Immunology 1977 33 109
The effect of sera from patients with connective tissue diseases on red cell binding and phagocytosis by monocytes
ANNE TEMPLE* & G. LOEWI MRC Rheumatism Unit, Canadian Red Cross Memorial Hospital, Taplow, Maidenhead, Berkshire
Received 4 November 1976; acceptedfor publication 9 December 1976
Summary. The uptake by human blood monocytes of sheep erythrocytes treated with rabbit anti-sheep antibody with or without mouse complement was assessed by a radioactive method to discover whether immune complexes would inhibit this reaction. It was found that sera from SLE patients inhibited uptake whereas normal sera enhanced. Some rheumatoid sera and rheumatoid joint fluids inhibited uptake whereas sera from juvenile rheumatoid patients did not.
pig macrophages can be inhibited by the presence of immune complexes. A system using human cells for the detection of immune complexes in human patients would appear to have advantages over an animal model. In the present experiments we have attempted to demonstrate whether human sera with a putative content of complexes can be detected by competitive inhibition of the uptake of radio-labelled sheep erythrocytes by human monocytes. Some sera and joint fluids caused inhibition, whereas others produced enhancement, as was reported by Onyewotu, Johnson, Johnson & Holborow (1975). Monocytes from rheumatoid patients were studied to determine whether the Fc or C3 receptors were blocked, and this was found not to be the case.
INTRODUCTION
Various methods for the detection of immune complexes in blood and joint fluids have been studied. In 1971, Agnello, Koffler, Eisenberg, Winchester & Kunkel used a Clq precipitin test using sera from systemic lupus erythematosus patients, and Winchester, Kunkel & Agnello (1971) used monoclonal rheumatoid factors as reagents for the detection of y-globulin complexes in the serum and joint fluid of rheumatoid arthritis patients. These reagents are difficult to obtain or prepare. Onyewotu, Holborow & Johnson (1974) reported a test in which the uptake of radio-labelled aggregated human IgG by guinea-
MATERIALS AND METHODS Monocyte preparation Twenty-five ml of heparinized human blood was sedimented with an equal volume of plasmagel (Roger Bellon, France). The supernatant plasma was centrifuged on a Ficoll-Triosil gradient and the interface cell layer was washed 3 times in tissue culture medium 199 containing HEPES buffer. Total and differential counts were made. If polymorphonuclear cells exceeded 1 %, the cells were discarded; otherwise they were resuspended at a concentration of 15 x 106 cells/ml in medium 199 containing 40%
Correspondence: Dr G. Loewi, Immunological Medicine, Clinical Research Centre, Watford Road, Harrow, Middlesex. * Present address: Division of Cell Pathology, Clinical Research Centre, Harrow, Middlesex.
109
110
Anne Temple and G. Loewi
foetal calf serum (FCS) (Flow Labs., Irvine, Scotland). Volumes of 0 1 ml were carefully smeared on glass cover slips which had been cleaned in 7X detergent (George T. Gurr, London) and dipped in I % bovine serum albumin before the final rinse. The cover slips were then incubated in a pre-warmed moist chamber for 20 min when the supernatant cells were removed by vigorous washing with medium 199 using a pasteur pipette. The cells adherent to the cover slip were then used in the assay for red cell binding and phagocytosis. Staining with Leishman showed that >95%o of the cells remaining on the cover slip could phagocytose antibody-coated erythrocytes within 1 h.
Erythrocyte coating with antibody and complement (EA and EAC) (a) Labelling with 5' Cr: Sheep erythrocytes (E) were washed and resuspended in medium 199 containing 4%O FCS at an approximate concentration of 2 x 109/ml. To 0-1 ml E was added 100 pCi IICr labelled sodium chromate (Radiochemical Centre, Amersham). After incubation at 37'C for 45 min the cells were washed 3 times in medium 199. (b) Antibody and complement coating: Sheep red cells labelled with 'ICr were incubated at 370C for 30 min in rabbit anti-sheep haemolysin (Wellcome Reagents, Beckenham) at a dilution of 1:500 and then washed 3 times (EA). An aliquot was then reincubated in mouse serum (C57 Bl or CBA mice) at a dilution of 1:15 at 370C for 15 min and then washed 3 times at 40C (EAC). The erythrocyte preparations were counted in a haemocytometer and the cell concentration adjusted to 2 x 106 cells per 0-2 ml for the assay. Adherence and phagocytosis assay Monolayers of monocytes on cover slips were covered with 0 2 ml 5'Cr labelled sheep cells, EA or EAC and incubated in a moist prewarmed chamber for 1 h. The cover slips were then placed in small petri dishes containing 4 ml medium 199 and washed for 2 min with slow rocking. The supernatant was removed and retained, and the cells were washed twice with 3 ml medium 199. The supernatants from each cover slip were pooled (sample A). The cover slips were then placed in small dishes containing medium 199, 20%Y fresh human serum and a further 1:250 anti-sheep haemolysin for 10 min to lyse all adherent SRC. Two further washes were done and the seuprnatants pooled (sample B). Each cover slip
was then placed in a tube (sample C). Radioactivity of all samples was counted. The total radioactivity in samples A + B + C was equivalent to the activity on 2 x 106 SRC. Sample B gave the proportion of adherent cells and sample C the phagocytosed cells. All tests were performed in duplicate.
Inhibition and enhancement of adherence and phagocytosis EA or EAC were suspended at a concentration of 5 x 106 per 0-45 ml to which was added 0 05 ml of the serum or joint fluid to be tested, giving a 10% serum concentration. The joint effusions were aspirated for therapeutic reasons. Monolayers were covered with 0 2 ml of the suspension. Tests were performed in duplicate and repeated at least once using a different monocyte donor. Inhibition or enhancement was calculated by comparing the results with those obtained in the absence of added serum. T-paired statistics were applied.
RESULTS An initial experiment was done to test the reproducibility of monocyte monolayers by labelling peripheral white cells with 5'Cr before Ficoll separation and incubating ten cover slips with 0-1-ml aliquots of cells. After vigorous washing the radioactivity of eight monolayers was within 6% of the mean and the others were 8% and 9 %Y respectively. Monocyte recovery after Ficoll separation was found to vary widely (15-50% of total mononuclear cells) and the actual number of monocytes on the cover slip varied between experiments. It was therefore necessary to use normal serum in each inhibition experiment and only to compare results within an experiment. The highest concentration of antibody that could reliably be used was 1:500 because sheep erythrocytes agglutinated at higher concentrations. When a SRC concentration of 2 x 107 was used for uptake studies, a greater number of E was bound but it was considered that inhibition might be easier to achieve at sub-optimal concentrations, so 2 x 106 sheep cells were used. Uncoated sheep cells showed very little binding to monocytes. The numbers of sheep cells bound varied widely between experiments. Fig. 1 shows the distribution of cells phagocytosed or adherent to monocytes using EA alone or EAC. EAC bound to monocytes 10 times more avidly than did EA.
III
Effect of sera from SLE patients on red cell binding lxlO6
r
'-I
*I S
r (f) 0
1*
X 105
GI
4.
E z
eV .A .
0D
.0
lx
lo Adh.
Phag. - Complement
Phag.
Adh.
+ Complement
Figure 1. Distribution of the binding of antibody and complement coated sheep erythrocytes by normal monocytes. Each point represents the mean of duplicate results in one experiment. The dotted line is the mean for the whole group of experiments.
lX106 r
030 *0
If_
*:
6@
u
en
x
105
0
00* 0
*0
-
0
6,
E
.9 z
l X 104
Normals
RA + JRA
Jt.
fluids RA
Normals
RA + JRA
Jt.
fluids RA
+ Complement -Complement Figure 2. Binding of antibody and complement coated sheep erythrocytes by monocytes from patients with rheumatoid arthritis and Juvenile rheumatoid arthritis. Each point represents the mean of duplicate results in one experiment. The dotted line is the mean for the whole group of experiments.
Anne Temple and G. Loewi
112
controls, so results are expressed as total E numbers bound. Cells from a few rheumatoid joint effusions were also tested in this system to determine whether their receptors were blocked. Joint effusions usually contained more adherent cells than an equal volume of Ficoll-separated blood, and many monolayers were discarded because they contained polymorpho-
Monocytes from twelve patients with sero-positive
rheumatoid arthritis (RA) or juvenile rheumatoid arthritis (JRA) were compared with normal controls to determine whether the Fc and C3 receptors gave normal binding of sheep cells (Fig. 2). No difference could be found either in binding or in the ratio of phagocytosis to adherence between patients and Ix
107r
Ix
16 F _
-I_
11
cr
U)
0 0)
X
105
E
I
-4--
--7--
I 0
z
x
0~~~~~~~~~~
104
Cells Without serum
+ 10% Normal serum
4-10% SLE serum
+ 10% RA serum
+ 10% JRA serum
+ 10% Jtfluids
Figure 3. Inhibition and enhancement of the binding of antibody-coated sheep erythrocytes by serum from SLE, rheumatoid arthritis and Juvenile rheumatoid arthritis. Experiments were performed in 10% fresh serum or joint fluid. Each point represents the mean of duplicate results in one experiment. The dotted line is the mean for the whole group of experiments.
Table 1. T-paired statistics on EA uptake by normal monocytes in the presence of normal or patients' sera Significance of results when compared to Total no
Additions toofsr of sera itiCr to
10% normal serum
10
binding by monocytes without serum additions 0-025
The effect of sera from patients with connective tissue diseases on red cell binding and phagocytosis by monocytes
ANNE TEMPLE* & G. LOEWI MRC Rheumatism Unit, Canadian Red Cross Memorial Hospital, Taplow, Maidenhead, Berkshire
Received 4 November 1976; acceptedfor publication 9 December 1976
Summary. The uptake by human blood monocytes of sheep erythrocytes treated with rabbit anti-sheep antibody with or without mouse complement was assessed by a radioactive method to discover whether immune complexes would inhibit this reaction. It was found that sera from SLE patients inhibited uptake whereas normal sera enhanced. Some rheumatoid sera and rheumatoid joint fluids inhibited uptake whereas sera from juvenile rheumatoid patients did not.
pig macrophages can be inhibited by the presence of immune complexes. A system using human cells for the detection of immune complexes in human patients would appear to have advantages over an animal model. In the present experiments we have attempted to demonstrate whether human sera with a putative content of complexes can be detected by competitive inhibition of the uptake of radio-labelled sheep erythrocytes by human monocytes. Some sera and joint fluids caused inhibition, whereas others produced enhancement, as was reported by Onyewotu, Johnson, Johnson & Holborow (1975). Monocytes from rheumatoid patients were studied to determine whether the Fc or C3 receptors were blocked, and this was found not to be the case.
INTRODUCTION
Various methods for the detection of immune complexes in blood and joint fluids have been studied. In 1971, Agnello, Koffler, Eisenberg, Winchester & Kunkel used a Clq precipitin test using sera from systemic lupus erythematosus patients, and Winchester, Kunkel & Agnello (1971) used monoclonal rheumatoid factors as reagents for the detection of y-globulin complexes in the serum and joint fluid of rheumatoid arthritis patients. These reagents are difficult to obtain or prepare. Onyewotu, Holborow & Johnson (1974) reported a test in which the uptake of radio-labelled aggregated human IgG by guinea-
MATERIALS AND METHODS Monocyte preparation Twenty-five ml of heparinized human blood was sedimented with an equal volume of plasmagel (Roger Bellon, France). The supernatant plasma was centrifuged on a Ficoll-Triosil gradient and the interface cell layer was washed 3 times in tissue culture medium 199 containing HEPES buffer. Total and differential counts were made. If polymorphonuclear cells exceeded 1 %, the cells were discarded; otherwise they were resuspended at a concentration of 15 x 106 cells/ml in medium 199 containing 40%
Correspondence: Dr G. Loewi, Immunological Medicine, Clinical Research Centre, Watford Road, Harrow, Middlesex. * Present address: Division of Cell Pathology, Clinical Research Centre, Harrow, Middlesex.
109
110
Anne Temple and G. Loewi
foetal calf serum (FCS) (Flow Labs., Irvine, Scotland). Volumes of 0 1 ml were carefully smeared on glass cover slips which had been cleaned in 7X detergent (George T. Gurr, London) and dipped in I % bovine serum albumin before the final rinse. The cover slips were then incubated in a pre-warmed moist chamber for 20 min when the supernatant cells were removed by vigorous washing with medium 199 using a pasteur pipette. The cells adherent to the cover slip were then used in the assay for red cell binding and phagocytosis. Staining with Leishman showed that >95%o of the cells remaining on the cover slip could phagocytose antibody-coated erythrocytes within 1 h.
Erythrocyte coating with antibody and complement (EA and EAC) (a) Labelling with 5' Cr: Sheep erythrocytes (E) were washed and resuspended in medium 199 containing 4%O FCS at an approximate concentration of 2 x 109/ml. To 0-1 ml E was added 100 pCi IICr labelled sodium chromate (Radiochemical Centre, Amersham). After incubation at 37'C for 45 min the cells were washed 3 times in medium 199. (b) Antibody and complement coating: Sheep red cells labelled with 'ICr were incubated at 370C for 30 min in rabbit anti-sheep haemolysin (Wellcome Reagents, Beckenham) at a dilution of 1:500 and then washed 3 times (EA). An aliquot was then reincubated in mouse serum (C57 Bl or CBA mice) at a dilution of 1:15 at 370C for 15 min and then washed 3 times at 40C (EAC). The erythrocyte preparations were counted in a haemocytometer and the cell concentration adjusted to 2 x 106 cells per 0-2 ml for the assay. Adherence and phagocytosis assay Monolayers of monocytes on cover slips were covered with 0 2 ml 5'Cr labelled sheep cells, EA or EAC and incubated in a moist prewarmed chamber for 1 h. The cover slips were then placed in small petri dishes containing 4 ml medium 199 and washed for 2 min with slow rocking. The supernatant was removed and retained, and the cells were washed twice with 3 ml medium 199. The supernatants from each cover slip were pooled (sample A). The cover slips were then placed in small dishes containing medium 199, 20%Y fresh human serum and a further 1:250 anti-sheep haemolysin for 10 min to lyse all adherent SRC. Two further washes were done and the seuprnatants pooled (sample B). Each cover slip
was then placed in a tube (sample C). Radioactivity of all samples was counted. The total radioactivity in samples A + B + C was equivalent to the activity on 2 x 106 SRC. Sample B gave the proportion of adherent cells and sample C the phagocytosed cells. All tests were performed in duplicate.
Inhibition and enhancement of adherence and phagocytosis EA or EAC were suspended at a concentration of 5 x 106 per 0-45 ml to which was added 0 05 ml of the serum or joint fluid to be tested, giving a 10% serum concentration. The joint effusions were aspirated for therapeutic reasons. Monolayers were covered with 0 2 ml of the suspension. Tests were performed in duplicate and repeated at least once using a different monocyte donor. Inhibition or enhancement was calculated by comparing the results with those obtained in the absence of added serum. T-paired statistics were applied.
RESULTS An initial experiment was done to test the reproducibility of monocyte monolayers by labelling peripheral white cells with 5'Cr before Ficoll separation and incubating ten cover slips with 0-1-ml aliquots of cells. After vigorous washing the radioactivity of eight monolayers was within 6% of the mean and the others were 8% and 9 %Y respectively. Monocyte recovery after Ficoll separation was found to vary widely (15-50% of total mononuclear cells) and the actual number of monocytes on the cover slip varied between experiments. It was therefore necessary to use normal serum in each inhibition experiment and only to compare results within an experiment. The highest concentration of antibody that could reliably be used was 1:500 because sheep erythrocytes agglutinated at higher concentrations. When a SRC concentration of 2 x 107 was used for uptake studies, a greater number of E was bound but it was considered that inhibition might be easier to achieve at sub-optimal concentrations, so 2 x 106 sheep cells were used. Uncoated sheep cells showed very little binding to monocytes. The numbers of sheep cells bound varied widely between experiments. Fig. 1 shows the distribution of cells phagocytosed or adherent to monocytes using EA alone or EAC. EAC bound to monocytes 10 times more avidly than did EA.
III
Effect of sera from SLE patients on red cell binding lxlO6
r
'-I
*I S
r (f) 0
1*
X 105
GI
4.
E z
eV .A .
0D
.0
lx
lo Adh.
Phag. - Complement
Phag.
Adh.
+ Complement
Figure 1. Distribution of the binding of antibody and complement coated sheep erythrocytes by normal monocytes. Each point represents the mean of duplicate results in one experiment. The dotted line is the mean for the whole group of experiments.
lX106 r
030 *0
If_
*:
6@
u
en
x
105
0
00* 0
*0
-
0
6,
E
.9 z
l X 104
Normals
RA + JRA
Jt.
fluids RA
Normals
RA + JRA
Jt.
fluids RA
+ Complement -Complement Figure 2. Binding of antibody and complement coated sheep erythrocytes by monocytes from patients with rheumatoid arthritis and Juvenile rheumatoid arthritis. Each point represents the mean of duplicate results in one experiment. The dotted line is the mean for the whole group of experiments.
Anne Temple and G. Loewi
112
controls, so results are expressed as total E numbers bound. Cells from a few rheumatoid joint effusions were also tested in this system to determine whether their receptors were blocked. Joint effusions usually contained more adherent cells than an equal volume of Ficoll-separated blood, and many monolayers were discarded because they contained polymorpho-
Monocytes from twelve patients with sero-positive
rheumatoid arthritis (RA) or juvenile rheumatoid arthritis (JRA) were compared with normal controls to determine whether the Fc and C3 receptors gave normal binding of sheep cells (Fig. 2). No difference could be found either in binding or in the ratio of phagocytosis to adherence between patients and Ix
107r
Ix
16 F _
-I_
11
cr
U)
0 0)
X
105
E
I
-4--
--7--
I 0
z
x
0~~~~~~~~~~
104
Cells Without serum
+ 10% Normal serum
4-10% SLE serum
+ 10% RA serum
+ 10% JRA serum
+ 10% Jtfluids
Figure 3. Inhibition and enhancement of the binding of antibody-coated sheep erythrocytes by serum from SLE, rheumatoid arthritis and Juvenile rheumatoid arthritis. Experiments were performed in 10% fresh serum or joint fluid. Each point represents the mean of duplicate results in one experiment. The dotted line is the mean for the whole group of experiments.
Table 1. T-paired statistics on EA uptake by normal monocytes in the presence of normal or patients' sera Significance of results when compared to Total no
Additions toofsr of sera itiCr to
10% normal serum
10
binding by monocytes without serum additions 0-025
