- Email: [email protected]
The identification of Fc and C3 receptors on human neutrophils
Journal of Immunological Methods 7 (1975) 69 76 © North-Holland Publishing Company
THE IDENTIFICATION
O F F c A N D C3 R E C E P T O R S O N H U M A N
NEUTROPHILS L. WONG and J.D. WILSON Section of Immunology, Department of Medicine and Department of Pathology, University of Auckland School of Medicine, A uekland, New Zealand
Received 24 September 1974,
accepted 10 October 1974
Rosette techniques are described for demonstrating receptors on human neutrophils for the 1,"c portion of immunoglobulin and the third component of complement C3. The two receptors are each present on up to 90% of neutrophils and optimal conditions have been established where binding occurs to one receptor but not to the other.
1. Introduction Human neutrophils have receptors on their membrane for the F c portion of immunoglobulin molecules (Henson, 1969; Messner and Jelinek, 1970) and receptors for the third component of complement-C3 (Henson, 1969; Pincus et al., 1972; Eden et al., 1973; Ross et al., 1973). There is tittle known of the role of these receptors in neutrophil physiology or pathology. As part of a study of this problem we have established optimal experimental conditions for the detection of F c and C3 receptors using rosette assays where the assay for one receptor will not detect binding to the other receptor. F c receptor is detected using red cells sensitized with antibody (EA) and C3 receptor by EAC 1423. Technical factors influencing the binding to the two receptors are described.
2. Materials and methods 2.1. Preparation o f two Ficoll Conray solutions
A and B
Solution A contained 11.2 g of Ficoll (mol. wt 400,000, Sigma), 20 ml of Conray 280 (Meglumine Iothalamate 60% w/v, May and Baker Ltd.) in 100 ml of distilled water. Solution B consisted of 7.2 g Ficoll, 20 ml of Conray 289, in 100 ml of distilled water. These solutions were kept at 4°C and used for up to a week after preparation. 69
70
L. Wong, ,I.D. Wilson, Fc and C3 receptors on neutrophils
2.2. Preparation o f polymorphonuclcar leucocyte suspensions Neutrophils were separated from whole blood by a modification of the method o f Boyum (1968). Venous blood was collected into heparinised Vacutainers. Ten ml of blood was gently layered onto 10 ml of F i c o l l - C o n r a y solution B in a universal container and the red cells were allowed to settle through the gradient over 50 60 rain. The leucocyte-rich layer was removed and layered onto a further double gradient in a plastic centrifuge tube. This gradient consisted of 3 ml of solution B layered onto 3 ml of solution A so that a distinct interface was formed between them. The tube was centrifuged at 1500 g for 15 min at 4°C. A neutrophil layer formed at the interface between the two F i c o l l - C o n r a y solutions, and lymphocytes and monocytes accumulated in a layer at the interface between the plasma and solution B. Red cells and eosinophils were spun to a pellet at the b o t t o m of the tube. The neutrophil layer was collected and washed twice in Earle's Balanced Salt Solution (Wellcome Reagents Ltd.) which had been buffered to pH 7.2 with HEPES (N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid). The cells were resuspended in the same solution to a concentration of 3 million per ml and kept at 4°C until used. 2.3. Sheep red blood cells (k7 Sheep blood was collected into Alsever's solution and stored at 4°C. Before use the red cells were washed 4 - 5 times in complement fixation diluent (CFD Oxoid) and adjusted to a concentration of 2.5% or 5% as required. 2.4. Sensitized sheep red blood cells (EA ) One volume of 5% E was mixed with one volume of rabbit anti-sheep red cell antibody (LEE U.S.A.) which had been diluted in CFD to 8 X 50% haemolytic doses. Both reagents were brought to 37°C before mixing and were then incubated at 37°C for 30 min. The sensitized sheep cells were then washed twice in CFD and resuspended to a final concentration of 2.5%. 2.5. Preparation of" human R 3 reagent One hundred milligrams of zymosan (Sigma) was suspended in 20 ml of saline and boiled for 1 hr, the volume being maintained at 20 ml by addition of distilled water. One volume of the boiled zymosan suspension was mixed with an equal volume of fresh human serum and incubated at 37°C for 1 hr with periodic agitation to keep the zymosan in suspension. The mixture was then centrifuged and the supernatant (R3) was removed and stored at 20°C until use. Human R3 prepared in this manner retains considerable C3 activity but shows more or less total depletion of C5 (Lachmann et al., 1973) and hence is non-haemolytic.
L. Wong, J.D. Wilson, F c and C3 receptors on neutrophils
71
2.6. Preparation o.f complement f i x e d EA (EA C) Human EAC 1423 was prepared according to the method o f Lachmann et al. (1973). Two millilitres o f 2.5% EA mixed with 0.5 ml of human R3 was shaken continuously at 37°C for 75 sec and then 0.1 ml o f Antrypol (Sumarin.BP, Bayer 205) solution (50 mg in 1.0 ml of distilled water) was added and the suspension shaken for a further 2 min at 37°C. The cells were washed twice in ice cold CFD and finally made up to 2.0 ml in CFD. The cells were kept at 4°C until use. 2. Z Preparation o f rosettes For the following experiments 0.25 ml o f the neutrophit suspension (3 million per ml) was mixed with 0.25 ml of 2.5% EA or EAC suspension in plastic conical centrifuge tubes. After preparation of EA or EAC rosettes a sample of the suspension was placed on a microscope slide and examined through a cover slip. A minimum of 100 neutrophils was counted and 'the number forming rosettes was expressed as a percentage. A rosette was taken as a neutrophil with three or more red cells adhering to it. If a stained and permanent preparation was required a drop of foetal calf serum was added to a drop of the suspension and a thick film made on a slide, dried, and then stained with a Romanovsky stain. 3. Results
3.1. Preliminary experiments established that EAC rosettes would form spontaneously at 37 °C while EA rosettes would not and required centrifugation. 3.2. Teehnical ¢~lctors influencing EA-neutrophil rosettes a) Following centri(ugation of neutrophils and EA at room temperature the pellet was incubated for 15 rain at selected temperatures. 81% of the neutrophils formed rosettes at 0°C, 82% at room temperature (approx. 20°C), but only 4% formed rosettes at 37 °C. The results are the mean of four separate experiments. b) With the neutrophil-EA pellet held at 20°C the duration of incubation was examined and the incidence of rosette formation is shown in fig. 1. An incubation time of 15 min produced maximal rosette numbers. c) Centrifugation at 6 0 g for 5, 10 and 15 min did not affect the incidence of neutrophils forming rosettes in experiments done in duplicate, but a centrifugation time of 10 min produced rosettes with more attached EA per neutrophil. d) The effect of incubating the EA-neutrophil suspension at different temperatures before centrifugation is exmnined. Results are shown in table 1. Incubation on ice or at 37°C had an adverse effect on rosette formation compared with incubation at room temperature. However, if the cells were at room temperature before prepa-
72
L. Wong, J.D. Wilson, b~, and C3 receptors on neutrophils
100
90
80
70
6O
/
5C G,.
o ~c '5 < 30 klJ
20
10
0
5
10 15 20 25 Duration of ,ncubat,on (m,nutes)
30
60
120
Fig. 1. EA-neutrophil rosettes: the effect of duration of incubation of the centrifuged pellet. The cell suspension was centrifuged to a pellet at 60 g for 10 rain;
ration there was no advantage in holding them at that temperature before centrifugation. In seven experiments where the rosettes were centrifuged immediately after mixing 81% of neutrophils formed rosettes compared with an average of 76% if the suspension was held at room temperature for 5 or 10 rain following mixing but before centrifugation. e) The stability of the EA rosettes after their resuspension was examined at different temperatures. In each case the rosettes had been formed by centrifugation at 6 0 g for 10 rain at room temperature followed by incubation of the pellet for 15 rain at room temperature. Results are shown in the fig. 2. The rosettes were stable on ice and at room temperature but at 37°C the rosettes rapidly disrupted. f) When non-sensitized sheep red cells were substituted for EA red cells no rosettes were formed under optimal conditions in a series of four experiments. g) Optimal conditions for the formation of EA-neutrophil rosettes were achieved by mixing 0.25 ml of the neutrophil suspension with 0.25 ml of the EA suspension
L. Wong, J.D. Wilson, b c and C3 receptors on neutrophils
73
Table 1 Effect on rosette formation of incubating EA-neutrophil suspensions at various temperatures before centrifugation. Duration of incubation (min)
Temperature
5 10 10 10
30 32 44 59
71 77 76 80
26 37 48 51
Average
41
76
41
Ice (%)
Room (%)
37°C (%)
After incubation the suspension was centrifuged at 60 g lbr 10 min and the pellet incubated at room temperature for 15 rain.
100
9o
• •
.--':
.............
A
•
.........
..,$
-
',
•
. v
, .II'"
70
•
6O
50
o
37 ° RT
o •
.......
ICE
•
---
40 .c c3. £ o °
,x w 20
10
5
10 15 20 25 30 Duration of incubation (minutes)
60
120
Fig. 2. The stability of EA-neutrophil rosettes at different temperatures. The cell suspension was centrifuged for 10 min at 6 0 g , incubated at room temperature for 15 min then resuspended and incubated.
74
L. Wong, J.D. Wilson, F c and C3 receptors on neutrophils
100
90
80
70
6O
2 =
50
o '5 ~: z,0
u.J 3O
20
10
o
W
2~ 3'0 ~ ~s 20 Duration of incubation {rnmdtes )
60
12fl
Fig. 3. Tile effect of incubation at 37°C on formation of EAC-neutrophil rosettes.
in a plastic conical centrifuge tube and, without preincubation, centrifuging at room temperature for 10 min at 60g. The centrifuged pellet was then allowed to stand at room temperature for 15 min, and completely but gently resuspended for examination. 3.3. Technical j i w t o r s i n f l u e n c i n g E A C - n e u t r o p h i l r o s e t t e s
a) EAC-neutrophil suspensions were incubated at different temperatures for 10 min. In a series of six experiments 8% of neutrophils formed rosettes at 4°C, 59% at room temperature and 90% at 37°C. b) The effect of time incubation at 37°C on rosette incidence is shown in fig. 3. It can be seen that an incubation time of 15 min is required for maximal rosette numbers. Phagocytosis was observed infrequently even after 2 hr incubation. c) Control experiments were performed to determine that the rosettes formed were complement-dependent. When EA was substituted for EAC, or when EAC were formed with heat inactivated R3 (R3 incubated at 56°C for 30 min) no rosettes formed during 10 rain incubation at 37 °C.
L. Wong, J.D. Wilson, F c and (2'3 receptors on neutrophils
75
d) Optimal conditions for the formation of EAC-neutrophil rosettes were obtained by mixing 0.25 ml of the neutrophil suspension with 0.25 ml of the EAC suspension in a plastic tube, incubating for 15 min at 37°C and then examining. After incubation the EAC neutrophil suspension was not allowed to stand at room temperature for more than a few minutes before examination (see Discussion).
4. Discussion Experimental conditions have been established for formation of EA and EAC rosettes by a maximal proportion of human neutrophils. Under optimal conditions up to 90% of neutrophils will form EA rosettes when none will form rosettes with unsensitized E. Similarly the best circumstances for EAC rosette formation demonstrate C3 receptors on 90% of neutrophils under conditions where EA, or EAC prepared with heat-inactivated R3 will not bind to the neutrophils. The techniques are simple and reproducible and provide tools for a closer examination of neutrophil function. Immunoglobulin-binding receptors (Fc) on human neutrophils have previously been demonstrated. Henson (1969) and Messner and Jelinek (1970) attached neutrophils to glass, exposed them to red cells sensitized with antibody and showed rosette formation between the two cell types. In these studies no attempt was made to establish conditions optimal for maximal rosette-formation. Henson (1969) also showed that EA binding occurred with both IgG and IgM antibody, but that the former was more effective. Jonas et al. (1965) and Huber et al. (1969) used both the glass-attached cell and a centrifugation suspension method to examine EA binding by a number of different cell types but not neutrophils. Huber et al. (1969) included incubation of the cells at 37°C, a temperature at which we have shown EA-neutrophil rosettes to be very unstable; Henson (1969) suggested that false union may occur with centrifugation, but in the present work substituting E for EA failed to produce rosettes. Instability of EA-neutrophil rosettes at 37°C may be a result of uncoupling of the immunoglobulin from the F c receptor, which could occur with shedding of the receptor into the medium or could arise from micropinocytosis of small pieces of the attached red cells, the remainder of the red cells then breaking away. Shedding of surface Ig or of specific antigen has been demonstrated from lymphocyte surface at 37°C (Cone et al., 1971; Wilson and Feldmann, 1972) and antigen-antibody complexes are also released from human lymphocyte surfaces but at a much slower rate than that at which EA are released from neutrophils (Pang and Wilson, 1974, unpublished). Micropinocytosis of pieces of red cells bound to monocytes through the F c receptor has been shown by Brown and Nelson (1973). Preliminary experiments with heat-aggregated human gammaglobulin and fluorescein-labelled antihuman Ig indicate that at 37°C some re-distribution of F c receptors occurs in some neutrophils with concentration of the aggregates towards a pseudopod at one pole of the cell.
76
L. WoJzg, J.D. Wilson, k c and C3 receptors on neutrophils
Human neutrophils have a receptor for the C3b but not C3d components of C3 (Eden et al., 1973; Ross et al., 1973). Serum contains an inactivator which converts C3b to C3d but the addition of Antrypol inhibits the inactivation of C3b (kachmann et al., 1973). Although EAC prepared without Anytrpol still produced good rosette numbers, presumably because inactivation of C3b is incomplete, EAC prepared with Antrypol and human R3 gave rosette numbers which were consistently about 5% higher. The use of purified complement components (Eden et al., 1973; Ross et al., 1973) avoids the presence of inactivator, but the use of human R3 and Antrypol is more economical. In a series of preliminary experiments whole mouse serum was used as a source of non-haemolytic complement in the preparation of EAC-neutrophil rosettes, but gave low and variable numbers. Pincus et al. (1972) found that EAC formed with mouse serum as a complement source bound almost entirely to mononuclear cells and not to neutrophils. Ross et al. (1973) suggested this was because the C3b was being converted to C3d by the inactivator present in the whole serum. Under the selected conditions lk~r preparation of EAC-neutrophil rosettes no rosettes formed with EA or EAC prepared with heat-inactivated R3 emphasizing that the C3 and F c receptors on human neutrophils are distinct entities. If however, EA-neutrophil suspension is allowed to stand without centrifugation for 1 hr at room temperature, occasional EA rosettes will form. If the EAC-neutrophil sample 3 ° C bath this complication is is read within a few nrinutes of taking from the _7 avoided. No attempt was made to separate the anti-sheep red blood cell antibody into lgG and IgM fractions, for the formation of EAC, but there is evidence that neutrophils have receptors for both (Henson, 1969).
References Boyum, A., 1968, J. Clin. Lab. Invest. Suppl. 21,97. Brown. D.C. and D.A. Nelson, t973, Brit. J. Ilaematol. 24, 301. Cone, R.k., J.J. Marchalonis and R.T. Rox~ley, 1971, J. l'xptl. Med. 134, 1373. Eden, A., G.W. Miller and V. Nussenzwcig, 1973, J. Clin. Invest. 52, 3239. llenson, P.M., 1969, hmnunology 16, 107. [tuber. tt., S.D. Douglas and H.It. Fudenberg, 1969. Immunology 17, 7. Jonas, W.I(., B.W. Gurner, D.S. Nelson and R.R.A. Coombe, 1965, Int. Arch. Allergy Appl. lmmunol. 28, 86. Lachmann, P.J., M.J. Hobart and W.P. Aston, 1973, in: Handbook of experimental immunology, ed. D.M. Weir, 2rid edition (Blackwell, Oxford) chapter 5, 5.1. Mcssner, R.P, and J. Jelinek, 19711, J. Clin. Invest. 49, 2165. Pincus. S., C. Bianco and V. Nussenzweig, 1972, Blood 40,303. Ross, G.D., M,J. Polley, F.M. Rabellino and ll.M. Grey, 1973, J. Exptl. Med. 138,798. Wilson, J.D. and M. Feldmann, 1972, Nature New Biol. 237, 3.
THE IDENTIFICATION
O F F c A N D C3 R E C E P T O R S O N H U M A N
NEUTROPHILS L. WONG and J.D. WILSON Section of Immunology, Department of Medicine and Department of Pathology, University of Auckland School of Medicine, A uekland, New Zealand
Received 24 September 1974,
accepted 10 October 1974
Rosette techniques are described for demonstrating receptors on human neutrophils for the 1,"c portion of immunoglobulin and the third component of complement C3. The two receptors are each present on up to 90% of neutrophils and optimal conditions have been established where binding occurs to one receptor but not to the other.
1. Introduction Human neutrophils have receptors on their membrane for the F c portion of immunoglobulin molecules (Henson, 1969; Messner and Jelinek, 1970) and receptors for the third component of complement-C3 (Henson, 1969; Pincus et al., 1972; Eden et al., 1973; Ross et al., 1973). There is tittle known of the role of these receptors in neutrophil physiology or pathology. As part of a study of this problem we have established optimal experimental conditions for the detection of F c and C3 receptors using rosette assays where the assay for one receptor will not detect binding to the other receptor. F c receptor is detected using red cells sensitized with antibody (EA) and C3 receptor by EAC 1423. Technical factors influencing the binding to the two receptors are described.
2. Materials and methods 2.1. Preparation o f two Ficoll Conray solutions
A and B
Solution A contained 11.2 g of Ficoll (mol. wt 400,000, Sigma), 20 ml of Conray 280 (Meglumine Iothalamate 60% w/v, May and Baker Ltd.) in 100 ml of distilled water. Solution B consisted of 7.2 g Ficoll, 20 ml of Conray 289, in 100 ml of distilled water. These solutions were kept at 4°C and used for up to a week after preparation. 69
70
L. Wong, ,I.D. Wilson, Fc and C3 receptors on neutrophils
2.2. Preparation o f polymorphonuclcar leucocyte suspensions Neutrophils were separated from whole blood by a modification of the method o f Boyum (1968). Venous blood was collected into heparinised Vacutainers. Ten ml of blood was gently layered onto 10 ml of F i c o l l - C o n r a y solution B in a universal container and the red cells were allowed to settle through the gradient over 50 60 rain. The leucocyte-rich layer was removed and layered onto a further double gradient in a plastic centrifuge tube. This gradient consisted of 3 ml of solution B layered onto 3 ml of solution A so that a distinct interface was formed between them. The tube was centrifuged at 1500 g for 15 min at 4°C. A neutrophil layer formed at the interface between the two F i c o l l - C o n r a y solutions, and lymphocytes and monocytes accumulated in a layer at the interface between the plasma and solution B. Red cells and eosinophils were spun to a pellet at the b o t t o m of the tube. The neutrophil layer was collected and washed twice in Earle's Balanced Salt Solution (Wellcome Reagents Ltd.) which had been buffered to pH 7.2 with HEPES (N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid). The cells were resuspended in the same solution to a concentration of 3 million per ml and kept at 4°C until used. 2.3. Sheep red blood cells (k7 Sheep blood was collected into Alsever's solution and stored at 4°C. Before use the red cells were washed 4 - 5 times in complement fixation diluent (CFD Oxoid) and adjusted to a concentration of 2.5% or 5% as required. 2.4. Sensitized sheep red blood cells (EA ) One volume of 5% E was mixed with one volume of rabbit anti-sheep red cell antibody (LEE U.S.A.) which had been diluted in CFD to 8 X 50% haemolytic doses. Both reagents were brought to 37°C before mixing and were then incubated at 37°C for 30 min. The sensitized sheep cells were then washed twice in CFD and resuspended to a final concentration of 2.5%. 2.5. Preparation of" human R 3 reagent One hundred milligrams of zymosan (Sigma) was suspended in 20 ml of saline and boiled for 1 hr, the volume being maintained at 20 ml by addition of distilled water. One volume of the boiled zymosan suspension was mixed with an equal volume of fresh human serum and incubated at 37°C for 1 hr with periodic agitation to keep the zymosan in suspension. The mixture was then centrifuged and the supernatant (R3) was removed and stored at 20°C until use. Human R3 prepared in this manner retains considerable C3 activity but shows more or less total depletion of C5 (Lachmann et al., 1973) and hence is non-haemolytic.
L. Wong, J.D. Wilson, F c and C3 receptors on neutrophils
71
2.6. Preparation o.f complement f i x e d EA (EA C) Human EAC 1423 was prepared according to the method o f Lachmann et al. (1973). Two millilitres o f 2.5% EA mixed with 0.5 ml of human R3 was shaken continuously at 37°C for 75 sec and then 0.1 ml o f Antrypol (Sumarin.BP, Bayer 205) solution (50 mg in 1.0 ml of distilled water) was added and the suspension shaken for a further 2 min at 37°C. The cells were washed twice in ice cold CFD and finally made up to 2.0 ml in CFD. The cells were kept at 4°C until use. 2. Z Preparation o f rosettes For the following experiments 0.25 ml o f the neutrophit suspension (3 million per ml) was mixed with 0.25 ml of 2.5% EA or EAC suspension in plastic conical centrifuge tubes. After preparation of EA or EAC rosettes a sample of the suspension was placed on a microscope slide and examined through a cover slip. A minimum of 100 neutrophils was counted and 'the number forming rosettes was expressed as a percentage. A rosette was taken as a neutrophil with three or more red cells adhering to it. If a stained and permanent preparation was required a drop of foetal calf serum was added to a drop of the suspension and a thick film made on a slide, dried, and then stained with a Romanovsky stain. 3. Results
3.1. Preliminary experiments established that EAC rosettes would form spontaneously at 37 °C while EA rosettes would not and required centrifugation. 3.2. Teehnical ¢~lctors influencing EA-neutrophil rosettes a) Following centri(ugation of neutrophils and EA at room temperature the pellet was incubated for 15 rain at selected temperatures. 81% of the neutrophils formed rosettes at 0°C, 82% at room temperature (approx. 20°C), but only 4% formed rosettes at 37 °C. The results are the mean of four separate experiments. b) With the neutrophil-EA pellet held at 20°C the duration of incubation was examined and the incidence of rosette formation is shown in fig. 1. An incubation time of 15 min produced maximal rosette numbers. c) Centrifugation at 6 0 g for 5, 10 and 15 min did not affect the incidence of neutrophils forming rosettes in experiments done in duplicate, but a centrifugation time of 10 min produced rosettes with more attached EA per neutrophil. d) The effect of incubating the EA-neutrophil suspension at different temperatures before centrifugation is exmnined. Results are shown in table 1. Incubation on ice or at 37°C had an adverse effect on rosette formation compared with incubation at room temperature. However, if the cells were at room temperature before prepa-
72
L. Wong, J.D. Wilson, b~, and C3 receptors on neutrophils
100
90
80
70
6O
/
5C G,.
o ~c '5 < 30 klJ
20
10
0
5
10 15 20 25 Duration of ,ncubat,on (m,nutes)
30
60
120
Fig. 1. EA-neutrophil rosettes: the effect of duration of incubation of the centrifuged pellet. The cell suspension was centrifuged to a pellet at 60 g for 10 rain;
ration there was no advantage in holding them at that temperature before centrifugation. In seven experiments where the rosettes were centrifuged immediately after mixing 81% of neutrophils formed rosettes compared with an average of 76% if the suspension was held at room temperature for 5 or 10 rain following mixing but before centrifugation. e) The stability of the EA rosettes after their resuspension was examined at different temperatures. In each case the rosettes had been formed by centrifugation at 6 0 g for 10 rain at room temperature followed by incubation of the pellet for 15 rain at room temperature. Results are shown in the fig. 2. The rosettes were stable on ice and at room temperature but at 37°C the rosettes rapidly disrupted. f) When non-sensitized sheep red cells were substituted for EA red cells no rosettes were formed under optimal conditions in a series of four experiments. g) Optimal conditions for the formation of EA-neutrophil rosettes were achieved by mixing 0.25 ml of the neutrophil suspension with 0.25 ml of the EA suspension
L. Wong, J.D. Wilson, b c and C3 receptors on neutrophils
73
Table 1 Effect on rosette formation of incubating EA-neutrophil suspensions at various temperatures before centrifugation. Duration of incubation (min)
Temperature
5 10 10 10
30 32 44 59
71 77 76 80
26 37 48 51
Average
41
76
41
Ice (%)
Room (%)
37°C (%)
After incubation the suspension was centrifuged at 60 g lbr 10 min and the pellet incubated at room temperature for 15 rain.
100
9o
• •
.--':
.............
A
•
.........
..,$
-
',
•
. v
, .II'"
70
•
6O
50
o
37 ° RT
o •
.......
ICE
•
---
40 .c c3. £ o °
,x w 20
10
5
10 15 20 25 30 Duration of incubation (minutes)
60
120
Fig. 2. The stability of EA-neutrophil rosettes at different temperatures. The cell suspension was centrifuged for 10 min at 6 0 g , incubated at room temperature for 15 min then resuspended and incubated.
74
L. Wong, J.D. Wilson, F c and C3 receptors on neutrophils
100
90
80
70
6O
2 =
50
o '5 ~: z,0
u.J 3O
20
10
o
W
2~ 3'0 ~ ~s 20 Duration of incubation {rnmdtes )
60
12fl
Fig. 3. Tile effect of incubation at 37°C on formation of EAC-neutrophil rosettes.
in a plastic conical centrifuge tube and, without preincubation, centrifuging at room temperature for 10 min at 60g. The centrifuged pellet was then allowed to stand at room temperature for 15 min, and completely but gently resuspended for examination. 3.3. Technical j i w t o r s i n f l u e n c i n g E A C - n e u t r o p h i l r o s e t t e s
a) EAC-neutrophil suspensions were incubated at different temperatures for 10 min. In a series of six experiments 8% of neutrophils formed rosettes at 4°C, 59% at room temperature and 90% at 37°C. b) The effect of time incubation at 37°C on rosette incidence is shown in fig. 3. It can be seen that an incubation time of 15 min is required for maximal rosette numbers. Phagocytosis was observed infrequently even after 2 hr incubation. c) Control experiments were performed to determine that the rosettes formed were complement-dependent. When EA was substituted for EAC, or when EAC were formed with heat inactivated R3 (R3 incubated at 56°C for 30 min) no rosettes formed during 10 rain incubation at 37 °C.
L. Wong, J.D. Wilson, F c and (2'3 receptors on neutrophils
75
d) Optimal conditions for the formation of EAC-neutrophil rosettes were obtained by mixing 0.25 ml of the neutrophil suspension with 0.25 ml of the EAC suspension in a plastic tube, incubating for 15 min at 37°C and then examining. After incubation the EAC neutrophil suspension was not allowed to stand at room temperature for more than a few minutes before examination (see Discussion).
4. Discussion Experimental conditions have been established for formation of EA and EAC rosettes by a maximal proportion of human neutrophils. Under optimal conditions up to 90% of neutrophils will form EA rosettes when none will form rosettes with unsensitized E. Similarly the best circumstances for EAC rosette formation demonstrate C3 receptors on 90% of neutrophils under conditions where EA, or EAC prepared with heat-inactivated R3 will not bind to the neutrophils. The techniques are simple and reproducible and provide tools for a closer examination of neutrophil function. Immunoglobulin-binding receptors (Fc) on human neutrophils have previously been demonstrated. Henson (1969) and Messner and Jelinek (1970) attached neutrophils to glass, exposed them to red cells sensitized with antibody and showed rosette formation between the two cell types. In these studies no attempt was made to establish conditions optimal for maximal rosette-formation. Henson (1969) also showed that EA binding occurred with both IgG and IgM antibody, but that the former was more effective. Jonas et al. (1965) and Huber et al. (1969) used both the glass-attached cell and a centrifugation suspension method to examine EA binding by a number of different cell types but not neutrophils. Huber et al. (1969) included incubation of the cells at 37°C, a temperature at which we have shown EA-neutrophil rosettes to be very unstable; Henson (1969) suggested that false union may occur with centrifugation, but in the present work substituting E for EA failed to produce rosettes. Instability of EA-neutrophil rosettes at 37°C may be a result of uncoupling of the immunoglobulin from the F c receptor, which could occur with shedding of the receptor into the medium or could arise from micropinocytosis of small pieces of the attached red cells, the remainder of the red cells then breaking away. Shedding of surface Ig or of specific antigen has been demonstrated from lymphocyte surface at 37°C (Cone et al., 1971; Wilson and Feldmann, 1972) and antigen-antibody complexes are also released from human lymphocyte surfaces but at a much slower rate than that at which EA are released from neutrophils (Pang and Wilson, 1974, unpublished). Micropinocytosis of pieces of red cells bound to monocytes through the F c receptor has been shown by Brown and Nelson (1973). Preliminary experiments with heat-aggregated human gammaglobulin and fluorescein-labelled antihuman Ig indicate that at 37°C some re-distribution of F c receptors occurs in some neutrophils with concentration of the aggregates towards a pseudopod at one pole of the cell.
76
L. WoJzg, J.D. Wilson, k c and C3 receptors on neutrophils
Human neutrophils have a receptor for the C3b but not C3d components of C3 (Eden et al., 1973; Ross et al., 1973). Serum contains an inactivator which converts C3b to C3d but the addition of Antrypol inhibits the inactivation of C3b (kachmann et al., 1973). Although EAC prepared without Anytrpol still produced good rosette numbers, presumably because inactivation of C3b is incomplete, EAC prepared with Antrypol and human R3 gave rosette numbers which were consistently about 5% higher. The use of purified complement components (Eden et al., 1973; Ross et al., 1973) avoids the presence of inactivator, but the use of human R3 and Antrypol is more economical. In a series of preliminary experiments whole mouse serum was used as a source of non-haemolytic complement in the preparation of EAC-neutrophil rosettes, but gave low and variable numbers. Pincus et al. (1972) found that EAC formed with mouse serum as a complement source bound almost entirely to mononuclear cells and not to neutrophils. Ross et al. (1973) suggested this was because the C3b was being converted to C3d by the inactivator present in the whole serum. Under the selected conditions lk~r preparation of EAC-neutrophil rosettes no rosettes formed with EA or EAC prepared with heat-inactivated R3 emphasizing that the C3 and F c receptors on human neutrophils are distinct entities. If however, EA-neutrophil suspension is allowed to stand without centrifugation for 1 hr at room temperature, occasional EA rosettes will form. If the EAC-neutrophil sample 3 ° C bath this complication is is read within a few nrinutes of taking from the _7 avoided. No attempt was made to separate the anti-sheep red blood cell antibody into lgG and IgM fractions, for the formation of EAC, but there is evidence that neutrophils have receptors for both (Henson, 1969).
References Boyum, A., 1968, J. Clin. Lab. Invest. Suppl. 21,97. Brown. D.C. and D.A. Nelson, t973, Brit. J. Ilaematol. 24, 301. Cone, R.k., J.J. Marchalonis and R.T. Rox~ley, 1971, J. l'xptl. Med. 134, 1373. Eden, A., G.W. Miller and V. Nussenzwcig, 1973, J. Clin. Invest. 52, 3239. llenson, P.M., 1969, hmnunology 16, 107. [tuber. tt., S.D. Douglas and H.It. Fudenberg, 1969. Immunology 17, 7. Jonas, W.I(., B.W. Gurner, D.S. Nelson and R.R.A. Coombe, 1965, Int. Arch. Allergy Appl. lmmunol. 28, 86. Lachmann, P.J., M.J. Hobart and W.P. Aston, 1973, in: Handbook of experimental immunology, ed. D.M. Weir, 2rid edition (Blackwell, Oxford) chapter 5, 5.1. Mcssner, R.P, and J. Jelinek, 19711, J. Clin. Invest. 49, 2165. Pincus. S., C. Bianco and V. Nussenzweig, 1972, Blood 40,303. Ross, G.D., M,J. Polley, F.M. Rabellino and ll.M. Grey, 1973, J. Exptl. Med. 138,798. Wilson, J.D. and M. Feldmann, 1972, Nature New Biol. 237, 3.