Clin. exp. Immunol. (1979) 38, 45-51.
Circulating immune complexes in IgA deficiency A. 0. KWITKO, P. E. MCKENZIE, D. J. C. SHEARMAN, A. A. GORMLY & A. J. WOODROFFE Department of Medicine, University of Adelaide and Renal Unit, Royal Adelaide Hospital, South Australia
(Acceptedfor publication 2 April 1979)
SUMMARY
Circulating immune complexes (IC) were demonstrated in patients with serum IgA deficiency. Sixteen of thirty-one IgA deficient patients had serum IC detected by solid phase Clq radioimmunoassay for IgG class complexes. The presence of cryoglobulins (thirteen out of thirty-one patients) and increased polyethylene glycol precipitation (ten out of thirty patients) provided additional evidence for the presence of IC. Fourteen patients were asymptomatic but seven had clinical evidence of disease which could have been IC mediated: two with glomerulonephritis, three with polyarthritis, one with vasculitis and one with thyroiditis. Serum IC remained detectable in multiple samples over several months but this correlated poorly with the presence or absence of disease. Serum antibody to IgA was detected in fifteen out of thirty-one patients. There was no direct relationship between the presence of IC and the level of serum anti-IgA antibody; however, this antibody was shown to be present in the IC isolate in eight patients. It is proposed that a considerable portion of the IC load in IgA deficiency results from defective antigen exclusion at the level of the mucosa. INTRODUCTION Isolated serum IgA deficiency is the most common form of immunodeficiency in man occurring in 1: 400 to 1: 700 individuals (Koistinen, 1975; Backman, 1965). While it is often asymptomatic it can predispose to a large number of clinical disorders (Horowitz & Hong, 1975). Asthma, atopy and sinopulmonary infection are most common and have been related to the reduction in secretory IgA (S-IgA) at mucosal surfaces (Heremans & Crabbe, 1968). However, autoimmune diseases also occur frequently. These include rheumatoid arthritis, systemic lupus erythematosus, vasculitis and thyroiditis. IgA-deficient patients have an increased incidence of autoantibodies when there is no evidence of overt disease (Wells, Michaeli & Fudenberg, 1975); these include antibody to smooth muscle, antinuclear antibodies and rheumatoid factor. Immune complexes (IC) have been implicated in both the pathogenesis and the course of many such autoimmune diseases. Another characteristic finding in IgA deficiency is the presence of serum antibody to IgA (Fudenberg, Vyas & Perkins, 1971; Koistinen & Sarna, 1975). Antibody to IgA coexists with the small amounts of serum IgA that are present but can cause anaphylaxis when exogenous IgA is administered (Vyas, Perkins & Fudenberg, 1968). Serum antibodies to dietary proteins, for example bovine serum albumin (BSA) and milk, are also more readily detected in IgA deficiency (Ammann & Hong, 1971), and many workers (Walker & Isselbacher, 1974; Andre et al., 1974; Walker, 1975) believe that in the absence of S-IgA significant amounts of foreign antigen such as BSA can pass into the circulation from mucosal surfaces. Correspondence: Professor D. J. C. Shearman, Department of Medicine, Royal Adelaide Hospital, Adelaide, South Australia 5000.
0099-9104/79/1000-0045$02.00 (C
1979 Blackwell Scientific Publications
45
46
A. 0. Kwitko et al.
With this large number of circulating antigen-antibody systems available, the potential exists for subsequent IC formation. In the present study we have examined sera from patients with IgA deficiency for the presence of circulating IC. MATERIALS AND METHODS Patient selection. This was primarily by screening a large population attending the emergency clinic of the Royal Adelaide Hospital for minor trauma, but four patients with known IgA deficiency were added (patients 3, 12, 13 and 23-see Table 1). Age- and sex-matched controls were selected from healthy nursing staff and volunteers. IgA measurement. Serum IgA was measured by standard single radial immunodiffusion (Mancini) technique using alphachain specific antiserum and appropriate standards (Behring). The sensitivity of this method was 1 mg 0 and the normal range was 219+ 100 mg % (mean± 1 s.d.) for the population studied. Patients with a serum IgA level persistently at or below 30 mg % which could not be attributed to any secondary cause were classed as IgA-deficient. Patients were followed for at least 1 year. Serum storage. Blood for IC determination was allowed to clot at room temperature, centrifuged at 500 g for 10 min and serum stored at -70'C until tested. Solid phase Clq radioimmunoassay (SP CIq RIA). This assay for detecting circulating IC was modified from that of Hay, Nineham & Roitt (1976) as described previously (Woodroffe et al., 1979). The only further modification was that 1% of human serum albumin was used instead of BSA as the carrier protein. Each test was performed in duplicate. Results were referred to a standard curve of the binding obtained with heat-aggregated human gammaglobulin (AHG) in normal human serum. Results were expressed as jig AHG equivalent per ml serum above the mean + 2 s.d. value of sera from fifty-seven healthy control subjects. IC size estimation. IC were sized by sucrose density gradient ultracentrifugation in three patients with high IC levels. A 3-4 ml 10-40% sucrose gradient was used with 200 pi of 1: 2 serum. After centrifugation for 18 hr (40C) at 300,000 g thirty-six 100 pi fractions were collected. Alternate samples were assayed for IC by SP Clq RIA and for serum IgG and IgM by laser nephelometry (Hyland). IC composition. The presence of anti-IgA antibody in the IC was assessed by isolating complexes in solid phase as for the Clq RIA and then measuring the binding of '25I-labelled S-IgA. Polyethylene glycol (PEG) precipitation assay. This assay for IC was performed according to the method of Digeon et al. (1977) and the results were expressed as OD280 of the precipitate dissolved in 0-1 N NaOH. S-IgA preparation. This was prepared from a pool of twenty colostral specimens. Colostrum was defatted by centrifugation, casein precipitated by acidification to pH 4-6 and diethylaminoethyl cellulose (DE-52) separation carried out according to the method of Newcomb, Normansell & Stanworth (1968). Further purification was by three sequential passages of IgA through Sephadex G-200. Serum anti IgA antibody, A solid phase radioimmunoassay was established and compared with the standard haemagglutination technique of Gold & Fudenberg (1967). Polystyrene tubes were coated with pure colostral IgA (10 gg/ml). 100 pi of the test serum was added to 900 jil of 0-15 M phosphate-buffered saline (PBS) at pH 7-2 in each tube in duplicate. Tubes were incubated for 60 min at 370C and then for 18 hr at 4VC. Results were expressed as the percentage binding of 125I-labelled goat anti-human IgG antibody. The class of antibody to IgA was determined in seven patients with the highest haemagglutination titres by fractionation of sera on Sephadex G-200 and retesting the concentrated IgM and IgG fractions. l25I-labelling ofproteins. This was performed by the chloramine-T method of McConahey & Dixon (1966).
RESULTS By screening sera, twenty-seven subjects (1: 387) were found to have a selective deficiency of serum IgA (< 30 mg %) and four (Nos 3, 12, 13, 23) subsequently found to be IgA-deficient were added later (Table 1). Of the whole group, twenty-five had serum levels of 10 mg % or less and nineteen had levels below 5 mg %. There were twenty-one females and ten males, ranging in age from twelve to seventythree years with a mean age of thirty-seven. Only three patients had chronic disease, two with histologically proven chronic glomerulonephritis and one with severe asthma. However, fourteen had had episodes of illness in the past of the type commonly associated with IgA deficiency, such as recurrent sino-pulmonary infection (five), asthma (four), atopy-eczema (two), polyarthritis (three), vasculitis (one), thyroiditis (one) and colitis (one). There was one patient with coeliac disease proven by small bowel biopsy. Using the SP Clq RIA, circulating IC were detected in sixteen out of thirty-one (52%) IgA-deficient patients, but only in six (19%) age- and sex-matched controls (Fig. 1). IgA-deficient patients had a mean serum IC level of 35 pg AHG equivalent per ml, whilst controls had a mean level of 0-8 Vg/ml.
Macrophage function in leprosy
47
examined. In addition, the ability of macrophages from non-responding patients to co-operate with T cells from healthy responding contacts has been studied. Preliminary results presented here suggest that the lack of response to in vitro stimulation by M. leprae is caused by an inability of macrophages from non-responding patients to present M. leprae antigens to T cells in an appropriate immunogen form. MATERIALS AND METHODS Cell donors. Eighteen previously untreated leprosy patients from the routine diagnostic clinic of the All Africa Leprosy and Rehabilitation Training Center (ALERT) in Addis Ababa were used in this study. They were selected and assessed on the basis of clinical diagnosis, skin smears for acid-fast bacilli, skin histology (Ridley & Jopling, 1966), and the results of lymphocyte transformation tests towards Mycobacterium leprae. Patients demonstrating a stimulation ratio (i.e. culture response cpm+M. leprae/cpm- M. leprae) of less than two were considered non-responding. These patients generally were in the LL/BL classification and are designated L in the tables and text. Patients and healthy contacts demonstrating a stimulation ratio greater than four were considered high responders and are designated T (tuberculoid) or N (normal) respectively. Macrophages. Mononuclear cells were separated from peripheral blood of leprosy patients and healthy contacts by FicollIsopaque floatation. Macrophages were obtained by incubating the isolated leucocytes in plastic flasks for 2 hr (Hirschberg, Kaakinen & Thorsby, 1976). The non-adherent cells, mainly lymphocytes, were decanted by repeated washing and collected. The adherent cells were allowed to continue in culture for an additional 3-5 days. In this period, many of the cultures developed confluent monolayers of mature macrophages. The tendency to develop monolayers appeared to be more pronounced for macrophages separated from patients compared to healthy contacts. The cells were released from the plastic flasks by a 90 min incubation at 370C in Medium RPMI 1640 containing 3-3 mg EDTA/ml and 30% foetal calf serum. Yields from each flask were in the order of 4 x 105- 1.5 x 106 cells. As a routine control of the purity of the macrophages obtained with this procedure, aliquots of the released cells were incubated with PHA for 3 days, labelled and harvested. None of the cell samples tested in this manner showed significant proliferation, indicating that few viable lymphocytes remained adherent to the macrophages. Lymphocyte/macrophage cultures. T cells were prepared by nylon-wool column filtration (Wisloff & Froland 1973). The filtered columns were incubated with peripheral blood mononuclear cells in medium RPMI 1640 supplemented with 20% pooled normal serum for 60 min at 370C, followed by a slow and careful elution with medium containing 5°/ serum. The cell fraction thus obtained ('T cells') contained 75-80% E-rosette-forming cells and less than 0-5% latex-ingesting cells. All cell cultures were performed in round-bottomed Lindbro tissue-culture trays, employing 75 x 103 enriched T cells/well and a variable number of autologous or allogeneic macrophages (see tables). The trays were incubated for 5 days at 37°C in a 5% CO2 atmosphere. The cultures were done in triplicate at two different concentrations of each antigen, and contained a total volume of 200 pl of medium RPMI 1640 supplemented with antibiotics and 20% pooled normal serum. Control cultures were incubated without antigen. 1 uCi of tritium-labelled thymidine was added 18 hr before harvest of the cultures. The cultures were harvested onto glass-fibre filters on a Skatron multiple-cell-culture harvester (Skatron A/S, Lierbyen, Norway) and counted in a liquid scintillation counter. The results are presented as the mean cpm of triplicate cultures ± s.e. In approximately 20% of all combinations tested, the mixed lymphocyte-macrophage culture response was over 15,000 cpm. These combinations have been excluded from this study. Antigens. Leprosy bacilli were prepared from a single subcutaneous nodule from a non-treated lepromatous leprosy patient (Bjune et al., 1976). This preparation is designated 'whole' M. leprae in the tables. A sonicated fraction of the whole M. leprae preparation was also prepared in a Bronson B-12 ultrasonicator (80 W for 1 hr), and is termed 'sonicated' in the tables. Purified protein derivative. PPD, was obtained from Statens Seruminstitutt, Copenhagen, Denmark, and diluted in medium RPMI 1640 containing 1% human serum albumin.
RESULTS Proliferative response of T cells to PPD and M. leprae in the presence and absence of autologous MO. Stimulation by both the soluble protein antigen PPD and M. leprae, either whole or sonicated, was dependent on the presence of macrophages (MO) in the culture (Table 1). Nylon-wool column purified T cells showed a relatively weak proliferation towards these antigens, while the addition of macrophages, representing 500 of the total number of cells, restored the response. Absorbing peripheral blood leucocytes (PBL) on plastic for 2 hr to reduce the number of adherent cells reduced the response to PPD to approximately 60% of the response observed with PBL. On the other hand, the response to Mycobacterium leprae was reduced to approximately 20-30% of control values. This indicates that the response to a weak antigen, like M. leprae, is more dependent on the number of macrophages present in the cultures than the response to a stronger antigen like PPD.
H. Hirschberg
48
TABLE 1. Effects of autologous macrophages on the response of T cells to antigen Antigen source
PPD 1 mg/ml
Responding cells
Control
A T cells A T cells+AMb B T cells B T cells+BMq
1430+ 158 138+22 345+61 33,757+3272 128+ 32 45+ 1 153+22 21,685+2626
M. leprae baccilli/culture 107 Whole 106
107 Sonicated 106
217+45 217+45 152+47 262+64 918+ 111 5116+842 3629+409 6471+718 176+ 11 55+ 5 58+ 18 75+ 6 725+ 150 5108+521 1169+66 135+4
75 x 10' nylon-wool-column purified T cells were incubated with or without autologous macrophages (M b) (3 5 x 103/well) for five days in the wells of round-bottomed micro plates containing 200 /1 of RPMI 1640 supplemented with 20% normal human serum and antibiotics. The plates were harvested following an 18 hr pulse of 3H-thymidine. Results are the mean cpm of triplicate cultures+ s.e. A and B are healthy contacts.
Proliferative response of T cells from L patients in the presence of macrophages from responding individuals T cells were prepared from patients demonstrating a weak response (stimulation ratio < 2) to M. leprae in in vitro tests. Macrophages were obtained from either L (lepromatous) patients, from other patient classifications (TT, BT designated T (tuberculoid) in the tables) showing a positive response to M. leprae in vitro as well as from healthy contacts (N). L (lepromatous) T cells co-incubated together with autologous macrophages failed to respond to M. leprae stimulation even at a T cell/macrophage ratio of 5:1. On the other hand, T cells from this class of patients gave a significant response towards M. leprae (Table 2) when macrophages from a healthy contact were used. This was the case even at T cell/macrophage ratios of 20:1 (50 macrophage mixtures). The response towards PPD was also significantly greater in many allogeneic T-cell/macrophage combinations compared to autologous combinations. Macrophages from polar or borderline tuberculoid (TT, BT) were also able to reconstitute the response, of T cells from L (lepromatous) patients (Table 2, lower half). In most of the combinations tested, macrophages from this patient category seemed to behave similarly to macrophages from normal individuals in combinations with T cells from the L (lepromatous patient group. On the other hand, the proliferative response towards M. leprae in combinations of T cells from the T (tuberculoid) patient group together with macrophages from normal contacts was usually somewhat greater than that observed in combinations of T cells and allogeneic macrophages from T (tuberculoid) patients.
Proliferative response of T cells from responding individuals in combination with macrophages from nonresponders The ability of macrophages from L (lepromatous) patients to co-operate with T cells from healthy contacts, was also studied. T cells were separated from donors who gave a strong positive response to M. leprae in vitro: macrophages were isolated from L (lepromatous) patients as well as from other healthy contacts to act as a source of normal allogeneic macrophages. In order to make comparisons as relevant as possible, the T cells were co-incubated with either L (lepromatous) macrophages or normal macrophages, both from allogeneic donors. Macrophages from both donors restored the response to PPD although cell combinations employing L (lepromatous) macrophages generally gave a weaker response (Table 3). T cells with allogeneic normal macrophages gave a potentiated response to M. leprae compared to unfractionated PBL, while, in the three combinations tested, macrophages from L (lepromatous) patients proved incapable of restoring the response towards M. leprae (Table 3). Furthermore, the ability of macrophages to stimulate allogeneic T cells was apparently inhibited by the presence of M. leprae antigen. A comparable effect (i.e., inhibition of the mixed lymphocyte-macrophage culture response by M. leprae) was also observed with normal macrophages at T cell/macrophage ratios of greater than 30:1.
49
Immune complexes in IgA deficiency IgG
IgM
8 7
CD A__
-4 E6-o £3
*\ I
.0
, 2 -i
4
0
>
8
J
(
t
*
f
20 24 28 12 16 Sucrose density gradient fractions
32
36
FIG. 3. Distribution of serum IC by sucrose density gradient ultracentrifugation. (O-*) IgA-deficient a) normal control. patient and (0 n =13
n =15
3*0 -X= 1-8±0-=221±0-4 P
Circulating immune complexes in IgA deficiency A. 0. KWITKO, P. E. MCKENZIE, D. J. C. SHEARMAN, A. A. GORMLY & A. J. WOODROFFE Department of Medicine, University of Adelaide and Renal Unit, Royal Adelaide Hospital, South Australia
(Acceptedfor publication 2 April 1979)
SUMMARY
Circulating immune complexes (IC) were demonstrated in patients with serum IgA deficiency. Sixteen of thirty-one IgA deficient patients had serum IC detected by solid phase Clq radioimmunoassay for IgG class complexes. The presence of cryoglobulins (thirteen out of thirty-one patients) and increased polyethylene glycol precipitation (ten out of thirty patients) provided additional evidence for the presence of IC. Fourteen patients were asymptomatic but seven had clinical evidence of disease which could have been IC mediated: two with glomerulonephritis, three with polyarthritis, one with vasculitis and one with thyroiditis. Serum IC remained detectable in multiple samples over several months but this correlated poorly with the presence or absence of disease. Serum antibody to IgA was detected in fifteen out of thirty-one patients. There was no direct relationship between the presence of IC and the level of serum anti-IgA antibody; however, this antibody was shown to be present in the IC isolate in eight patients. It is proposed that a considerable portion of the IC load in IgA deficiency results from defective antigen exclusion at the level of the mucosa. INTRODUCTION Isolated serum IgA deficiency is the most common form of immunodeficiency in man occurring in 1: 400 to 1: 700 individuals (Koistinen, 1975; Backman, 1965). While it is often asymptomatic it can predispose to a large number of clinical disorders (Horowitz & Hong, 1975). Asthma, atopy and sinopulmonary infection are most common and have been related to the reduction in secretory IgA (S-IgA) at mucosal surfaces (Heremans & Crabbe, 1968). However, autoimmune diseases also occur frequently. These include rheumatoid arthritis, systemic lupus erythematosus, vasculitis and thyroiditis. IgA-deficient patients have an increased incidence of autoantibodies when there is no evidence of overt disease (Wells, Michaeli & Fudenberg, 1975); these include antibody to smooth muscle, antinuclear antibodies and rheumatoid factor. Immune complexes (IC) have been implicated in both the pathogenesis and the course of many such autoimmune diseases. Another characteristic finding in IgA deficiency is the presence of serum antibody to IgA (Fudenberg, Vyas & Perkins, 1971; Koistinen & Sarna, 1975). Antibody to IgA coexists with the small amounts of serum IgA that are present but can cause anaphylaxis when exogenous IgA is administered (Vyas, Perkins & Fudenberg, 1968). Serum antibodies to dietary proteins, for example bovine serum albumin (BSA) and milk, are also more readily detected in IgA deficiency (Ammann & Hong, 1971), and many workers (Walker & Isselbacher, 1974; Andre et al., 1974; Walker, 1975) believe that in the absence of S-IgA significant amounts of foreign antigen such as BSA can pass into the circulation from mucosal surfaces. Correspondence: Professor D. J. C. Shearman, Department of Medicine, Royal Adelaide Hospital, Adelaide, South Australia 5000.
0099-9104/79/1000-0045$02.00 (C
1979 Blackwell Scientific Publications
45
46
A. 0. Kwitko et al.
With this large number of circulating antigen-antibody systems available, the potential exists for subsequent IC formation. In the present study we have examined sera from patients with IgA deficiency for the presence of circulating IC. MATERIALS AND METHODS Patient selection. This was primarily by screening a large population attending the emergency clinic of the Royal Adelaide Hospital for minor trauma, but four patients with known IgA deficiency were added (patients 3, 12, 13 and 23-see Table 1). Age- and sex-matched controls were selected from healthy nursing staff and volunteers. IgA measurement. Serum IgA was measured by standard single radial immunodiffusion (Mancini) technique using alphachain specific antiserum and appropriate standards (Behring). The sensitivity of this method was 1 mg 0 and the normal range was 219+ 100 mg % (mean± 1 s.d.) for the population studied. Patients with a serum IgA level persistently at or below 30 mg % which could not be attributed to any secondary cause were classed as IgA-deficient. Patients were followed for at least 1 year. Serum storage. Blood for IC determination was allowed to clot at room temperature, centrifuged at 500 g for 10 min and serum stored at -70'C until tested. Solid phase Clq radioimmunoassay (SP CIq RIA). This assay for detecting circulating IC was modified from that of Hay, Nineham & Roitt (1976) as described previously (Woodroffe et al., 1979). The only further modification was that 1% of human serum albumin was used instead of BSA as the carrier protein. Each test was performed in duplicate. Results were referred to a standard curve of the binding obtained with heat-aggregated human gammaglobulin (AHG) in normal human serum. Results were expressed as jig AHG equivalent per ml serum above the mean + 2 s.d. value of sera from fifty-seven healthy control subjects. IC size estimation. IC were sized by sucrose density gradient ultracentrifugation in three patients with high IC levels. A 3-4 ml 10-40% sucrose gradient was used with 200 pi of 1: 2 serum. After centrifugation for 18 hr (40C) at 300,000 g thirty-six 100 pi fractions were collected. Alternate samples were assayed for IC by SP Clq RIA and for serum IgG and IgM by laser nephelometry (Hyland). IC composition. The presence of anti-IgA antibody in the IC was assessed by isolating complexes in solid phase as for the Clq RIA and then measuring the binding of '25I-labelled S-IgA. Polyethylene glycol (PEG) precipitation assay. This assay for IC was performed according to the method of Digeon et al. (1977) and the results were expressed as OD280 of the precipitate dissolved in 0-1 N NaOH. S-IgA preparation. This was prepared from a pool of twenty colostral specimens. Colostrum was defatted by centrifugation, casein precipitated by acidification to pH 4-6 and diethylaminoethyl cellulose (DE-52) separation carried out according to the method of Newcomb, Normansell & Stanworth (1968). Further purification was by three sequential passages of IgA through Sephadex G-200. Serum anti IgA antibody, A solid phase radioimmunoassay was established and compared with the standard haemagglutination technique of Gold & Fudenberg (1967). Polystyrene tubes were coated with pure colostral IgA (10 gg/ml). 100 pi of the test serum was added to 900 jil of 0-15 M phosphate-buffered saline (PBS) at pH 7-2 in each tube in duplicate. Tubes were incubated for 60 min at 370C and then for 18 hr at 4VC. Results were expressed as the percentage binding of 125I-labelled goat anti-human IgG antibody. The class of antibody to IgA was determined in seven patients with the highest haemagglutination titres by fractionation of sera on Sephadex G-200 and retesting the concentrated IgM and IgG fractions. l25I-labelling ofproteins. This was performed by the chloramine-T method of McConahey & Dixon (1966).
RESULTS By screening sera, twenty-seven subjects (1: 387) were found to have a selective deficiency of serum IgA (< 30 mg %) and four (Nos 3, 12, 13, 23) subsequently found to be IgA-deficient were added later (Table 1). Of the whole group, twenty-five had serum levels of 10 mg % or less and nineteen had levels below 5 mg %. There were twenty-one females and ten males, ranging in age from twelve to seventythree years with a mean age of thirty-seven. Only three patients had chronic disease, two with histologically proven chronic glomerulonephritis and one with severe asthma. However, fourteen had had episodes of illness in the past of the type commonly associated with IgA deficiency, such as recurrent sino-pulmonary infection (five), asthma (four), atopy-eczema (two), polyarthritis (three), vasculitis (one), thyroiditis (one) and colitis (one). There was one patient with coeliac disease proven by small bowel biopsy. Using the SP Clq RIA, circulating IC were detected in sixteen out of thirty-one (52%) IgA-deficient patients, but only in six (19%) age- and sex-matched controls (Fig. 1). IgA-deficient patients had a mean serum IC level of 35 pg AHG equivalent per ml, whilst controls had a mean level of 0-8 Vg/ml.
Macrophage function in leprosy
47
examined. In addition, the ability of macrophages from non-responding patients to co-operate with T cells from healthy responding contacts has been studied. Preliminary results presented here suggest that the lack of response to in vitro stimulation by M. leprae is caused by an inability of macrophages from non-responding patients to present M. leprae antigens to T cells in an appropriate immunogen form. MATERIALS AND METHODS Cell donors. Eighteen previously untreated leprosy patients from the routine diagnostic clinic of the All Africa Leprosy and Rehabilitation Training Center (ALERT) in Addis Ababa were used in this study. They were selected and assessed on the basis of clinical diagnosis, skin smears for acid-fast bacilli, skin histology (Ridley & Jopling, 1966), and the results of lymphocyte transformation tests towards Mycobacterium leprae. Patients demonstrating a stimulation ratio (i.e. culture response cpm+M. leprae/cpm- M. leprae) of less than two were considered non-responding. These patients generally were in the LL/BL classification and are designated L in the tables and text. Patients and healthy contacts demonstrating a stimulation ratio greater than four were considered high responders and are designated T (tuberculoid) or N (normal) respectively. Macrophages. Mononuclear cells were separated from peripheral blood of leprosy patients and healthy contacts by FicollIsopaque floatation. Macrophages were obtained by incubating the isolated leucocytes in plastic flasks for 2 hr (Hirschberg, Kaakinen & Thorsby, 1976). The non-adherent cells, mainly lymphocytes, were decanted by repeated washing and collected. The adherent cells were allowed to continue in culture for an additional 3-5 days. In this period, many of the cultures developed confluent monolayers of mature macrophages. The tendency to develop monolayers appeared to be more pronounced for macrophages separated from patients compared to healthy contacts. The cells were released from the plastic flasks by a 90 min incubation at 370C in Medium RPMI 1640 containing 3-3 mg EDTA/ml and 30% foetal calf serum. Yields from each flask were in the order of 4 x 105- 1.5 x 106 cells. As a routine control of the purity of the macrophages obtained with this procedure, aliquots of the released cells were incubated with PHA for 3 days, labelled and harvested. None of the cell samples tested in this manner showed significant proliferation, indicating that few viable lymphocytes remained adherent to the macrophages. Lymphocyte/macrophage cultures. T cells were prepared by nylon-wool column filtration (Wisloff & Froland 1973). The filtered columns were incubated with peripheral blood mononuclear cells in medium RPMI 1640 supplemented with 20% pooled normal serum for 60 min at 370C, followed by a slow and careful elution with medium containing 5°/ serum. The cell fraction thus obtained ('T cells') contained 75-80% E-rosette-forming cells and less than 0-5% latex-ingesting cells. All cell cultures were performed in round-bottomed Lindbro tissue-culture trays, employing 75 x 103 enriched T cells/well and a variable number of autologous or allogeneic macrophages (see tables). The trays were incubated for 5 days at 37°C in a 5% CO2 atmosphere. The cultures were done in triplicate at two different concentrations of each antigen, and contained a total volume of 200 pl of medium RPMI 1640 supplemented with antibiotics and 20% pooled normal serum. Control cultures were incubated without antigen. 1 uCi of tritium-labelled thymidine was added 18 hr before harvest of the cultures. The cultures were harvested onto glass-fibre filters on a Skatron multiple-cell-culture harvester (Skatron A/S, Lierbyen, Norway) and counted in a liquid scintillation counter. The results are presented as the mean cpm of triplicate cultures ± s.e. In approximately 20% of all combinations tested, the mixed lymphocyte-macrophage culture response was over 15,000 cpm. These combinations have been excluded from this study. Antigens. Leprosy bacilli were prepared from a single subcutaneous nodule from a non-treated lepromatous leprosy patient (Bjune et al., 1976). This preparation is designated 'whole' M. leprae in the tables. A sonicated fraction of the whole M. leprae preparation was also prepared in a Bronson B-12 ultrasonicator (80 W for 1 hr), and is termed 'sonicated' in the tables. Purified protein derivative. PPD, was obtained from Statens Seruminstitutt, Copenhagen, Denmark, and diluted in medium RPMI 1640 containing 1% human serum albumin.
RESULTS Proliferative response of T cells to PPD and M. leprae in the presence and absence of autologous MO. Stimulation by both the soluble protein antigen PPD and M. leprae, either whole or sonicated, was dependent on the presence of macrophages (MO) in the culture (Table 1). Nylon-wool column purified T cells showed a relatively weak proliferation towards these antigens, while the addition of macrophages, representing 500 of the total number of cells, restored the response. Absorbing peripheral blood leucocytes (PBL) on plastic for 2 hr to reduce the number of adherent cells reduced the response to PPD to approximately 60% of the response observed with PBL. On the other hand, the response to Mycobacterium leprae was reduced to approximately 20-30% of control values. This indicates that the response to a weak antigen, like M. leprae, is more dependent on the number of macrophages present in the cultures than the response to a stronger antigen like PPD.
H. Hirschberg
48
TABLE 1. Effects of autologous macrophages on the response of T cells to antigen Antigen source
PPD 1 mg/ml
Responding cells
Control
A T cells A T cells+AMb B T cells B T cells+BMq
1430+ 158 138+22 345+61 33,757+3272 128+ 32 45+ 1 153+22 21,685+2626
M. leprae baccilli/culture 107 Whole 106
107 Sonicated 106
217+45 217+45 152+47 262+64 918+ 111 5116+842 3629+409 6471+718 176+ 11 55+ 5 58+ 18 75+ 6 725+ 150 5108+521 1169+66 135+4
75 x 10' nylon-wool-column purified T cells were incubated with or without autologous macrophages (M b) (3 5 x 103/well) for five days in the wells of round-bottomed micro plates containing 200 /1 of RPMI 1640 supplemented with 20% normal human serum and antibiotics. The plates were harvested following an 18 hr pulse of 3H-thymidine. Results are the mean cpm of triplicate cultures+ s.e. A and B are healthy contacts.
Proliferative response of T cells from L patients in the presence of macrophages from responding individuals T cells were prepared from patients demonstrating a weak response (stimulation ratio < 2) to M. leprae in in vitro tests. Macrophages were obtained from either L (lepromatous) patients, from other patient classifications (TT, BT designated T (tuberculoid) in the tables) showing a positive response to M. leprae in vitro as well as from healthy contacts (N). L (lepromatous) T cells co-incubated together with autologous macrophages failed to respond to M. leprae stimulation even at a T cell/macrophage ratio of 5:1. On the other hand, T cells from this class of patients gave a significant response towards M. leprae (Table 2) when macrophages from a healthy contact were used. This was the case even at T cell/macrophage ratios of 20:1 (50 macrophage mixtures). The response towards PPD was also significantly greater in many allogeneic T-cell/macrophage combinations compared to autologous combinations. Macrophages from polar or borderline tuberculoid (TT, BT) were also able to reconstitute the response, of T cells from L (lepromatous) patients (Table 2, lower half). In most of the combinations tested, macrophages from this patient category seemed to behave similarly to macrophages from normal individuals in combinations with T cells from the L (lepromatous patient group. On the other hand, the proliferative response towards M. leprae in combinations of T cells from the T (tuberculoid) patient group together with macrophages from normal contacts was usually somewhat greater than that observed in combinations of T cells and allogeneic macrophages from T (tuberculoid) patients.
Proliferative response of T cells from responding individuals in combination with macrophages from nonresponders The ability of macrophages from L (lepromatous) patients to co-operate with T cells from healthy contacts, was also studied. T cells were separated from donors who gave a strong positive response to M. leprae in vitro: macrophages were isolated from L (lepromatous) patients as well as from other healthy contacts to act as a source of normal allogeneic macrophages. In order to make comparisons as relevant as possible, the T cells were co-incubated with either L (lepromatous) macrophages or normal macrophages, both from allogeneic donors. Macrophages from both donors restored the response to PPD although cell combinations employing L (lepromatous) macrophages generally gave a weaker response (Table 3). T cells with allogeneic normal macrophages gave a potentiated response to M. leprae compared to unfractionated PBL, while, in the three combinations tested, macrophages from L (lepromatous) patients proved incapable of restoring the response towards M. leprae (Table 3). Furthermore, the ability of macrophages to stimulate allogeneic T cells was apparently inhibited by the presence of M. leprae antigen. A comparable effect (i.e., inhibition of the mixed lymphocyte-macrophage culture response by M. leprae) was also observed with normal macrophages at T cell/macrophage ratios of greater than 30:1.
49
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