -20°C. Antisera to normal ferritin were raised by an identical immunization schedule. Specificity of the antisera was demonstrated by immunoelectrophoresis against ferritin standards. For the leucocyte migration inhibition experiment an extract of y-globulin fraction from the antiserum was obtained by adding ammonium sulphate to 35% saturation with dialysis of the resulting precipitate against normal saline. Antihuman spleen sera were prepared (in a similar manner to that of the antiferritin antisera) from rabbits immunized with normal and Hodgkin’s disease involved spleen extracts. Preparation of splenic extracts. In the experiments with normal and Hodgkin’s disease spleen tissue (a different spleen from that used for the ferritin preparation) 1 g of tissue was homogenized with 5 ml of water and passed through a millipore filter (pore size 0-22,pm) to remove bacteria. The protein concentration of the samples was approximately 200-400 mg/l. Freeze-dried fetal spleen tissue (wet weight 1 g) was reconstituted in 5 ml Weymouth’s medium (Gibco Bio-Cult Ltd). In leucocyte migration experiments high concentrations of ferritin and ferritin antiserum were toxic; best results were obtained using 100 ng/l and 1/100 dilution respectively of these
Ferritin in M a l i p a n t Lymphoma
225
preparations. Spleen extracts used in a dilution of 1/40 gave the best results. Ferritin concentrations were measured by the Folin method (Cowry et at, 1951) using a horse spleen ferritin standard (Koch Light Co.) in which protein concentration was measured by total nitrogen content (Kjeldahl method).) Polyacrylamidegel electrophoresis. Running gels (100 mm x 5 mm) were prepared to contain 5% w/v polyacrylamide and 0.15% w/v ammonium persulphate in tris-HC1 buffer, pH 8.9. Electrophoresis was performed in a Shandon tank and gels were stained for protein (2% amido black in 7% w/v acetic acid) and iron (2% w/v potassium ferrocyanide, 2% HC1). Electrophoresis on cellulose acetate paper. Electrophoresis on cellulose acetate and in 1.5% agar plates was performed in pH 8.6 veronal buffer on protein extracts from normal spleen (NS) and Hodgkin's disease involved spleen (HS) with normal ferritin and Hodgkin's spleen ferritin run as standards. Strips and plates were stained for protein with Ponceau S and for non-haem iron with 0.2% potassium ferrocyanide. Immunodi8usion and immunoelectrophoresis. Immunodiffusion in 1.5% agar of NS extract, HS extract and ferritin derived from NS and HS homogenates was performed against antibodies raised against normal and Hodgkin's disease ferritins. Studies were repeated using 1/10 dilutions of NS and HS extracts. Immunoelectrophoresis of ferritins, NS extract and HS extract against all the raised antibodies was carried out in 1.5% agar plates (pH 8.6 Verona120 mA/cm). Plates were stained by incubation for 20 min in 0.1% Ponceau S in 5% trichloracetic acid and destained in 5% acetic acid. ImvnunofZuorescence.lmmunofluorescence was carried out according to the method of Order er a1 (1972) using antisera raised to HS ferritin and to HS extract with antisera to NS extract as a control. Cryostat sections of HS and NS (6-10 pm) were incubated for 15 min at room temperature with each of the antisera diluted 1/20 after absorption overnight at 4°C with freshly prepared normal spleen cells. Sections were then washed with tissue culture Tyrode solution, blotted dry and then incubated for 15 min a t room temperature with fluorescein conjugated goat anti-rabbit globulin diluted 1/8. Sections were then washed with tissue culture Tyrode solution and examined under fluorescence microscopy. Tumour-bearing regions were identified by 0.1?An thionin (Ehrlich) staining of sections (Gurr, 1956). Nuclei stained blue to purple, collagen red and elastic tissues green. I ~ m u n a r u ~ i a m e ~arsay r i c ( I M R A ) and rudiaimmttnaussuy. Immunoradiometric assay of ferritin was performed by the method of Addison et a1 (1972).Antiserum was raised against Hodgkin's disease spleen ferritin by the above method. Standard curves were obtained using a normal and Hodgkin's disease spleen ferritin. In the IMRA an increase in affinity of the antigen for the antibody will lead to an increase in radioactivity (counts per minute, cpm) in the supernatant. Radioimmunoassay of ferritins was carried out using a Gamma DabTMcommercial assay kit (Travenol Laboratories) in which crystalline human liver (CL) ferritin is used as immunogen, tracer and reference standard. Standard curves were obtained for crystalline liver ferritin (Travenol) normal and Hodgkin's spleen ferritin. In the RIA an increase in affinity of the antigen for the antibody leads to a reduction in the number of counts obtained in the bound fraction (counts bound in sample tube as a percentage of the counts bound at zero antigen dose, "/.BIB,).
Isoelectricfocusing. Isoelectric focusing was carried out on 100-200 pg of purified ferritins in 5% acrylamide with 2% w/v ampholines (LKB Broma) with equal quantities of pH 4-6 and
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5-7 and booster quantities of pH 3.5-10.0. Gels were run until equilibrium was reached and stained for protein with Coomassie blue. Leucocyte migration test. The leucocyte migration inhibition technique was that of Soberg & Bendixen (1967) with minor modifications (Hancock et al, 1976a). Separated washed peripheral blood leucocytes were allowed to migrate from microcapillary tubes in wells containing the various ‘antigens’ under test. Migration areas with antigen were statistically compared with control migration areas and significant migration inhibition was said to have occurred if W 0 . 0 5 . A migration index was also obtained by dividing the area of migration with antigen by the area of migration in control medium. Patients. (a) In initial assessments 74 patients with malignant lymphoma were studied (48 Hodgkin’s disease, 25 non-Hodgkin’s lymphoma) for sensitization to HS extract, HS ferritin, Candida antigen (50 mg/l) and purified protein derivative (50 mg/l). Twenty-one of these patients were tested with NS extract. Twenty of those patients reacting to HS extract and HS ferritin were tested against ferritin obtained from NS homogenate. In eight patients known to react to HS extract further investigations were performed in which migration indices were simultaneously determined with: (i) HS extract alone, (ii) HS extract+ HS ferritin antiserum, (iii) HS extract+ HS ferritin antiserum previously absorbed with HS ferritin, (iv) HS ferritin alone, (v) HS ferritin+HS ferritin antiserum, (vi) HS ferritin +HS ferritin antiserum previously absorbed with HS ferritin. HS ferritin antiserum was tested alone in the leucocyte migration inhibition test in 18 patients and was added to the migration wells containing PPD and Candida antigens in five patients reacting to these antigens. NS ferritin antiserum was tested alone in five patients known to react to HS extract. (b) A second series of 46 patients was tested simultaneously with HS extract and fetal spleen. (c) In order to eliminate the possibility of third party immune complexes abolishing the leucocyte migration inhibition 10 further patients known to react to HS extract were tested with: (i) HS extract alone, (ii) bovine serum albumin (BSA) +anti-BSA, (iii) HS+anti-BSA. Two of these patients were also simultaneously tested with BSA alone, anti-BSA alone and HS+BSA anti-BSA complex. RESULTS
Polyacrylamide Gel Electrophoresis Monomeric and dimeric forms of ferritin were observed on protein and iron staining. NO difference was apparent in the mobilities of NS and HS ferritins in this buffer system (Fig 1). Electrophoresis on Cellulose Acetate Paper A single band staining for iron and for protein was found in the ferritin standards and this corresponded to a similar band in both the NS and HS extracts. Extracts were produced from the same weight of tissue in both cases and from the intensity of protein staining it was evident that ferritin was present in higher concentration in the HS extract. The yields of ferritin from Hodgkin’s and normal spleens confirmed this observation. Yields were 0.5 mg/g of tissue and 0.08 mg/g of tissue respectively. Immunodtflusion and Immunoelectrophoresis After immunodiffusion lines of complete identity were found between NS extract, HS
Ferritin in Malignant Lymphoma
227
FIG 1. Polyacrylarnide gel electrophoresis of Hodgkin’s disease spleen ferritin (left) and normal spleen ferritin (right) stained for protein with Coomassieblue (bands obtained also stained positive for iron).
extract, HS ferritin and NS ferritin with each of the prepared antisera. Ferritin in the HS extract was detected at higher dilutions than that of the NS extract with HS ferritin antisera. Thus it would appear, from the lines of complete identity (Fig 2), that there is no qualitative immunological difference between the ferritins from HS and NS which may be detected by this technique. The lines of identity seen after immunoelectrophoresis showed that a ferritin antibody was present in the antisera raised to HS homogenate and was also present, though to a lesser extent, in the antiserum raised to NS homogenate. The single precipitation line (Fig 3) obtained on immunoelectrophoresis of the NS ferritin and HS ferritin against the antisera to both demonstrates the specificity of the antisera.
Immunojluorescence In areas of tumour involvement pericellular fluorescence was more intense than in surrounding non-involved tissue both with HS antibody and HS ferritin antibody, indicating the presence of ferritin as a tumour associated substance. Little fluorescence was seen in the NS sections.
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FIG2. Immunodiffusion of normal spleen ferritin (NSF) and Hodgkin’s disease spleen ferritin (HSF) against antisera raised against Hodgkin’s disease spleen ferritin (A) and against normal spleen ferritin (B).
FIG 3. Immunoelectrophoresis of normal spleen ferritin against normal spleen ferritin antiserum (above) and of Hodgkin’s disease spleen ferritin against Hodgkin’s disease spleen ferritin antiserum (below).
Immunoradiometric Assay (IMRA) and Radioimmmoassay From the IMRA standard curves (Fig 4) obtained with HS ferritin and NS ferritin against HS ferritin antibody it is evident that there is a difference in the quantitative immunological reaction of the ferritins isolated from NS and HS. HS ferritin shows a greater affinity than the NS ferritin for the antibodies to HS ferritin. From the RIA standard curves (Fig 5) obtained with HS ferritin and NS ferritin it is evident that there is a quantitative difference in the
Ferritin in Malignant Lymphoma
229
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FIG 4 FIG 5 FIG4.Standard curves obtained on immunoradiometric assay of ferritins prepared from Hodgkin’s disease spleen (HSF) and normal spleen (NSF) against Hodgkin’s disease spleen ferritin antiserum. Values for counts per minute (cpm) x lo2 Iz5Iare given as meanfstandard deviation. FIG 5. Standard curves obtained on RIA (Travenol) of crystalline livcr ferritin (CLF) and ferritins prepared from Hodgkin’s disease spleen (HSF) and normal spleen (NSF). %BIB, (counts bound in sample tube as percentage of counts bound at zero antigen dose) given as mean of three determinations.
cross-reactivity with anti-liver ferritin. NS ferritin shows a greater affinity than the HS ferritin for the antibodies to liver ferritin. The affinities are thus reversed compared with those obtained on IMRA against HS ferritin antibody demonstrating immunological differences between the two ferritins. Isoelectric Focusing Additional acidic bands were found on isoelectric focusing in the HS, adenocarcinoma liver (CaL), Hodgkin’s lymph node (HLN) and fetal liver (FL) ferritin preparations which were not found in the normal spleen ferritin preparation (Fig 6), although the extra bands in HS ferritins were relatively weak. Leucocyte Migration Studies (a) In preliminary experiments with 74 patients with malignant lymphoma there was excellent correlation between migration indices with HS extract and HS ferritin (I =0.76, P< 0.001). There was no significant correlation between migration indices with HS extract 4 6 0.44). None of the 20 patients with and those with Candida and PPD antigens ( ~ ~ 0 . and malignant lymphorna, known to react to HS extract and HS ferritin, tested against equivalent
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FIG6. Isoelectric focusing of ferritins prepared from normal spleen (NSF), Hodgkin’s disease spleens (HSF 1, 2, 3), Hodgkin’s disease lymph nodes (HLNF), fetal liver (FLF) and adenocarcinoma liver (CaLF) stained for protein with Coomassie blue.
concentrations of NS ferritin showed significant inhibition. No migration inhibition was shown with NS extract. In the simultaneous tests on the leucocytes from patients reacting to HS and HS ferritin addition of HS ferritin antiserum abolished the leucocyte migration inhibition (Fig 7). No such effect was shown by NS ferritin antisera. This effect on leucocyte migration was not observed if the HS antiserum was first absorbed with HS ferritin. HS ferritin antiserum alone caused migration inhibition in only one of 18 patients tested. Migration indices with Candida and PPD were not significantly altered by the addition of HS ferritin antiserum. NS ferritin antiserum alone caused no migration inhibition. (b) Migration indices with fetal spleen correlated well with those obtained with HS and HS ferritin (r=0.74, P
Ferritin, a Sensitizing Substance in the Leucocyte Migration Inhibition Test in Patients with Malignant Lymphoma B. W. HANCOCK, LESLEY BRUCE,K. MAYA N D J. RICHMOND University of S h d j e l d Department $Medicine, Hallamshire Hospital, Shejjeld (Received 28 February 1978; acceptedfor publication 3 0 February 1979) SUMMARY. The nature of the factor in Hodgkin’s disease involved spleen to which many patients with malignant lymphoma react in the leucocyte migration inhibition test has been investigated. Our results suggest that ferritin from Hodgkin’s disease involved spleens is antigenically different to that prepared from normal spleen. Isoelectric focusing shows the presence of more acidic ‘isoferritins’in ferritin prepared from Hodgkin’s disease involved spleen than in that prepared from normal spleen. Further observations using the leucocyte migration inhibition test suggest that sensitization to the abnormal ferritin, acting as an onco-fetal tumour associated substance, may be responsible for the reaction of patients with malignant lymphoma in this test. The immunological derangements found in malignant lymphoma and the varying prognosis seen in relationship to the proportion of lymphocytes in tissue biopsies in Hodgkin’s disease suggest the possibility of an immunological response in the host against the tumour. Recent reports indicate that there may be tumour associated ‘antigens’ in the spleen in Hodgkin’s disease (Order et al, 1972; Long et al, 1973). Elevated levels of carcinoembryonic antigen have also been found in spleens from such patients (Khoo et al, 1973); in addition elevated serum levels of other onco-fetal substances have been recorded in malignant lymphoma (Lawrence & Neville, 1972). Using the leucocyte migration inhibition test we have demonstrated sensitization to a factor present in Hodgkin’s disease involved spleen obtained at necropsy in approximately 50% of patients with malignant lymphoma tested (Hancock et al, 1976a) and it appears that follow-up assessment of sensitization may be of prognostic value (Hancock et al, 1976b). The nature of the Hodgkin’s disease tissue culture ‘antigen’ ofLong et a1 (1973, 1974a, b) has not been established. Eshaar et al(1974) have concluded that the antigen that they detected with fast electrophoretic activity (F antigen) in non-cultured spleen is normal tissue ferritin. High serum ferritin levels have been found in patients with leukaemia and with Hodgkin’s disease (Jones et al, 1973) but may often be related to the non-specific anaemia of neoplastic disease with its associated increase in the reticulo-endothelial iron stores and decreased serum Correspondence: Dr B. W. Hancock, Department of Medicine, Floor M, Hallamshire Hospital, Sheffield S10 2JF. 0007-1048/79/1ooCr0223$02.00 01979 Blackwekl Scientific Publications
223
224
B. W . Hancock or a1
iron concentrations (Jacobs& Worwood, 1975).Increased ferritin synthesisby neoplastic tissue has been shown in acute leukaemia (White et al, 1974) but may reflect an overall increase in protein synthesis rather than tumour cell over-production of a specific ferritin. Increased ferritin synthesis and release by peripheral blood lymphocytes has also been demonstrated in Hodgkin’s disease (Sarcione et al, 1977) and ferritin has also been implicated as a possible blocking substance in Hodgkin’s disease (Moroz t f al, 1977). There have been reports of abnormal isoferritin profiles in malignancy (Drysdale & Alpert, 1975; Halliday et al, 1976)and differences in the quantitative immunological reactions of various isoferritins have also been documented (Worwood et al, 1976; Hazard et al, 1977a, b). It has been suggested that abnormal tissue isoferritins are onco-fetal substances. It was decided to determine whether ferritin was concerned in the sensitization which we had demonstrated to Hodgkin’s disease involved spleen in the leucocyte migration inhibition test. We also investigated the difference between ferritins from normal and Hodgkin’s disease involved spleens. METHODS Preparation offerritin. Ferritin was prepared from normal and Hodgkin’s disease involved human spleens obtained at necropsy by the method of Worwood et a1 (1975). Briefly, the homogenate was heated to 70°C, cooled, acidified, centrifuged and the supernatant collected. Protein was then precipitated by adding solid ammonium sulphate to 50% saturation. The precipitate containing ferritin was dissolved in phosphate buffer, dialysed and subjected to gel filtration, on Sepharose 6B (Pharmacia GI3 Ltd) the gel filtration then being repeated. Ferritins were similarly prepared from necropsy specimens of Hodgkin’s disease involved lymph nodes, of adenocarcinoma involved liver and of fetal livers. Raising ofantisera. Antisera to Hodgkin’s disease spleen ferritin were raised by injecting New Zealand white rabbits with 1 mg of ferritin together with Freund’s complete adjuvant. Injection of ferritin was repeated three times at 2-3 week intervals and 2 weeks after the final injection the rabbits were bled and the antisera stored at -20°C. Antisera to normal ferritin were raised by an identical immunization schedule. Specificity of the antisera was demonstrated by immunoelectrophoresis against ferritin standards. For the leucocyte migration inhibition experiment an extract of y-globulin fraction from the antiserum was obtained by adding ammonium sulphate to 35% saturation with dialysis of the resulting precipitate against normal saline. Antihuman spleen sera were prepared (in a similar manner to that of the antiferritin antisera) from rabbits immunized with normal and Hodgkin’s disease involved spleen extracts. Preparation of splenic extracts. In the experiments with normal and Hodgkin’s disease spleen tissue (a different spleen from that used for the ferritin preparation) 1 g of tissue was homogenized with 5 ml of water and passed through a millipore filter (pore size 0-22,pm) to remove bacteria. The protein concentration of the samples was approximately 200-400 mg/l. Freeze-dried fetal spleen tissue (wet weight 1 g) was reconstituted in 5 ml Weymouth’s medium (Gibco Bio-Cult Ltd). In leucocyte migration experiments high concentrations of ferritin and ferritin antiserum were toxic; best results were obtained using 100 ng/l and 1/100 dilution respectively of these
Ferritin in M a l i p a n t Lymphoma
225
preparations. Spleen extracts used in a dilution of 1/40 gave the best results. Ferritin concentrations were measured by the Folin method (Cowry et at, 1951) using a horse spleen ferritin standard (Koch Light Co.) in which protein concentration was measured by total nitrogen content (Kjeldahl method).) Polyacrylamidegel electrophoresis. Running gels (100 mm x 5 mm) were prepared to contain 5% w/v polyacrylamide and 0.15% w/v ammonium persulphate in tris-HC1 buffer, pH 8.9. Electrophoresis was performed in a Shandon tank and gels were stained for protein (2% amido black in 7% w/v acetic acid) and iron (2% w/v potassium ferrocyanide, 2% HC1). Electrophoresis on cellulose acetate paper. Electrophoresis on cellulose acetate and in 1.5% agar plates was performed in pH 8.6 veronal buffer on protein extracts from normal spleen (NS) and Hodgkin's disease involved spleen (HS) with normal ferritin and Hodgkin's spleen ferritin run as standards. Strips and plates were stained for protein with Ponceau S and for non-haem iron with 0.2% potassium ferrocyanide. Immunodi8usion and immunoelectrophoresis. Immunodiffusion in 1.5% agar of NS extract, HS extract and ferritin derived from NS and HS homogenates was performed against antibodies raised against normal and Hodgkin's disease ferritins. Studies were repeated using 1/10 dilutions of NS and HS extracts. Immunoelectrophoresis of ferritins, NS extract and HS extract against all the raised antibodies was carried out in 1.5% agar plates (pH 8.6 Verona120 mA/cm). Plates were stained by incubation for 20 min in 0.1% Ponceau S in 5% trichloracetic acid and destained in 5% acetic acid. ImvnunofZuorescence.lmmunofluorescence was carried out according to the method of Order er a1 (1972) using antisera raised to HS ferritin and to HS extract with antisera to NS extract as a control. Cryostat sections of HS and NS (6-10 pm) were incubated for 15 min at room temperature with each of the antisera diluted 1/20 after absorption overnight at 4°C with freshly prepared normal spleen cells. Sections were then washed with tissue culture Tyrode solution, blotted dry and then incubated for 15 min a t room temperature with fluorescein conjugated goat anti-rabbit globulin diluted 1/8. Sections were then washed with tissue culture Tyrode solution and examined under fluorescence microscopy. Tumour-bearing regions were identified by 0.1?An thionin (Ehrlich) staining of sections (Gurr, 1956). Nuclei stained blue to purple, collagen red and elastic tissues green. I ~ m u n a r u ~ i a m e ~arsay r i c ( I M R A ) and rudiaimmttnaussuy. Immunoradiometric assay of ferritin was performed by the method of Addison et a1 (1972).Antiserum was raised against Hodgkin's disease spleen ferritin by the above method. Standard curves were obtained using a normal and Hodgkin's disease spleen ferritin. In the IMRA an increase in affinity of the antigen for the antibody will lead to an increase in radioactivity (counts per minute, cpm) in the supernatant. Radioimmunoassay of ferritins was carried out using a Gamma DabTMcommercial assay kit (Travenol Laboratories) in which crystalline human liver (CL) ferritin is used as immunogen, tracer and reference standard. Standard curves were obtained for crystalline liver ferritin (Travenol) normal and Hodgkin's spleen ferritin. In the RIA an increase in affinity of the antigen for the antibody leads to a reduction in the number of counts obtained in the bound fraction (counts bound in sample tube as a percentage of the counts bound at zero antigen dose, "/.BIB,).
Isoelectricfocusing. Isoelectric focusing was carried out on 100-200 pg of purified ferritins in 5% acrylamide with 2% w/v ampholines (LKB Broma) with equal quantities of pH 4-6 and
226
B . W . Hancock et a1
5-7 and booster quantities of pH 3.5-10.0. Gels were run until equilibrium was reached and stained for protein with Coomassie blue. Leucocyte migration test. The leucocyte migration inhibition technique was that of Soberg & Bendixen (1967) with minor modifications (Hancock et al, 1976a). Separated washed peripheral blood leucocytes were allowed to migrate from microcapillary tubes in wells containing the various ‘antigens’ under test. Migration areas with antigen were statistically compared with control migration areas and significant migration inhibition was said to have occurred if W 0 . 0 5 . A migration index was also obtained by dividing the area of migration with antigen by the area of migration in control medium. Patients. (a) In initial assessments 74 patients with malignant lymphoma were studied (48 Hodgkin’s disease, 25 non-Hodgkin’s lymphoma) for sensitization to HS extract, HS ferritin, Candida antigen (50 mg/l) and purified protein derivative (50 mg/l). Twenty-one of these patients were tested with NS extract. Twenty of those patients reacting to HS extract and HS ferritin were tested against ferritin obtained from NS homogenate. In eight patients known to react to HS extract further investigations were performed in which migration indices were simultaneously determined with: (i) HS extract alone, (ii) HS extract+ HS ferritin antiserum, (iii) HS extract+ HS ferritin antiserum previously absorbed with HS ferritin, (iv) HS ferritin alone, (v) HS ferritin+HS ferritin antiserum, (vi) HS ferritin +HS ferritin antiserum previously absorbed with HS ferritin. HS ferritin antiserum was tested alone in the leucocyte migration inhibition test in 18 patients and was added to the migration wells containing PPD and Candida antigens in five patients reacting to these antigens. NS ferritin antiserum was tested alone in five patients known to react to HS extract. (b) A second series of 46 patients was tested simultaneously with HS extract and fetal spleen. (c) In order to eliminate the possibility of third party immune complexes abolishing the leucocyte migration inhibition 10 further patients known to react to HS extract were tested with: (i) HS extract alone, (ii) bovine serum albumin (BSA) +anti-BSA, (iii) HS+anti-BSA. Two of these patients were also simultaneously tested with BSA alone, anti-BSA alone and HS+BSA anti-BSA complex. RESULTS
Polyacrylamide Gel Electrophoresis Monomeric and dimeric forms of ferritin were observed on protein and iron staining. NO difference was apparent in the mobilities of NS and HS ferritins in this buffer system (Fig 1). Electrophoresis on Cellulose Acetate Paper A single band staining for iron and for protein was found in the ferritin standards and this corresponded to a similar band in both the NS and HS extracts. Extracts were produced from the same weight of tissue in both cases and from the intensity of protein staining it was evident that ferritin was present in higher concentration in the HS extract. The yields of ferritin from Hodgkin’s and normal spleens confirmed this observation. Yields were 0.5 mg/g of tissue and 0.08 mg/g of tissue respectively. Immunodtflusion and Immunoelectrophoresis After immunodiffusion lines of complete identity were found between NS extract, HS
Ferritin in Malignant Lymphoma
227
FIG 1. Polyacrylarnide gel electrophoresis of Hodgkin’s disease spleen ferritin (left) and normal spleen ferritin (right) stained for protein with Coomassieblue (bands obtained also stained positive for iron).
extract, HS ferritin and NS ferritin with each of the prepared antisera. Ferritin in the HS extract was detected at higher dilutions than that of the NS extract with HS ferritin antisera. Thus it would appear, from the lines of complete identity (Fig 2), that there is no qualitative immunological difference between the ferritins from HS and NS which may be detected by this technique. The lines of identity seen after immunoelectrophoresis showed that a ferritin antibody was present in the antisera raised to HS homogenate and was also present, though to a lesser extent, in the antiserum raised to NS homogenate. The single precipitation line (Fig 3) obtained on immunoelectrophoresis of the NS ferritin and HS ferritin against the antisera to both demonstrates the specificity of the antisera.
Immunojluorescence In areas of tumour involvement pericellular fluorescence was more intense than in surrounding non-involved tissue both with HS antibody and HS ferritin antibody, indicating the presence of ferritin as a tumour associated substance. Little fluorescence was seen in the NS sections.
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FIG2. Immunodiffusion of normal spleen ferritin (NSF) and Hodgkin’s disease spleen ferritin (HSF) against antisera raised against Hodgkin’s disease spleen ferritin (A) and against normal spleen ferritin (B).
FIG 3. Immunoelectrophoresis of normal spleen ferritin against normal spleen ferritin antiserum (above) and of Hodgkin’s disease spleen ferritin against Hodgkin’s disease spleen ferritin antiserum (below).
Immunoradiometric Assay (IMRA) and Radioimmmoassay From the IMRA standard curves (Fig 4) obtained with HS ferritin and NS ferritin against HS ferritin antibody it is evident that there is a difference in the quantitative immunological reaction of the ferritins isolated from NS and HS. HS ferritin shows a greater affinity than the NS ferritin for the antibodies to HS ferritin. From the RIA standard curves (Fig 5) obtained with HS ferritin and NS ferritin it is evident that there is a quantitative difference in the
Ferritin in Malignant Lymphoma
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FIG 4 FIG 5 FIG4.Standard curves obtained on immunoradiometric assay of ferritins prepared from Hodgkin’s disease spleen (HSF) and normal spleen (NSF) against Hodgkin’s disease spleen ferritin antiserum. Values for counts per minute (cpm) x lo2 Iz5Iare given as meanfstandard deviation. FIG 5. Standard curves obtained on RIA (Travenol) of crystalline livcr ferritin (CLF) and ferritins prepared from Hodgkin’s disease spleen (HSF) and normal spleen (NSF). %BIB, (counts bound in sample tube as percentage of counts bound at zero antigen dose) given as mean of three determinations.
cross-reactivity with anti-liver ferritin. NS ferritin shows a greater affinity than the HS ferritin for the antibodies to liver ferritin. The affinities are thus reversed compared with those obtained on IMRA against HS ferritin antibody demonstrating immunological differences between the two ferritins. Isoelectric Focusing Additional acidic bands were found on isoelectric focusing in the HS, adenocarcinoma liver (CaL), Hodgkin’s lymph node (HLN) and fetal liver (FL) ferritin preparations which were not found in the normal spleen ferritin preparation (Fig 6), although the extra bands in HS ferritins were relatively weak. Leucocyte Migration Studies (a) In preliminary experiments with 74 patients with malignant lymphoma there was excellent correlation between migration indices with HS extract and HS ferritin (I =0.76, P< 0.001). There was no significant correlation between migration indices with HS extract 4 6 0.44). None of the 20 patients with and those with Candida and PPD antigens ( ~ ~ 0 . and malignant lymphorna, known to react to HS extract and HS ferritin, tested against equivalent
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FIG6. Isoelectric focusing of ferritins prepared from normal spleen (NSF), Hodgkin’s disease spleens (HSF 1, 2, 3), Hodgkin’s disease lymph nodes (HLNF), fetal liver (FLF) and adenocarcinoma liver (CaLF) stained for protein with Coomassie blue.
concentrations of NS ferritin showed significant inhibition. No migration inhibition was shown with NS extract. In the simultaneous tests on the leucocytes from patients reacting to HS and HS ferritin addition of HS ferritin antiserum abolished the leucocyte migration inhibition (Fig 7). No such effect was shown by NS ferritin antisera. This effect on leucocyte migration was not observed if the HS antiserum was first absorbed with HS ferritin. HS ferritin antiserum alone caused migration inhibition in only one of 18 patients tested. Migration indices with Candida and PPD were not significantly altered by the addition of HS ferritin antiserum. NS ferritin antiserum alone caused no migration inhibition. (b) Migration indices with fetal spleen correlated well with those obtained with HS and HS ferritin (r=0.74, P
