Clin. exp. Immunol. (1975) 19, 399-406.
SPECIFIC CONCENTRATION OF ANTILYMPHOCYTE ANTIBODIES IN THE SERUM CRYOPRECIPITATES OF PATIENTS WITH SYSTEMIC LUPUS ERYTHEMATOSUS J. B. WINFIELD,* R. J. WINCHESTER,t P. WERNET AND H. G. KUNKEL
Rockefeller University, New York City, New York, U.S.A. (Received 3 July 1974) SUMMARY
Antibodies to surface determinants ofhuman lymphocytes, recognized both by cytotoxicity and fluorescent antibody analysis, were shown to be specifically enriched over the serum levels in cryoprecipitates from patients with systemic lupus erythematosus (SLE). The antilymphocyte antibody was shown to be cold reactive and was exclusively IgM. It was distinct from IgM anti-IgG, which was also variably concentrated in the cryoprecipitates. The question whether the antilymphocyte antibodies appear in the cryoprecipitates as complexes because of interaction with surface membrane antigens, or simply because of cold reactive properties, remains to be determined. The possible clinical relevance of the cryoprecipitation of these antibodies in systemic lupus erythematosus is discussed. INTRODUCTION Cytotoxic antibodies to lymphocytes have been noted in the serums of patients with systemic lupus erythematosus (SLE) for a number of years (Terasaki, Mottironi & Barnett, 1970). Recently the marked cold reactivity of these antibodies has been demonstrated by absorption experiments and fluorescent antibody analysis (Winfield et al., 1974, 1975; Winchester et al., 1974). The immunopathological significance and clinical relevance of these antibodies remains unclear. Certain types of IgM anti-y-globulins show a similar cold reactivity (Capra, Winchester & Kunkel, 1969) and some of these have been demonstrated in the cryoprecipitates in this disease (Hanauer & Christian, 1967; Agnello et al., 1971). The presence of cryoprecipitates in SLE serums has been associated with hypocomplementaemia and nephritis (Koffier et al., 1970; Agnello et al., 1971). The latter authors have suggested that the anti-IgG may be important functionally, both in cryoprecipitate formation and in the development of nephritis in certain patients. However, not all SLE cryoprecipitates *
Postdoctoral fellow of the Arthritis Foundation.
t Senior Investigator of the Arthritis Foundation. Correspondence: Dr J. B. Winfield, Rockefeller University, New York, N.Y. 10021, U.S.A.
399
400
J. B. Winfield et al.
have detectable IgM anti-IgG, even though IgM is invariably a significant component. Because of the cold reactivity of the IgM antibody to lymphocytes, it seemed possible that certain cryoprecipitates might contain antibody of this specificity. The present data indicate that both types of antibody are present in SLE cryoprecipitates and are enriched over the serum levels.
MATERIALS AND METHODS Collection of serum and isolation of cryoprecipitates Serum from patients with SLE and other diseases was collected at 370C. Cryoprecipitates were obtained by allowing 20 ml of serum to stand at 40C for 1-7 days, followed by centrifugation at 1000 g for 30 min in the cold. In most experiments 1 ml of cold phosphatebuffered saline (PBS), pH 7-6, was layered over the cryoprecipitate. The saline was carefully drawn off after 30 min, and the cryoprecipitate was dispersed in 50 volumes of distilled water and washed three times. This method minimized entrapment of serum proteins (estimated to be less than 2% of the protein in the cryoprecipitate) and reduced loss of cryoprotein during the washing steps. In addition, certain cryoprecipitates were washed with cold PBS throughout, with results identical to those washed in distilled water. The cryoprecipitate was resolubilized in 1 ml of PBS at 370C or, for fractionation studies, in glycineacetate buffer, pH 3*5. Protein content of the solubilized cryoprecipitate was determined by the Folin-Ciacolteau method. Fractionation of serum cryoprecipitates was performed by sucrose density gradient ultracentrifugation in glycine-acetate buffer, pH 3.5. 125I-labelled IgM which lacked antibody activity, was used as a 19S marker.
Quantitation of immunoglobulin in serum and cryoprecipitates IgM and IgG in serums, in the whole resolubilized cryoprecipitates, and in fractions obtained by sucrose gradient centrifugation, were quantitated by radial immunodiffusion in 1% agarose, veronal buffer, pH 8 6, using WHO standards (Meloy) and immunoglobulin class-specific rabbit antisera (Mancini, Carbonara & Heremans, 1965; Fahey & McKelvey, 1965). Determinations made at 25°C and 37°C were identical. Anti-IgG antibody Anti-IgG in serum and cryoprecipitates was determined by haemagglutination of chromic chloride-sensitized type 0 human erythrocytes coated with human y-globulin (Cohn Fraction II). The assay was performed at 4°C and 25°C; titres were usually 1-2 dilutions lower at the warmer temperature. The results are expressed as the log2 of the reciprocal of the haemagglutination titre.
Lymphocytes These were obtained by initially incubating heparinized whole venous blood on nylon wool columns (0-1 g of nylon wool/ml of blood) at 37°C for 30 min to remove phagocytic cells. The cells were eluted from the column with Hanks's balanced salt solution. Lymphocytes were obtained by flotation on Ficoll-Hypaque (Boyum, 1968). The final cell population obtained consisted of 75-90%o sheep erythrocyte rosette-forming cells (T cells) and 525% aggregated IgG-binding or immunoglobulin-bearing cells (B cells).
Antilymphocyte antibodies in SLE cryoprecipitates
401
Microcytotoxicity assay A modification of the two-stage microdroplet method was used (Terasaki & McClelland, 1964; Wernet, Feizi & Kunkel 1972). Rabbit serum (Pel Freeze Biologicals, Rogers, Arkansas) served as a complement source. Assays were routinely performed at 15'C. Immunofluorescent studies These were performed as previously described (Fu, Winchester & Kunkel, 1974; Winfield et al., 1974). Briefly, 5 x 105 PBL in 0-025 ml of HBSS were incubated at 40C for 30 min with 0-025 ml of serum or solubilized cryoprecipitate. The cells were washed three times in 2% BSA in PBS, pH 7-4, containing sodium azide. Class-specific rhodamineconjugated rabbit antibody to human IgM or IgG was then added and the cells were incubated for 30 min at 40C and washed three times as before. Slides were examined as wet mounts with incident illumination.
Differential absorption experiments Certain SLE and non-SLE cryoprecipitates, or their immunoglobulin fractions, were absorbed with human thymus or peripheral blood lymphocytes, erythrocytes, or an IgGagarose immunoabsorbant to determine antibody specificity. The cryoprecipitates, resolubilized by incubation at 370C, were incubated at 40C for 30 min with an equal volume of packed cells or IgG-agarose. After centrifugation, IgG and IgM concentration, cytotoxic antibody, and anti-IgG titre were determined in absorbed and sham-absorbed samples. RESULTS Cryoprecipitates from seven patients with SLE and eight with other diseases: monoclonal 'mixed' cryoglobulinaemia, Sjogren's syndrome, chronic active hepatitis and hyperlipidaemia-were examined. Using the microcytotoxicity assay, antilymphocyte antibody was found in five out of seven SLE cryoprecipitates and in one cryoprecipitate from a patient with Sjogren's syndrome. The presence of antibody in the cryoprecipitates was associated with similar antibody in the serum in each SLE case. The antilymphocyte antibody in the cryoprecipitates was cold reactive in the same fashion as the serum antibody described previously (Winfield et al., 1974). It was limited to the 19S fractions obtained by sucrose density gradient centrifugation (Fig. 1). Association of antilymphocyte activity with IgM was confirmed by indirect immunofluorescence experiments using class-specific, rhodamine-conjugated antiserums. Incubation of normal PBL with cytotoxic SLE cryoprecipitates resulted in bright staining of a high percentage of cells (56-100%) with anti-IgM. Such was not observed with the non-cytotoxic SLE cryoglobulins, which gave the expected background of 5-25% positive cells. Staining with anti-IgG was similarly low for all cryoprecipitates, approximating the number of B cells in the lymphocyte preparations used. The quality of the staining resembled that seen with immune complexes bound to the Fc receptor on the B cells (Winchester et al., 1974). Sixty-one per cent of PBL were stained with anti-IgM after incubation with a non-cytotoxic cryoprecipitate from one patient with Sjogren's syndrome. None of the other non-SLE cryoprecipitates gave indirect IgM staining of PBL above the background levels. Specific enrichment of IgM antilymphocyte antibody in the SLE cryoprecipitates was
J. B. Winfield et al.
402 o
_11
_ 100 (0
( a10
100
80
_8 -
80
_8
60
6
60 -6a
40
4
40
~~~~2
20
E~~~~~20
27 I2
Negativee
i (b) tate.
relavNegative
_j f07\
G
0 p7
I-0
i
/e
L-r
I-
E
obFIG 1.Lclzto fatlmhct nioy
0
0 0
-0-5
0-5
0
.-2
igM
n
niIGt
h 19S9
rcino
L
0-3
0-3
~~19 S0
c5
0.1
0.1
25
50
75
100
25
50
75
100
Percentage of gradient volume
FIG. 1. Localization of antilymphocyte antibody and anti-IgG to the 19S fraction of SLE cryoprecipitates fractionated by sucrose gradient centrifugation at acid pH. (a) Esp cryoprecipitate. (b) Harv cryoprecipitate. Neither antibody was present in the 7S fraction. The relative of antilymphocyte antibody and anti-IgG vary in these cryoprecipitates. tB ade proportion o
based upon increased cytotoxic activity per milligram of cryo-IgM relative to serum IgM (Table 1). Antibody activity in the cryoprecipitates was increased from 3-37 times over that of serum. Cytotoxic activity was similar in preparations isolated under conditions dissociain the unfractionated cryoof ting immune complexes. Thus for patient Esp, 0e023 mglgM of killed fraction the isolated in 19S mg 0c03 and 50% the test PBL in the microprecipitate This of test. enrichment antilymphocyte activity was also demonstrated by cytotoxicity immunofluorescence indirect experiments in which dilutions of the cryoprecipitates were added to PBL. Certain SLE cryoprecipitates (Esp, Ite) were absorbed with PBL, human thymus lymphocytes, and human erythrocytes to estimate how much of the IgM in the cryoprecipitate was antilymphocyte antibody. Absorption with lymphocytes in the cold removed 41-45%/ of the IgM and more than 90o. of the cytotoxic activity. Erythrocytes removed less IgM (7-12%) and very little antilymphocyte cytotoxic activity (8-14%). The IgG concentration was essentially unchanged after absorption with either lymphocytes or erythrocytes. Five of six SLE cryoprecipitates also contained IgM with anti-IgG activity. Specific enrichment of this 1gM rheumatoid factor also was demonstrated (Table 2). However, in contrast to cytotoxic activity which was equivalent for the IgM in unfractionated cryo-
Antilymphocyte antibodies in SLE cryoprecipitates TABLE 1. Specific enrichment of antilymphocyte antibody in SLE precipitates
CTX5o* (mg of IgM/ml)
Subject
Sam Esp Ite Harv Dep Abb Gross
403
cryo-
Serum
Cryoprecipitate
Relative enrichment of lymphocytotoxicity in cryoprecipitate
0.60 0 60 0 70 2-20 0-78
0-016 0-023 0 07 0-25 0-22
37 26 10 9 3
Negative Negative
Negativet Negativet
* CTX50 is the concentration of IgM killing 50%. of PBL in the microcytotoxicity assay.
t Cryoprecipitate
was not
cytotoxic at
a
protein concentration of 0 50
mg/ml. TABLE 2. Enrichment of anti-IgG activity in SLE cryoprecipitates
Anti-IgG haemagglutination titre (1/log2)
Subject Ite Esp Dep Harv
Serum IgM* 0 1 2 2
Relative enrichment of anti-IgG activity in
Cryo-IgM
cryoprecipitate
0 5 7 10
16 32 256
* Based on IgM, 0 1 mg/ml isolated by sucrose density gradient centrifugation at pH 3-5.
precipitates and that isolated under conditions dissociating immune complexes, anti-IgG activity was considerably increased when IgM was isolated at acid pH. Thus for Dep, the anti-IgG titre for 0.1 mg IgM/ml in the unfractionated cryoglobulin was 2; the titre of the dissociated IgM (0 1 mg/ml) was 7. Differential absorptions with lymphocytes and an IgG-agarose immunoabsorbent of such IgM fractions from both SLE and non-SLE cryoprecipitates further documented the presence of separate anti-IgG and antilymphocyte antibodies (Table 3). Lymphocytes removed cytotoxic activity from the SLE material, but did not reduce the anti-IgG titre. Anti-IgG antibody was absorbed from both types of cryoprecipitates by insoluble IgG without decreasing antilymphocyte antibody. Eighty-four per cent of the IgM was removed by absorption with insoluble IgG from a non-SLE cryoprecipitate (Mil); 46% was absorbed from an SLE cryoprecipitate (Harv).
J. B. Winfield et al.
404
TABLE 3. Presence of distinct IgM anti-IgG and antilymphocyte antibodies in SLE cryoprecipitate
Anti-IgG titre (1/log2) Absorption
SLE cryoprecipitate Esp
None Lymphocytes IgG-agarose
6 6 0
Non-SLE
Harv McD
11 10 0
8 7 0
Lymphocytotoxicity (% cells killed) SLE cryoprecipitate
Non-SLE
Mil
Esp
Harv
McD
Mil
6 6 0
56
SPECIFIC CONCENTRATION OF ANTILYMPHOCYTE ANTIBODIES IN THE SERUM CRYOPRECIPITATES OF PATIENTS WITH SYSTEMIC LUPUS ERYTHEMATOSUS J. B. WINFIELD,* R. J. WINCHESTER,t P. WERNET AND H. G. KUNKEL
Rockefeller University, New York City, New York, U.S.A. (Received 3 July 1974) SUMMARY
Antibodies to surface determinants ofhuman lymphocytes, recognized both by cytotoxicity and fluorescent antibody analysis, were shown to be specifically enriched over the serum levels in cryoprecipitates from patients with systemic lupus erythematosus (SLE). The antilymphocyte antibody was shown to be cold reactive and was exclusively IgM. It was distinct from IgM anti-IgG, which was also variably concentrated in the cryoprecipitates. The question whether the antilymphocyte antibodies appear in the cryoprecipitates as complexes because of interaction with surface membrane antigens, or simply because of cold reactive properties, remains to be determined. The possible clinical relevance of the cryoprecipitation of these antibodies in systemic lupus erythematosus is discussed. INTRODUCTION Cytotoxic antibodies to lymphocytes have been noted in the serums of patients with systemic lupus erythematosus (SLE) for a number of years (Terasaki, Mottironi & Barnett, 1970). Recently the marked cold reactivity of these antibodies has been demonstrated by absorption experiments and fluorescent antibody analysis (Winfield et al., 1974, 1975; Winchester et al., 1974). The immunopathological significance and clinical relevance of these antibodies remains unclear. Certain types of IgM anti-y-globulins show a similar cold reactivity (Capra, Winchester & Kunkel, 1969) and some of these have been demonstrated in the cryoprecipitates in this disease (Hanauer & Christian, 1967; Agnello et al., 1971). The presence of cryoprecipitates in SLE serums has been associated with hypocomplementaemia and nephritis (Koffier et al., 1970; Agnello et al., 1971). The latter authors have suggested that the anti-IgG may be important functionally, both in cryoprecipitate formation and in the development of nephritis in certain patients. However, not all SLE cryoprecipitates *
Postdoctoral fellow of the Arthritis Foundation.
t Senior Investigator of the Arthritis Foundation. Correspondence: Dr J. B. Winfield, Rockefeller University, New York, N.Y. 10021, U.S.A.
399
400
J. B. Winfield et al.
have detectable IgM anti-IgG, even though IgM is invariably a significant component. Because of the cold reactivity of the IgM antibody to lymphocytes, it seemed possible that certain cryoprecipitates might contain antibody of this specificity. The present data indicate that both types of antibody are present in SLE cryoprecipitates and are enriched over the serum levels.
MATERIALS AND METHODS Collection of serum and isolation of cryoprecipitates Serum from patients with SLE and other diseases was collected at 370C. Cryoprecipitates were obtained by allowing 20 ml of serum to stand at 40C for 1-7 days, followed by centrifugation at 1000 g for 30 min in the cold. In most experiments 1 ml of cold phosphatebuffered saline (PBS), pH 7-6, was layered over the cryoprecipitate. The saline was carefully drawn off after 30 min, and the cryoprecipitate was dispersed in 50 volumes of distilled water and washed three times. This method minimized entrapment of serum proteins (estimated to be less than 2% of the protein in the cryoprecipitate) and reduced loss of cryoprotein during the washing steps. In addition, certain cryoprecipitates were washed with cold PBS throughout, with results identical to those washed in distilled water. The cryoprecipitate was resolubilized in 1 ml of PBS at 370C or, for fractionation studies, in glycineacetate buffer, pH 3*5. Protein content of the solubilized cryoprecipitate was determined by the Folin-Ciacolteau method. Fractionation of serum cryoprecipitates was performed by sucrose density gradient ultracentrifugation in glycine-acetate buffer, pH 3.5. 125I-labelled IgM which lacked antibody activity, was used as a 19S marker.
Quantitation of immunoglobulin in serum and cryoprecipitates IgM and IgG in serums, in the whole resolubilized cryoprecipitates, and in fractions obtained by sucrose gradient centrifugation, were quantitated by radial immunodiffusion in 1% agarose, veronal buffer, pH 8 6, using WHO standards (Meloy) and immunoglobulin class-specific rabbit antisera (Mancini, Carbonara & Heremans, 1965; Fahey & McKelvey, 1965). Determinations made at 25°C and 37°C were identical. Anti-IgG antibody Anti-IgG in serum and cryoprecipitates was determined by haemagglutination of chromic chloride-sensitized type 0 human erythrocytes coated with human y-globulin (Cohn Fraction II). The assay was performed at 4°C and 25°C; titres were usually 1-2 dilutions lower at the warmer temperature. The results are expressed as the log2 of the reciprocal of the haemagglutination titre.
Lymphocytes These were obtained by initially incubating heparinized whole venous blood on nylon wool columns (0-1 g of nylon wool/ml of blood) at 37°C for 30 min to remove phagocytic cells. The cells were eluted from the column with Hanks's balanced salt solution. Lymphocytes were obtained by flotation on Ficoll-Hypaque (Boyum, 1968). The final cell population obtained consisted of 75-90%o sheep erythrocyte rosette-forming cells (T cells) and 525% aggregated IgG-binding or immunoglobulin-bearing cells (B cells).
Antilymphocyte antibodies in SLE cryoprecipitates
401
Microcytotoxicity assay A modification of the two-stage microdroplet method was used (Terasaki & McClelland, 1964; Wernet, Feizi & Kunkel 1972). Rabbit serum (Pel Freeze Biologicals, Rogers, Arkansas) served as a complement source. Assays were routinely performed at 15'C. Immunofluorescent studies These were performed as previously described (Fu, Winchester & Kunkel, 1974; Winfield et al., 1974). Briefly, 5 x 105 PBL in 0-025 ml of HBSS were incubated at 40C for 30 min with 0-025 ml of serum or solubilized cryoprecipitate. The cells were washed three times in 2% BSA in PBS, pH 7-4, containing sodium azide. Class-specific rhodamineconjugated rabbit antibody to human IgM or IgG was then added and the cells were incubated for 30 min at 40C and washed three times as before. Slides were examined as wet mounts with incident illumination.
Differential absorption experiments Certain SLE and non-SLE cryoprecipitates, or their immunoglobulin fractions, were absorbed with human thymus or peripheral blood lymphocytes, erythrocytes, or an IgGagarose immunoabsorbant to determine antibody specificity. The cryoprecipitates, resolubilized by incubation at 370C, were incubated at 40C for 30 min with an equal volume of packed cells or IgG-agarose. After centrifugation, IgG and IgM concentration, cytotoxic antibody, and anti-IgG titre were determined in absorbed and sham-absorbed samples. RESULTS Cryoprecipitates from seven patients with SLE and eight with other diseases: monoclonal 'mixed' cryoglobulinaemia, Sjogren's syndrome, chronic active hepatitis and hyperlipidaemia-were examined. Using the microcytotoxicity assay, antilymphocyte antibody was found in five out of seven SLE cryoprecipitates and in one cryoprecipitate from a patient with Sjogren's syndrome. The presence of antibody in the cryoprecipitates was associated with similar antibody in the serum in each SLE case. The antilymphocyte antibody in the cryoprecipitates was cold reactive in the same fashion as the serum antibody described previously (Winfield et al., 1974). It was limited to the 19S fractions obtained by sucrose density gradient centrifugation (Fig. 1). Association of antilymphocyte activity with IgM was confirmed by indirect immunofluorescence experiments using class-specific, rhodamine-conjugated antiserums. Incubation of normal PBL with cytotoxic SLE cryoprecipitates resulted in bright staining of a high percentage of cells (56-100%) with anti-IgM. Such was not observed with the non-cytotoxic SLE cryoglobulins, which gave the expected background of 5-25% positive cells. Staining with anti-IgG was similarly low for all cryoprecipitates, approximating the number of B cells in the lymphocyte preparations used. The quality of the staining resembled that seen with immune complexes bound to the Fc receptor on the B cells (Winchester et al., 1974). Sixty-one per cent of PBL were stained with anti-IgM after incubation with a non-cytotoxic cryoprecipitate from one patient with Sjogren's syndrome. None of the other non-SLE cryoprecipitates gave indirect IgM staining of PBL above the background levels. Specific enrichment of IgM antilymphocyte antibody in the SLE cryoprecipitates was
J. B. Winfield et al.
402 o
_11
_ 100 (0
( a10
100
80
_8 -
80
_8
60
6
60 -6a
40
4
40
~~~~2
20
E~~~~~20
27 I2
Negativee
i (b) tate.
relavNegative
_j f07\
G
0 p7
I-0
i
/e
L-r
I-
E
obFIG 1.Lclzto fatlmhct nioy
0
0 0
-0-5
0-5
0
.-2
igM
n
niIGt
h 19S9
rcino
L
0-3
0-3
~~19 S0
c5
0.1
0.1
25
50
75
100
25
50
75
100
Percentage of gradient volume
FIG. 1. Localization of antilymphocyte antibody and anti-IgG to the 19S fraction of SLE cryoprecipitates fractionated by sucrose gradient centrifugation at acid pH. (a) Esp cryoprecipitate. (b) Harv cryoprecipitate. Neither antibody was present in the 7S fraction. The relative of antilymphocyte antibody and anti-IgG vary in these cryoprecipitates. tB ade proportion o
based upon increased cytotoxic activity per milligram of cryo-IgM relative to serum IgM (Table 1). Antibody activity in the cryoprecipitates was increased from 3-37 times over that of serum. Cytotoxic activity was similar in preparations isolated under conditions dissociain the unfractionated cryoof ting immune complexes. Thus for patient Esp, 0e023 mglgM of killed fraction the isolated in 19S mg 0c03 and 50% the test PBL in the microprecipitate This of test. enrichment antilymphocyte activity was also demonstrated by cytotoxicity immunofluorescence indirect experiments in which dilutions of the cryoprecipitates were added to PBL. Certain SLE cryoprecipitates (Esp, Ite) were absorbed with PBL, human thymus lymphocytes, and human erythrocytes to estimate how much of the IgM in the cryoprecipitate was antilymphocyte antibody. Absorption with lymphocytes in the cold removed 41-45%/ of the IgM and more than 90o. of the cytotoxic activity. Erythrocytes removed less IgM (7-12%) and very little antilymphocyte cytotoxic activity (8-14%). The IgG concentration was essentially unchanged after absorption with either lymphocytes or erythrocytes. Five of six SLE cryoprecipitates also contained IgM with anti-IgG activity. Specific enrichment of this 1gM rheumatoid factor also was demonstrated (Table 2). However, in contrast to cytotoxic activity which was equivalent for the IgM in unfractionated cryo-
Antilymphocyte antibodies in SLE cryoprecipitates TABLE 1. Specific enrichment of antilymphocyte antibody in SLE precipitates
CTX5o* (mg of IgM/ml)
Subject
Sam Esp Ite Harv Dep Abb Gross
403
cryo-
Serum
Cryoprecipitate
Relative enrichment of lymphocytotoxicity in cryoprecipitate
0.60 0 60 0 70 2-20 0-78
0-016 0-023 0 07 0-25 0-22
37 26 10 9 3
Negative Negative
Negativet Negativet
* CTX50 is the concentration of IgM killing 50%. of PBL in the microcytotoxicity assay.
t Cryoprecipitate
was not
cytotoxic at
a
protein concentration of 0 50
mg/ml. TABLE 2. Enrichment of anti-IgG activity in SLE cryoprecipitates
Anti-IgG haemagglutination titre (1/log2)
Subject Ite Esp Dep Harv
Serum IgM* 0 1 2 2
Relative enrichment of anti-IgG activity in
Cryo-IgM
cryoprecipitate
0 5 7 10
16 32 256
* Based on IgM, 0 1 mg/ml isolated by sucrose density gradient centrifugation at pH 3-5.
precipitates and that isolated under conditions dissociating immune complexes, anti-IgG activity was considerably increased when IgM was isolated at acid pH. Thus for Dep, the anti-IgG titre for 0.1 mg IgM/ml in the unfractionated cryoglobulin was 2; the titre of the dissociated IgM (0 1 mg/ml) was 7. Differential absorptions with lymphocytes and an IgG-agarose immunoabsorbent of such IgM fractions from both SLE and non-SLE cryoprecipitates further documented the presence of separate anti-IgG and antilymphocyte antibodies (Table 3). Lymphocytes removed cytotoxic activity from the SLE material, but did not reduce the anti-IgG titre. Anti-IgG antibody was absorbed from both types of cryoprecipitates by insoluble IgG without decreasing antilymphocyte antibody. Eighty-four per cent of the IgM was removed by absorption with insoluble IgG from a non-SLE cryoprecipitate (Mil); 46% was absorbed from an SLE cryoprecipitate (Harv).
J. B. Winfield et al.
404
TABLE 3. Presence of distinct IgM anti-IgG and antilymphocyte antibodies in SLE cryoprecipitate
Anti-IgG titre (1/log2) Absorption
SLE cryoprecipitate Esp
None Lymphocytes IgG-agarose
6 6 0
Non-SLE
Harv McD
11 10 0
8 7 0
Lymphocytotoxicity (% cells killed) SLE cryoprecipitate
Non-SLE
Mil
Esp
Harv
McD
Mil
6 6 0
56
