Immunology 1979 37 25
In vitro complement activation by rabbit lymphocytes and thymocytes in autologous serum
ANNE B. WILSON, P. J. LACHMANN* & R. R. A. COOMBS Division of Immunology, Department of Pathology, University of Cambridge and *MRC Group on Mechanisms in Tumour Immunity, clo Laboratory of Molecular Biology, The Medical School, Cambridge
Received 19 June 1978; acceptedfor publication 13 September 1978
Summary. The activation of C3, previously demonstrated to occur during the isolation of lymphocytes from rabbit blood, has been investigated further by in vitro exposure of blood and lymph node lymphocytes and thymocytes to autologous serum. Activated C3 deposited on the lymphoid cell-surface was detected by (i) anti-C3, (ii) immune adherence reaction, and (iii) blocking of receptors for C3. An affinity shown by guinea-pig erythrocytes for activated complement was also investigated. Using EGTA, which inhibits the classical complement pathway while leaving the alternative pathway unaffected, it was shown that either pathway may be activated by rabbit cells depending on the temperature of incubation. The classical pathway was triggered at 40, probably by the reaction of cold auto-antibodies with lymphoid cell-surface determinants. At 370, however, complement activation followed the alternative pathway in a similar manner to that reported by other workers with human lymphoblastoid cell lines.
ment is activated during the procedures used for the purification of lymphocytes (Wilson, Kanski & Coombs, 1978). Activated C3 deposited on the lymphocyte surface was detected by a direct rosette forming reaction with trypsin-treated ox erythrocytes (Eox) carrying antibodies to rabbit C3 and by immune adherence ofhuman 0 erythrocytes (Ehuman). The lymphocytes were not killed by complement activation, but surface receptors for C3 were usually partially blocked. Little, if any, C3 was detectable on lymphocytes from blood taken in citrate or EDTA which inhibit complement activation by chelating Ca2 + and Mg2 +, thus indicating that the complement was activated in vitro. Platts-Mills & Ishizaka (1974) found that human complement was activated by rabbit lymphocytes, thymocytes and erythrocytes. Activation followed the alternative complement pathway and was thought to be antibody-independent, although more recent studies indicate that Ig is probably implicated (Nelson & Ruddy, 1978). Alternative pathway activation has also been shown in an homologous system composed of human lymphoblastoid cells and normal human serum (Budzko, Lachmann & McConnell, 1976; Theofilopoulos & Perrin, 1976; Yefenof, Klein & Kvarnung, 1977). In the present study, the mechanism of complement activation by rabbit lymphoid cells in autologous serum was examined by reaction with antibodies to C3 (direct rosette forming test) or with Ehuman (immune adherence). Large numbers of lym-
INTRODUCTION Previous work on rabbit blood showed that compleCorrespondence: Dr A. B. Wilson, Division of Immunology, Laboratories' Block, Addenbrooke's Hospital, Hills Road, Cambridge, CB2 2QQ. 00 1 9-2805/79/0500-0025$02.00
©) 1979 Blackwell Scientific Publications 25
26
Anne B. Wilson, P. J. Lachmann & R. R. A. Coombs
phocytes or thymocytes were found to have acquired activated C3 which was bound to the cell surface either by the C3 receptors or hydrophobically. Evidence for the involvement of auto-antibodies in classical complement activation at low temperatures led to the use of guinea-pig erythrocytes for the detection of immune complexes on the lymphoid cell surface. These erythrocytes are agglutinated in the presence of antigenantibody complexes, giving rise to the 'co-agglutination' reaction of Bordet & Gengou (191 1). Rosettes were formed with guinea-pig erythrocytes (Eg.pig) by lymphoid cells pre-exposed to autologous serum, and they were greatly enhanced by treating the erythrocytes with neuraminidase. Evidence will be presented that the affinity of Eg.pig is for one or more activated complement components rather than for Ig.
iron, respectively. The purified lymphocytes (95% or more of total cells) were washed three times at room temperature (22-26°, RT) and adjusted to a concentration of 2 x 106 per ml. Contaminating erythrocytes were removed by brief exposure (20 s) to distilled water before the penultimate wash.
MATERIALS AND METHODS
(b) Lymph node and thymus cells. The tissues were removed, cleaned and teased into RPMI 1640 medium containing 10% iFCS. The cell suspensions were filtered through a fine wire gauze and treated with carbonyl iron to remove macrophages. The lymphocytes or thymocytes were then washed three times at RT, and adjusted to 2 x 106 cells per ml.
Animals The rabbits were normal, uninjected animals of the New Zealand White strain, weighing between 2 and 4 kg. Lymphocyte and thymocyte preparations Unless otherwise stated, the medium used for washing lymphoid cells was RPMI (Flow) containing 20 mM HEPES (Sigma) and 5% heat-inactivated foetal calf serum (iFCS, serum supplied by Flow). The medium was adjusted to pH 7-2. (a) Blood lymphocytes. Blood was drawn into either ethylenediamine tetra-acetate (EDTA) or acid-citratedextrose (ACD) so as to obtain lymphocytes initially free of surface C3. Full details of the lymphocyte separation procedures were given by Wilson et al. (1978). EDTA-blood. The blood was taken into 0-01 M EDTA, diluted with phosphate buffered saline (PBS) and separated by centrifugation at 200 o-n FicollHypaque (51 088) for 35 min at 400g. This procedure leads to a loss of T lymphocytes which are killed by EDTA and migrate to the bottom of the tube. The lymphocytes at the Ficoll-Hypaque interface were therefore at least 90% Ig-bearing cells with 10% or fewer T cells. Contaminating platelets and monocytes were removed by slow centrifugation and by carbonyl
A CD-blood. Blood was drawn into ACD, and erythrocytes were sedimented at 370 with methyl cellulose. The supernate leucocytes were centrifuged and washed twice at RT to remove ACD and platelets. They were then treated with carbonyl iron and the lymphocytes washed as described above. The lymphocytes were only 80 90% pure because citrate inhibits phagocytosis and this effect persists after washing the cells. The method, however, has the advantage of retaining both B and T lymphocytes.
Enumeration of Ig-bearing and T lymphocytes (a) Total Ig-bearing cells. These were enumerated by the direct antiglobulin rosette forming reaction (DARR) as described by Coombs, Wilson, Eremin, Gurner, Haegert, Lawson, Bright & Munro (1977) and used in previous studies of rabbit lymphocytes (Wilson et al., 1978). Essentially, the IgG fraction of a goat antiserum to rabbit IgG was coupled by chromic chloride to E., which had been treated with trypsin to make them more agglutinable and thus give a more sensitive rosette-forming reaction. The IgG-coupled indicator cells were prepared freshly each week and stored in PBS at 4°. They were tested for effective coupling by (i) antiglobulin titration, (ii) reverse passive haemagglutination with rabbit Ig. Lymphocytes to be tested for surface Ig were washed twice at 40 with PBS containing 1% bovine plasma albumin and resuspended in this medium for rosette formation.
(b) Detection ofcell-surface IgM and IgG. Lymphocytes or thymocytes bearing IgM and IgG were enumerated by the DARR technique using E., coupled to the IgG fraction of sheep antiserum to rabbit p-Fc or to rabbit y-Fc, respectively.
27
Complement activation by rabbit lymphocytes
The specificity of the coupled E., was confirmed by reverse passive agglutination with rabbit purified IgM and IgG. (c) Tlymphocytes. The T lymphocytes were detected by rosette formation with papain-treated rabbit erythrocytes (Wilson, Gurner & Coombs, 1975; Wilson et al.,1978). Both the lymphocytes and Erabbit (1% suspension) were prepared in medium with 5% iFCS.
Incubation of lymphocytes or thymocytes in autologous serum The serum was always taken from the same animal as the lymphocytes or thymocytes being tested, i.e. it was autologous. The blood was usually drawn on the day of the experiment and left to clot at RT for at least 2h before separating the serum. The lymphoid cells were divided into 400 .ul aliquots in 2 ml siliconized glass tubes. They were deposited by centrifugation and the supernatant replaced with autologous serum equilibrated at the appropriate temperature (4 or 370). The tubes were sealed and rotated for 1 h, after which the cells were centrifuged and washed three times before testing for surface C3.
Rosette formation with guinea-pig erythrocytes Initial experiments showed that guinea-pig erythrocytes would adhere to serum-incubated lymphoid cells if they were mixed and centrifuged at 350 g for 2 min. This reaction was greatly strengthened when the erythrocytes were pre-treated with neuraminidase using the method of Wilson et al. (1975). The Eg.pig were used as a 1% suspension in RPMI 1640/0-2% iFCS, and stored for up to 1 week at 4°. RESULTS
Tests for C3 on the lymphoid cell-surface (a) Reaction with anti-rabbit C3. The IgG fraction of a sheep (T297) antiserum to rabbit C3 was coupled by chromic chloride to trypsin-treated E., Full details of the anti-C3 reagent and rosette-formation have been given previously (Wilson et al., 1978). The anti-C3 coupled E., were routinely tested by agglutination with (a) anti-sheep Ig and (b) normal rabbit serum which contains 'native' C3. (b) Immune adherence reaction. Lymphocytes and thymocytes were tested for the presence of surfacebound C3b by centrifuging (350 g, 2 min) with equal volumes of 1% washed Ehuman and incubating the cell pellets at 370 for 15 min.
1. Preliminary experiments on the in vitro activation of complement and the inhibitory effects of EGTA and EDTA It had earlier been shown that complement was activated during the isolation of lymphocytes from defibrinated rabbit blood. In order to study the mechanism of this reaction, lymphocytes isolated from EDTA-blood and initially free of adsorbed C3 were incubated in autologous serum under conditions similar to those used in separating lymphocytes from defibrinated blood. This involved incubating the cells in neat serum at 370, rotating for 1 h. The cells were then cooled to 4° and washed three times at 40 before testing for the presence of cell-membrane bound C3. Complement activation in this system led to a deposition of C3 on the surface of the blood lymphocytes, and further investigations showed similar activity with both lymph node lymphocytes and thymocytes. In addition to C3, thymocytes also acquired surface Ig during the incubation period, so that the number of cells reacting by DARR, normally less than 3%, were often increased to 50% or more. Results of representative tests are shown in Table 1, together with the effects of adding either EGTA (inhibitor of classical complement pathway) or EDTA (inhibitor of classical and alternative complement pathways).
(c) Blocking of receptors for C3. Lymphocytes were tested, before and after incubation with serum, for rosetting with indicator erythrocytes carrying either C3b, C3d or a mixture of both C3b and C3d (see Wilson et al., 1978). These indicator cells (EAC) were prepared by coating sheep erythrocytes with rabbit IgM antibodies and then with complement from C6-deficient rabbit serum (pre-absorbed with sheep erythrocytes at 4°). Control indicator cells were treated with heat-inactivated rabbit serum (560, 40 min).
(a) Binding of C3 to lymphocytes and thymocytes Reaction with anti-C3. Considerable numbers (up to 80%) of lymphoid cells from blood, lymph node and thymus acquired membrane-bound C3 when incubated in autologous serum either alone or with added Mg'. The most sensitive test for C3 proved to be rosette formation with E., coupled to anti-C3 antibodies. These indicator cells gave negative or very weak reactions (0-3%) with untreated lymphocytes and thymocytes, indicating that the C3 was acquired in vitro.
Anne B. Wilson, P. J. Lachmann & R. R. A. Coombs
28
Table 1. Activation of complement by blood and lymph node lymphocytes and thymocytes. Uptake of C3 on to the membrane detected by reaction with anti-C3, immune adherence and blocking of receptors for C3b and C3d. Inhibitory effects effects of EGTA/Mg2 + and EDTA also shown Percentage of reacting lymphocytes After incubation in autologous serum§ containing
Origin of lymphoid cells
Peripheral* blood
Lymph nodet
Thymus
Indicator erythrocytes
Untreated
Ox carrying anti-C3 Human (immune adherence) Nase-guinea-pig C3b + C3d EAC carrying C3b C3d Ox carrying anti-C3 Human (immune adherence) Nase-guinea-pig C3b + C3d EAC carrying C3b C3d Ox carrying anti-C3 Human (immune adherence) Nase-guinea-pig Ox carrying anti-Ig
0 0 3 92 56 77 0 0 0 69 37 57 0 0 0 0
Mg2+ (035 mM)t 80 33 52 68 19 59 69 22 14 48 12 21 63 11 19 36
(10) (0) (0)
(5) (0) (0)
(0) (0) (0)
EGTA( lOmM)+ Mg2+ (035 mM)
EDTA (10 mM)
65 1 0 92 38 79 51 4 0 62 24 50 48 0 0 29
6 0 1 90 50 80 12 0 0 68 28 54 0 0 0 22
* Peripheral blood lymphocytes isolated from EDTA blood; 94% Ig-bearing. t Lymph node lymphocytes (mesenteric) 64% Ig-bearing t Results in parentheses were obtained by incubation in serum heated to 56° for 30 min.
§ Lymphoid cells (8 x 105) were suspended in 400 p1 serum and rotated for 1 h at 37°. EGTA/Mg2 + or EDTA were added to serum prior to mixing with lymphocytes. After incubation, cells were washed three times at 4°.
Immune adherence reaction. Human erythrocytes have receptors for C3b, and the positive immune adherence tests on serum-incubated lymphocytes and thymocytes (Table 1) showed that the C3 on these cells had been activated and was not merely 'native' C3 passively adsorbed from serum. The numbers of cells rosetting with Ehuman were always lower than those reacting with anti-C3, presumably due to the conversion of some C3b to C3bi and C3d by the action of KAF and other serum enzymes during the incubation period.
Blocking of C3 receptors. The uptake of C3 by both blood and lymph node lymphocytes was accompanied by a reduction in the numbers of cells forming rosettes with EAC (Table 1), indicating a blocking of the surface receptors for C3. As expected, the greatest reduction was in C3b receptors, although receptors for C3d were also reduced.
(b) Effect ofheating the serum When the serum was pre-heated to 560 for 30 min the uptake of C3 on to blood and lymph node lymphocytes and thymocytes was almost completely abolished (see Table 1). In tests with anti-C3 coated E.,, 10% or less of the lymphoid cells reacted weakly compared with up to 80% of the cells incubated in nonheated serum. (c) Inhibition of C3-binding in the presence of EGTA or EDTA The uptake of C3 by lymphocytes was partially inhibited by 10 mm ethylene glycol tetra-acetate (EGTA), as indicated by a fall in the percentage of lymphocytes reacting with anti-C3 and by the abolition of the immune adherence reaction. There was no reduction in receptors for C3d, although C3b receptors were still partially blocked. Doubling the concentration of
Complement activation by rabbit lymphocytes EGTA to 20 mM did not produce any further inhibition of complement activation. EDTA (10 mM) almost completely inhibited C3 uptake by the lymphocytes. The few lymphocytes (usually less than 10%) reacting with anti-C3-coated EO, produced weak rosettes, and blocking of receptors for C3 was limited to a small reduction in cells reacting with C3b. The trace amount of activated C3 in this EDTA-serum was not detectable in similar tests with plasma from EDTA-blood, and is probably produced by the action of factors such as thrombin and plasmin released during the clotting process (Ratnoff & Naff, 1967). The finding of small percentages of C3-bearing lymphocytes after incubation in heated serum (see above) appears to support this conclusion. EGTA/Mg2+ and EDTA had similar inhibitory effects on the uptake of C3 by thymocytes. EDTA, however, which is cytotoxic for rabbit T cells (Wilson et al., 1978), killed over 90% of the thymocytes, making the interpretation of results difficult.
29
serum containing either EGTA/Mg2 + or EDTA, indicating that the factor(s) responsible for the adherence of Eg.pig may be associated with those which trigger or are a part of the classical complement pathway. They are probably not linked with the co-agglutination phenomenon which does not appear to require complement.
(e) Tests for inhibition of the lymphocyte reactions with anti-C3, human 0 erythrocytes and guinea-pig erythrocytes by antisera to rabbit IgG, C3 and whole serum Lymphocytes from EDTA blood were incubated with serum so that they reacted strongly with anti-C3, and with Ehuman and Eg.pig. They were subsequently treated for lh at 40 with sheep antisera to either rabbit IgG or rabbit whole serum, or with the IgG fraction of sheep anti-rabbit C3. Normal sheep serum was used as a control, and a DARR test for surface Ig was included to measure the inhibitory capacity of the anti-IgG and anti-whole serum reagents. From the results (Table 2), the lymphocyte reactions with both anti-C3 coated EO, and Eh.an were similar in that both were abolished by anti-C3 and by antirabbit whole serum, but were not affected by anti-Ig. This showed that the rosette formation with Ehuman was a true immune adherence reaction dependent on
(d) Rosette-formation with guinea-pig erythrocytes Considerable numbers of lymphocytes and thymocytes incubated in autologous serum were found to have acquired an affinity for Eg.pig which formed strong, stable rosettes (Table 1). This reactivity was not acquired from heated serum or from unheated
the presence of C3.
Table 2. Tests for inhibition of the rosette-forming reactions given by EDTA-blood lymphocytes* (pre-incubated in autologous serumt) by antisera to (a) rabbit IgG and (b) rabbit whole serum, and by the IgG fraction of sheep anti-rabbit C3
Percentage of lymphocytes reacting with erythrocytes Final dilution Ox carrying Human Nase-treated Ox carrying Reagent used to treat lymphocytest of reagent anti-C3 (immune adherence) guinea-pig anti-Ig None Normal sheep serum Sheep (C 1 7/1 1 1 F) anti-rabbit IgG Sheep (T297) IgG anti-rabbit C3
Sheep (207, bleed 5) anti-rabbit whole serum
-
1/20 1/5 1/4 (3mg protein/ml) 1/20
69 69 67
56 58 59
72 79 80
95 95 lOw
0
0
75
89
0
0
24
44
*Lymphocytes isolated from EDTA-blood were 94% Ig-bearing cells. tLymphocytes incubated in autologous serum and washed as described in subscript of Table 1. tAll reagents were heat-inactivated and absorbed with rabbit erythrocytes. Lymphocytes which had been incubated with autologous serum were well washed and mixed with the appropriate reagent for 1 h at 40. They were then centrifuged and washed three times in the cold with RPMI 1640/5% iFCS before rosetting. w = Weak rosettes.
30
Anne B. Wilson, P. J. Lachmann & R. R. A. Coombs
The adherence of Eg.pig, however, was quite different and was affected neither by anti-C3 nor by anti-Ig, although it was partially inhibited by anti-rabbit whole serum. Unfortunately, no antisera to other purified components of rabbit complement were available to extend these inhibition tests, apart from anti-C6 (raised in C6-deficient rabbits) which had no effect on rosette-formation. 2.Effect of temperature on complement activation by lymphocytes and thymocytes The experiments with EGTA and EDTA indicated that both the classical and the alternative complement pathways were involved. In those tests, lymphoid cells were incubated with serum at 370 and then cooled and washed at 40, and it was not possible to know at which temperature complement activation occurred. Aliquots of lymphoid cells were, therefore, rotated in serum for 1 h either at 40 with subsequent washing at 40 or 370, or at 370 followed by washing at 40 or 37. The results of experiments on blood and thymus are given in Table 3. Other tests on lymph node lymphocytes (not reported) showed similar reactivity to those from blood. The temperature of incubation and washing had little effect on the numbers of lymphocytes reacting with anti-C3, although thymocytes gave a stronger reaction after incubation at 40. Complement
activation, therefore, took place even at low temperatures, and lymphoid cells incubated and washed at 40 always gave the maximum number of rosettes with Ehuman. This was probably because the immune adherence receptor is for C3b, most of which would be converted to C3bi or C3d during the 1 h incubation at 370. In short-term incubation experiments at 370 (not shown), immune adherence reactions were positive after only 1 min, and over 70% of lymphocytes reacted with anti-C3. The reaction of lymphoid cells with Eg.pig was also dependent on contact with serum at low temperatures; the strongest reactions being obtained on lymphocytes and thymocytes incubated and washed at 40. The reactivity was removed when lymphoid cells were warmed at 370 in the presence of serum, although in other tests (not shown) incubation of positive lymphocytes in serum-free medium did not affect their reaction with Eg.pig.
3. The in vitro uptake of Ig by lymphocytes and thymocytes
In some experiments, lymphocytes and thymocytes were tested by DARR for surface Ig both before and after incubation in serum. Tests with reagents specific
for y-Fc and ,u-Fc indicated that most normal Ig-bearing rabbit lymphocytes in blood and lymph nodes
Table 3. Incubation of blood lymphocytes and thymocytes in autologous serum: effect of temperature
Temperature (0)
Percentage of lymphoid cells reacting with erythrocytes
Incubation Wash*
Ox carrying Human Nase-treated Ox carrying anti-C3 (immune adherence) guinea-pig anti-Ig
Origin of lymphoid cells
Bloodt
Thymus
4 4 4 37 37 4 37 37 Untreated 4 4 4 37 4 37 37 37 Untreated
60 66 62 58 3 34 33 24 16 0
37 2
13 14 0 29 0 10 1 0
78 0 25 2 1 61 0 14 0 0
NT NT NI NT NT 73 2 35 8 0
* Where cells were washed at a different temperature from that of the incubation, they were equilibrated at the second temperature for 5 min before centrifuging. t Lymphocytes isolated from ACD blood. NT, Not tested.
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Complement activation by rabbit lymphocytes IgM, but IgG was detected on only a minority (0-2%) of untreated cells and no evidence was found for the uptake of IgG during in vitro treatment with serum at either 40 or 37°. An increase in total Ig-bearing lymphocytes was found in some experiments on lymph node lymphocytes. One test showed a rise in Ig-positive lymphocytes of from 70 to 80% after incubation with serum at 370 and washing at 40, although the same cells washed at 370 showed no increase in Ig-bearing lymphocytes. Another experiment carried out entirely at 40 produced an increase in Ig-bearing lymphocytes from 63 to 84% in a cell suspension containing 42% T lymphocarry
cytes.
Much clearer evidence for the in vitro uptake of Ig obtained in tests with thymocytes, since untreated suspensions had a much lower 'background' of 0-3% cells with detectable surface Ig. Preliminary experiments had shown that thymocytes will pick up Ig from autologous serum (Table 1), and when tested at different temperatures, this was found to be a cold-dependent phenomenon (Table 3). Immunoglobulin was deposited on thymocytes at 40, but could be removed by washing at 37°. Further investigations showed that at least some of this Ig was IgM (e.g. in one experiment, 76% of thymocytes became Ig-positive and 25% reacted with antibody to u-Fc), but as with lymphocytes, no evidence was found for the acquisition of IgG. All rabbits tested were found to have circulating, was
cold auto-agglutinins, and the properties of the Ig acquired by lymphoid cells indicate that it may be complement-fixing, cold auto-antibody similar to those described in pathological conditions in man (Shumak, Rachkewich & Greaves, 1975; Pruzanski, Farid, Keystone, Armstrong & Greaves, 1975). 4. Effect of EGTA on complement activation at 40 and 370 The probable link between cold auto-antibodies and activation of the classical complement pathway was investigated by examining the inhibitory effect of EGTA at 40 and 370. Experiments on blood lymphocytes and thymocytes (Table 4) showed that complement activation at 40 was strongly inhibited by EGTA, whilst activation at 370 was relatively little affected. Also, EGTA had no effect on the uptake of Ig by thymocytes at 4°. It was thus apparent that complement activation at 40 was principally by the classical pathway, and was probably initiated by the interaction of (IgM?) cold auto-antibodies with lymphocyte or thymocyte cellmembrane antigens. At 370, however, complement was apparently activated by the alternative pathway.
5. Test to find whether peripheral lymphocytes carrying C3 were T or B cells Lymphocytes isolated from ACD blood were incu-
Table 4. Effect of EGTA on the uptake of C3 by peripheral blood lymphocytes and thymocytes (also uptake of Ig on thymocytes) incubated in autologous serum at 40 and 370
Percentage of/cells reacting with erythrocytes Origin of Temperature (0) Ox carrying Human Ox carrying lymphoid anti-C3 (immune adherence) cells Incubation Wash EGTA/Mg2 + anti-Ig
Blood*
4
4
37
37
Absent Present Absent Present
Thymus
Untreated 4
4
37
37
Untreated
Absent Present Absent Present -
70 17 74 80 2 70 14 47 39 2
*Lymphocytes separated from EDTA-blood
36 0 4 1 0 72 1 0 0 0
NT NT NT NT NT 52 57 1 2 2
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Anne B. Wilson, P. J. Lachmann & R. R. A. Coombs
bated in serum at either 4 or 37°. They were then tested by mixed rosette forming reactions, using fluoresceinconjugated, papain-treated Erabbit as a T-cell marker (Wilson et al, 1975). Lymphocytes not reacting with the Erabbit were regarded as B cells since few 'null' cells are found in the rabbit. After incubation in serum at 40, between 90% and 100% of the B lymphocytes reacted with anti-C3 and were positive for immune adherence. The same proportion of B cells also formed rosettes with Eg.pig. The percentages of T lymphocytes reacting, however, were much lower; 60% combining with anti-C3, 17% with Ehuman and 37% with Eg.pig. The C3 was thus bound preferentially to B lymphocytes. A similar result was obtained at 370 when C3 was detected on 97% of B cells and on 70% of T cells.
DISCUSSION
Complement activation occurred when rabbit blood or lymph node lymphocytes or thymocytes were incubated in autologous serum. The mechanism of activation varied with the temperature of incubation, proceeding by the classical pathway at 4° and by the alternative pathway at 37°. Activated C3 deposited on the surface of lymphoid cells was detected by rosetteformation with E., carrying antibodies to C3 and by immune adherence of Ehuman. In suspensions containing both T and B lymphocytes, C3 was preferentially bound to B lymphocytes and there was blocking of cell-surface receptors for C3b and C3d. The presence of these receptors was not essential for the uptake of C3 since it was also detected on thymocytes and some peripheral T cells which lacked C3-receptors. The C3 on these T cells was presumably attached by hydrophobic bonds as 'nascent' C3b (McConnell & Lachmann, 1977). There was no evidence for a reduction in cell viability following activation of complement. At 40, complement activation was almost exclusively by the classical pathway, as shown by inhibition with EGTA/Mg2 +. In experiments with thymocytes, C3 activation at low temperatures was accompanied by an uptake of Ig, which included IgM but not IgG. There was also evidence that some peripheral T lymphocytes can acquire surface Ig when incubated with serum at 40. It is possible that the Ig was passively adsorbed by reaction with receptors for IgM such as those shown on T cells of man (McConnel & Hurd, 1976) and mouse (Santana, 1977). The rabbit Ig, however, shares properties such as its reactivity at 40 and
release at 370, with the complement activating, cold auto-antibodies described in man (Pruzanski et al, 1975; Shumak et al, 1975). These human antibodies may react with lymphocyte surface determinants antigenically identical to erythrocyte membrane antigens (Shumak et al, 1975). The finding of cold auto-agglutinins in rabbit sera favours the hypothesis that the reaction of such antibodies with lymphocyte antigens is the trigger for complement activation at 40. In contrast to the reaction at 40, complement activation by rabbit lymphocytes and thymocytes at 37° was not inhibited by EGTA/Mg2 +, and was therefore by the alternative pathway. A similar mechanism of complement fixation has also been described in homologous systems involving human tumour and other cell lines incubated in normal human serum (Okada & Baba, 1974; Budzko et al, 1976; Theofilopoulos & Perrin, 1976; Yefenof et al, 1977). These authors found no evidence of complement activation by normal human lymphocytes, although other workers claim that there is a blocking of C3 receptors when these cells are incubated in normal human serum (Abrahamsohn, Nilsson & Abdou, 1974; Erdei, Fiist, Sarmay, Medgyesi & Gergely, 1977). Neo-antigens from the terminal sequence of complement proteins (C5b-C9) have recently been demonstrated on normal human peripheral blood lymphocytes isolated from defibrinated blood by Ficoll-Hypaque centrifugation (Sundsmo, Kolb & Muller-Eberhard, 1978). As with rabbit blood (Wilson et al, 1978), the complement components on these human lymphocytes were acquired in vitro since they were not present on cells isolated from blood collected in EDTA. Unlike the present findings with rabbit C3, the acquisition of C5b-C9 neo-antigens by human lymphocytes during in vitro incubation with serum was inhibited neither by EDTA nor by heating the serum to 560 (Sundsmo, Curd, Kolb & Muller-Eberhard, 1978). As these human cells were not examined for C3, it is not clear whether the generation of activated terminal complement components is linked in any way with alternative pathway activation such as that described in the present investigation. Many agents are known to trigger the alternative complement pathway. These include carbohydrates such as zymosan, inulin and bacterial endotoxins, certain types of antibodies and C3-nephritic factor. C3b itself can activate the alternative pathway by means of the feedback cycle in which the C3 convertase, C3b-Bb, is generated. This process is normally regulated by the action of conglutinogen activation factor
Complement activation by rabbit lymphocytes (C3b inactivator) (KAF) and B1 H which destroy the C3b-Bb complex. Recent work by Fearon & Austen (1977), however, showed that the membranes of certain cells, in this case rabbit erythrocytes, protect bound C3b and C3b-Bb from the action of KAF and ,I3H. The C3b feedback cycle therefore proceeds more or less uncontrolled and C3 conversion is greatly increased. As mentioned above, trace amounts of activated C3 may be present in rabbit serum. It is conceivable, therefore, that rabbit lymphocytes and thymocytes also have 'protected' surfaces and that cell-membrane binding of small amounts of C3b from the serum is sufficient to trigger the rapid and extensive activation of C3 observed in the present investigations. The nature of the reaction between serum-incubated lymphoid cells and Eg.pig remains undetermined. Strong rosette-formation with Eg.pig was produced by lymphocytes and thymocytes incubated in serum at 40, but not at 37°. Rosettes were not formed after incubation in serum containing EGTA/Mg2 + or EDTA, nor when the serum had been heat-inactivated. This indicated that the receptors on Eg.pig may react with one or more components associated with the classical, but not with the alternative, pathway of complement activation (i.e. activated C 1, C4 or C2). Further characterization of the factor(s) responsible for this affinity reaction was not possible in the present study as no purified components of rabbit complement were available. The present findings with rabbit lymphocytes are important in two respects. The first of these has been largely dealt with in an earlier report (Wilson et al, 1978) and involves the practical aspects of isolating lymphocytes, especially from blood. The activation of complement, leading to blocking of receptors for C3, and the uptake of Ig at low temperatures must be avoided in order to retain, as far as possible, the in vivo characteristics and reactivity of the purified lymphocytes. For this reason, it is essential to take blood into an anticoagulant such as citrate which also inhibits complement activation (EDTA kills rabbit T cells), and to avoid cooling the lymphocytes while in contact with serum. The second point for consideration must be the possible in vivo activation of the alternative complement pathway by lymphoid cells. Although in the present study on normal animals only a small percentage (up to 3%) of untreated lymphocytes or thymocytes had detectable surface C3, it appears that only slight changes in cell properties might be necessary to enable them to trigger complement activation. For
33
example, the infection of human acute myeloid leukemia cells with Moloney Sarcoma virus enables them to activate complement by the alternative pathway (Okada & Baba, 1974), and enhanced activation has been demonstrated with lymphoblastoid cell lines infected with EB virus (McConnell & Lachmann, 1977). Changes in lymphocytes brought about in vivo by infectious agents or by other alterations in their environment might therefore be sufficient to initiate complement activation.
ACKNOWLEDGMENTS We wish to thank Mrs A. Fieldes for able technical assistance, and the Medical Research Council for financial support. We are also grateful to Dr A. Feinstein for the gift of sheep anti-rabbit ,i-Fc and y-Fc reagents.
REFERENCES ABRAHAMSOHN I., NILSSON U. & ABDOU N.J. (1974) Relationship of immunoglobulin to complement receptors of human B cells J. immunol. 112, 1931. BORDET J. & GENGOU 0. (1911) La coagglutination des globules rouges par les melanges des anticorps et des antigenes albumineux. Zbl. Bakt., I. Abt. Orig. 58, 330 (See also Coombs et al, 1955, Brit. J. exp. Path. 36, 179.) BUDZKO DELIA B., LACHMANN P.J. & MCCONNELL I. (1976) Activation of the alternative complement pathway by lymphoblastoid cell lines derived from patients with Burkitt's lymphoma and infectious mononucleosis. Cell. Immunol. 22, 98. COOMBS R.R.A., WILSON ANNE B., EREMIN 0., GURNER B.W., HAEGERT D.G., LAWSON YVONNE A., BRIGHT SUSAN & MUNRO A.J. (1977) Comparison of the direct antiglobulin rosetting reaction with the mixed antiglobulin rosetting reaction for the detection of immunoglobulin on lymphocytes. J. immunol. Meth. 18, 45. ERDEI ANNA, FUST G., SARMAY GABRIELLA, MEDGYESI G.A. & GERGELY J. (1977) Studies on the mechanism of the complement-mediated inhibition of the Fc and C3 receptors of B lymphocytes. Clin. Immunol. Immunopathol. 8, 367. FEARON D.T. & AUSTEN K.F. (1977) Activation of the alternative complement pathway with rabbit erythrocytes by circumvention of the regulatory action of endogenous control proteins. J. exp. Med. 146, 22. MCCONNELL I. & HURD C.H. (1976) Lymphocyte receptors. II. Receptors for rabbit IgM on human T lymphocytes. Immunology, 30, 835. MCCONNELL 1. & LACHMANN P.J. (1977) Complement receptors and cell associated complement components. Immunol. Commun. 6, 111. NELSON B. & RUDDY S. (1978) A role for IgG in the activation of the human alternatlve pathway by rabbit erythrocytes
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Anne B. Wilson, P. J. Lachmann & R. R. A. Coombs
(Abstract). In: Report of the 7th Int. Complement Workshop, J. Immunol. 120, 1788. OKADA H. & BABA T. (1974) Rosette formation of human erythrocytes on cultured cells of tumour origin and activation of complement by cell membrane. Nature (Lond), 248, 521. PLATTS-MILLs T.A.E. & ISHIZAKA K. (1974) Activation of the alternative pathway of human complement by rabbit cells. J. Immunol. 113, 348. PRUZANSKI W., FARID N., KEYSTONE E., ARMSTRONG M. & GREAVES M.F. (1975) The influence of homogeneous cold agglutinins on human B and T lymphocytes. Clin. Immunol. Immunopathol. 4, 248. RATNOFF O.D. & NAFF G.B. (1967) The conversion of C'ls to C'l esterase by plasmin and trypsin. J. exp. Med. 125, 337. SANTANA VILMA (1977) Receptors for IgM on murine lymphoid cells. Immunology, 32, 273. SHUMAK K.H., RACHKEWICH ROSE A. & GREAVES M.F. (1975) 1 and i antigens on normal human T and B lymphocytes and on lymphocytes from patients with chronic lymphocytic leukemia. Clin. Immunol. Immunopathol. 4, 241. SUNDSMO J.S., KOLB W.D. & MuLLER-EBERHARD H.J. (1978) Leukocyte complement: neoantigens of the membrane
attack complex on the surface of human leukocytes prepared from defibrinated blood. J. Immunol. 120, 850. SUNDSMO J.S., CURD J.G., KOLB W.P. & MuLLER-EBERHARD H.J. (1978) Leukocyte complement: assembly of the membrane attack complex of complement by human peripheral blood leukocytes in the presence and absence of serum. J. Immunol. 120, 855. THEOFILOPOULOS A.N. & PERRIN L.H. (1976) Lysis of human lymphoblastoid cells by self-activation of the alternative complement pathway. Fed. Proc. 35, 493. WILSON ANNE B., GURNER B.W. & COOMBS R.R.A. (1975) Observations on rabbit thymocytes and peripheral T cells. II. Rosette formation with rabbit erythrocytes. Int. Arch. Allergy, 48, 383. WILSON ANNE B., KANSKI A. & COOMBS R.R.A. (1978) Artefactual variations in the B and T sub-populations of rabbit blood lymphocytes depending on method of isolation, and blocking of C3 receptors due to in vitro activation of complement. J. Immunol. Meth. 24, 201. YEFENOF E., KLEIN G. & KVARNUNG K. (1977) Relationships between complement activation, complement binding and EBV absorption by human hematopoietic cell lines. Cell. Immunol. 31, 225.
In vitro complement activation by rabbit lymphocytes and thymocytes in autologous serum
ANNE B. WILSON, P. J. LACHMANN* & R. R. A. COOMBS Division of Immunology, Department of Pathology, University of Cambridge and *MRC Group on Mechanisms in Tumour Immunity, clo Laboratory of Molecular Biology, The Medical School, Cambridge
Received 19 June 1978; acceptedfor publication 13 September 1978
Summary. The activation of C3, previously demonstrated to occur during the isolation of lymphocytes from rabbit blood, has been investigated further by in vitro exposure of blood and lymph node lymphocytes and thymocytes to autologous serum. Activated C3 deposited on the lymphoid cell-surface was detected by (i) anti-C3, (ii) immune adherence reaction, and (iii) blocking of receptors for C3. An affinity shown by guinea-pig erythrocytes for activated complement was also investigated. Using EGTA, which inhibits the classical complement pathway while leaving the alternative pathway unaffected, it was shown that either pathway may be activated by rabbit cells depending on the temperature of incubation. The classical pathway was triggered at 40, probably by the reaction of cold auto-antibodies with lymphoid cell-surface determinants. At 370, however, complement activation followed the alternative pathway in a similar manner to that reported by other workers with human lymphoblastoid cell lines.
ment is activated during the procedures used for the purification of lymphocytes (Wilson, Kanski & Coombs, 1978). Activated C3 deposited on the lymphocyte surface was detected by a direct rosette forming reaction with trypsin-treated ox erythrocytes (Eox) carrying antibodies to rabbit C3 and by immune adherence ofhuman 0 erythrocytes (Ehuman). The lymphocytes were not killed by complement activation, but surface receptors for C3 were usually partially blocked. Little, if any, C3 was detectable on lymphocytes from blood taken in citrate or EDTA which inhibit complement activation by chelating Ca2 + and Mg2 +, thus indicating that the complement was activated in vitro. Platts-Mills & Ishizaka (1974) found that human complement was activated by rabbit lymphocytes, thymocytes and erythrocytes. Activation followed the alternative complement pathway and was thought to be antibody-independent, although more recent studies indicate that Ig is probably implicated (Nelson & Ruddy, 1978). Alternative pathway activation has also been shown in an homologous system composed of human lymphoblastoid cells and normal human serum (Budzko, Lachmann & McConnell, 1976; Theofilopoulos & Perrin, 1976; Yefenof, Klein & Kvarnung, 1977). In the present study, the mechanism of complement activation by rabbit lymphoid cells in autologous serum was examined by reaction with antibodies to C3 (direct rosette forming test) or with Ehuman (immune adherence). Large numbers of lym-
INTRODUCTION Previous work on rabbit blood showed that compleCorrespondence: Dr A. B. Wilson, Division of Immunology, Laboratories' Block, Addenbrooke's Hospital, Hills Road, Cambridge, CB2 2QQ. 00 1 9-2805/79/0500-0025$02.00
©) 1979 Blackwell Scientific Publications 25
26
Anne B. Wilson, P. J. Lachmann & R. R. A. Coombs
phocytes or thymocytes were found to have acquired activated C3 which was bound to the cell surface either by the C3 receptors or hydrophobically. Evidence for the involvement of auto-antibodies in classical complement activation at low temperatures led to the use of guinea-pig erythrocytes for the detection of immune complexes on the lymphoid cell surface. These erythrocytes are agglutinated in the presence of antigenantibody complexes, giving rise to the 'co-agglutination' reaction of Bordet & Gengou (191 1). Rosettes were formed with guinea-pig erythrocytes (Eg.pig) by lymphoid cells pre-exposed to autologous serum, and they were greatly enhanced by treating the erythrocytes with neuraminidase. Evidence will be presented that the affinity of Eg.pig is for one or more activated complement components rather than for Ig.
iron, respectively. The purified lymphocytes (95% or more of total cells) were washed three times at room temperature (22-26°, RT) and adjusted to a concentration of 2 x 106 per ml. Contaminating erythrocytes were removed by brief exposure (20 s) to distilled water before the penultimate wash.
MATERIALS AND METHODS
(b) Lymph node and thymus cells. The tissues were removed, cleaned and teased into RPMI 1640 medium containing 10% iFCS. The cell suspensions were filtered through a fine wire gauze and treated with carbonyl iron to remove macrophages. The lymphocytes or thymocytes were then washed three times at RT, and adjusted to 2 x 106 cells per ml.
Animals The rabbits were normal, uninjected animals of the New Zealand White strain, weighing between 2 and 4 kg. Lymphocyte and thymocyte preparations Unless otherwise stated, the medium used for washing lymphoid cells was RPMI (Flow) containing 20 mM HEPES (Sigma) and 5% heat-inactivated foetal calf serum (iFCS, serum supplied by Flow). The medium was adjusted to pH 7-2. (a) Blood lymphocytes. Blood was drawn into either ethylenediamine tetra-acetate (EDTA) or acid-citratedextrose (ACD) so as to obtain lymphocytes initially free of surface C3. Full details of the lymphocyte separation procedures were given by Wilson et al. (1978). EDTA-blood. The blood was taken into 0-01 M EDTA, diluted with phosphate buffered saline (PBS) and separated by centrifugation at 200 o-n FicollHypaque (51 088) for 35 min at 400g. This procedure leads to a loss of T lymphocytes which are killed by EDTA and migrate to the bottom of the tube. The lymphocytes at the Ficoll-Hypaque interface were therefore at least 90% Ig-bearing cells with 10% or fewer T cells. Contaminating platelets and monocytes were removed by slow centrifugation and by carbonyl
A CD-blood. Blood was drawn into ACD, and erythrocytes were sedimented at 370 with methyl cellulose. The supernate leucocytes were centrifuged and washed twice at RT to remove ACD and platelets. They were then treated with carbonyl iron and the lymphocytes washed as described above. The lymphocytes were only 80 90% pure because citrate inhibits phagocytosis and this effect persists after washing the cells. The method, however, has the advantage of retaining both B and T lymphocytes.
Enumeration of Ig-bearing and T lymphocytes (a) Total Ig-bearing cells. These were enumerated by the direct antiglobulin rosette forming reaction (DARR) as described by Coombs, Wilson, Eremin, Gurner, Haegert, Lawson, Bright & Munro (1977) and used in previous studies of rabbit lymphocytes (Wilson et al., 1978). Essentially, the IgG fraction of a goat antiserum to rabbit IgG was coupled by chromic chloride to E., which had been treated with trypsin to make them more agglutinable and thus give a more sensitive rosette-forming reaction. The IgG-coupled indicator cells were prepared freshly each week and stored in PBS at 4°. They were tested for effective coupling by (i) antiglobulin titration, (ii) reverse passive haemagglutination with rabbit Ig. Lymphocytes to be tested for surface Ig were washed twice at 40 with PBS containing 1% bovine plasma albumin and resuspended in this medium for rosette formation.
(b) Detection ofcell-surface IgM and IgG. Lymphocytes or thymocytes bearing IgM and IgG were enumerated by the DARR technique using E., coupled to the IgG fraction of sheep antiserum to rabbit p-Fc or to rabbit y-Fc, respectively.
27
Complement activation by rabbit lymphocytes
The specificity of the coupled E., was confirmed by reverse passive agglutination with rabbit purified IgM and IgG. (c) Tlymphocytes. The T lymphocytes were detected by rosette formation with papain-treated rabbit erythrocytes (Wilson, Gurner & Coombs, 1975; Wilson et al.,1978). Both the lymphocytes and Erabbit (1% suspension) were prepared in medium with 5% iFCS.
Incubation of lymphocytes or thymocytes in autologous serum The serum was always taken from the same animal as the lymphocytes or thymocytes being tested, i.e. it was autologous. The blood was usually drawn on the day of the experiment and left to clot at RT for at least 2h before separating the serum. The lymphoid cells were divided into 400 .ul aliquots in 2 ml siliconized glass tubes. They were deposited by centrifugation and the supernatant replaced with autologous serum equilibrated at the appropriate temperature (4 or 370). The tubes were sealed and rotated for 1 h, after which the cells were centrifuged and washed three times before testing for surface C3.
Rosette formation with guinea-pig erythrocytes Initial experiments showed that guinea-pig erythrocytes would adhere to serum-incubated lymphoid cells if they were mixed and centrifuged at 350 g for 2 min. This reaction was greatly strengthened when the erythrocytes were pre-treated with neuraminidase using the method of Wilson et al. (1975). The Eg.pig were used as a 1% suspension in RPMI 1640/0-2% iFCS, and stored for up to 1 week at 4°. RESULTS
Tests for C3 on the lymphoid cell-surface (a) Reaction with anti-rabbit C3. The IgG fraction of a sheep (T297) antiserum to rabbit C3 was coupled by chromic chloride to trypsin-treated E., Full details of the anti-C3 reagent and rosette-formation have been given previously (Wilson et al., 1978). The anti-C3 coupled E., were routinely tested by agglutination with (a) anti-sheep Ig and (b) normal rabbit serum which contains 'native' C3. (b) Immune adherence reaction. Lymphocytes and thymocytes were tested for the presence of surfacebound C3b by centrifuging (350 g, 2 min) with equal volumes of 1% washed Ehuman and incubating the cell pellets at 370 for 15 min.
1. Preliminary experiments on the in vitro activation of complement and the inhibitory effects of EGTA and EDTA It had earlier been shown that complement was activated during the isolation of lymphocytes from defibrinated rabbit blood. In order to study the mechanism of this reaction, lymphocytes isolated from EDTA-blood and initially free of adsorbed C3 were incubated in autologous serum under conditions similar to those used in separating lymphocytes from defibrinated blood. This involved incubating the cells in neat serum at 370, rotating for 1 h. The cells were then cooled to 4° and washed three times at 40 before testing for the presence of cell-membrane bound C3. Complement activation in this system led to a deposition of C3 on the surface of the blood lymphocytes, and further investigations showed similar activity with both lymph node lymphocytes and thymocytes. In addition to C3, thymocytes also acquired surface Ig during the incubation period, so that the number of cells reacting by DARR, normally less than 3%, were often increased to 50% or more. Results of representative tests are shown in Table 1, together with the effects of adding either EGTA (inhibitor of classical complement pathway) or EDTA (inhibitor of classical and alternative complement pathways).
(c) Blocking of receptors for C3. Lymphocytes were tested, before and after incubation with serum, for rosetting with indicator erythrocytes carrying either C3b, C3d or a mixture of both C3b and C3d (see Wilson et al., 1978). These indicator cells (EAC) were prepared by coating sheep erythrocytes with rabbit IgM antibodies and then with complement from C6-deficient rabbit serum (pre-absorbed with sheep erythrocytes at 4°). Control indicator cells were treated with heat-inactivated rabbit serum (560, 40 min).
(a) Binding of C3 to lymphocytes and thymocytes Reaction with anti-C3. Considerable numbers (up to 80%) of lymphoid cells from blood, lymph node and thymus acquired membrane-bound C3 when incubated in autologous serum either alone or with added Mg'. The most sensitive test for C3 proved to be rosette formation with E., coupled to anti-C3 antibodies. These indicator cells gave negative or very weak reactions (0-3%) with untreated lymphocytes and thymocytes, indicating that the C3 was acquired in vitro.
Anne B. Wilson, P. J. Lachmann & R. R. A. Coombs
28
Table 1. Activation of complement by blood and lymph node lymphocytes and thymocytes. Uptake of C3 on to the membrane detected by reaction with anti-C3, immune adherence and blocking of receptors for C3b and C3d. Inhibitory effects effects of EGTA/Mg2 + and EDTA also shown Percentage of reacting lymphocytes After incubation in autologous serum§ containing
Origin of lymphoid cells
Peripheral* blood
Lymph nodet
Thymus
Indicator erythrocytes
Untreated
Ox carrying anti-C3 Human (immune adherence) Nase-guinea-pig C3b + C3d EAC carrying C3b C3d Ox carrying anti-C3 Human (immune adherence) Nase-guinea-pig C3b + C3d EAC carrying C3b C3d Ox carrying anti-C3 Human (immune adherence) Nase-guinea-pig Ox carrying anti-Ig
0 0 3 92 56 77 0 0 0 69 37 57 0 0 0 0
Mg2+ (035 mM)t 80 33 52 68 19 59 69 22 14 48 12 21 63 11 19 36
(10) (0) (0)
(5) (0) (0)
(0) (0) (0)
EGTA( lOmM)+ Mg2+ (035 mM)
EDTA (10 mM)
65 1 0 92 38 79 51 4 0 62 24 50 48 0 0 29
6 0 1 90 50 80 12 0 0 68 28 54 0 0 0 22
* Peripheral blood lymphocytes isolated from EDTA blood; 94% Ig-bearing. t Lymph node lymphocytes (mesenteric) 64% Ig-bearing t Results in parentheses were obtained by incubation in serum heated to 56° for 30 min.
§ Lymphoid cells (8 x 105) were suspended in 400 p1 serum and rotated for 1 h at 37°. EGTA/Mg2 + or EDTA were added to serum prior to mixing with lymphocytes. After incubation, cells were washed three times at 4°.
Immune adherence reaction. Human erythrocytes have receptors for C3b, and the positive immune adherence tests on serum-incubated lymphocytes and thymocytes (Table 1) showed that the C3 on these cells had been activated and was not merely 'native' C3 passively adsorbed from serum. The numbers of cells rosetting with Ehuman were always lower than those reacting with anti-C3, presumably due to the conversion of some C3b to C3bi and C3d by the action of KAF and other serum enzymes during the incubation period.
Blocking of C3 receptors. The uptake of C3 by both blood and lymph node lymphocytes was accompanied by a reduction in the numbers of cells forming rosettes with EAC (Table 1), indicating a blocking of the surface receptors for C3. As expected, the greatest reduction was in C3b receptors, although receptors for C3d were also reduced.
(b) Effect ofheating the serum When the serum was pre-heated to 560 for 30 min the uptake of C3 on to blood and lymph node lymphocytes and thymocytes was almost completely abolished (see Table 1). In tests with anti-C3 coated E.,, 10% or less of the lymphoid cells reacted weakly compared with up to 80% of the cells incubated in nonheated serum. (c) Inhibition of C3-binding in the presence of EGTA or EDTA The uptake of C3 by lymphocytes was partially inhibited by 10 mm ethylene glycol tetra-acetate (EGTA), as indicated by a fall in the percentage of lymphocytes reacting with anti-C3 and by the abolition of the immune adherence reaction. There was no reduction in receptors for C3d, although C3b receptors were still partially blocked. Doubling the concentration of
Complement activation by rabbit lymphocytes EGTA to 20 mM did not produce any further inhibition of complement activation. EDTA (10 mM) almost completely inhibited C3 uptake by the lymphocytes. The few lymphocytes (usually less than 10%) reacting with anti-C3-coated EO, produced weak rosettes, and blocking of receptors for C3 was limited to a small reduction in cells reacting with C3b. The trace amount of activated C3 in this EDTA-serum was not detectable in similar tests with plasma from EDTA-blood, and is probably produced by the action of factors such as thrombin and plasmin released during the clotting process (Ratnoff & Naff, 1967). The finding of small percentages of C3-bearing lymphocytes after incubation in heated serum (see above) appears to support this conclusion. EGTA/Mg2+ and EDTA had similar inhibitory effects on the uptake of C3 by thymocytes. EDTA, however, which is cytotoxic for rabbit T cells (Wilson et al., 1978), killed over 90% of the thymocytes, making the interpretation of results difficult.
29
serum containing either EGTA/Mg2 + or EDTA, indicating that the factor(s) responsible for the adherence of Eg.pig may be associated with those which trigger or are a part of the classical complement pathway. They are probably not linked with the co-agglutination phenomenon which does not appear to require complement.
(e) Tests for inhibition of the lymphocyte reactions with anti-C3, human 0 erythrocytes and guinea-pig erythrocytes by antisera to rabbit IgG, C3 and whole serum Lymphocytes from EDTA blood were incubated with serum so that they reacted strongly with anti-C3, and with Ehuman and Eg.pig. They were subsequently treated for lh at 40 with sheep antisera to either rabbit IgG or rabbit whole serum, or with the IgG fraction of sheep anti-rabbit C3. Normal sheep serum was used as a control, and a DARR test for surface Ig was included to measure the inhibitory capacity of the anti-IgG and anti-whole serum reagents. From the results (Table 2), the lymphocyte reactions with both anti-C3 coated EO, and Eh.an were similar in that both were abolished by anti-C3 and by antirabbit whole serum, but were not affected by anti-Ig. This showed that the rosette formation with Ehuman was a true immune adherence reaction dependent on
(d) Rosette-formation with guinea-pig erythrocytes Considerable numbers of lymphocytes and thymocytes incubated in autologous serum were found to have acquired an affinity for Eg.pig which formed strong, stable rosettes (Table 1). This reactivity was not acquired from heated serum or from unheated
the presence of C3.
Table 2. Tests for inhibition of the rosette-forming reactions given by EDTA-blood lymphocytes* (pre-incubated in autologous serumt) by antisera to (a) rabbit IgG and (b) rabbit whole serum, and by the IgG fraction of sheep anti-rabbit C3
Percentage of lymphocytes reacting with erythrocytes Final dilution Ox carrying Human Nase-treated Ox carrying Reagent used to treat lymphocytest of reagent anti-C3 (immune adherence) guinea-pig anti-Ig None Normal sheep serum Sheep (C 1 7/1 1 1 F) anti-rabbit IgG Sheep (T297) IgG anti-rabbit C3
Sheep (207, bleed 5) anti-rabbit whole serum
-
1/20 1/5 1/4 (3mg protein/ml) 1/20
69 69 67
56 58 59
72 79 80
95 95 lOw
0
0
75
89
0
0
24
44
*Lymphocytes isolated from EDTA-blood were 94% Ig-bearing cells. tLymphocytes incubated in autologous serum and washed as described in subscript of Table 1. tAll reagents were heat-inactivated and absorbed with rabbit erythrocytes. Lymphocytes which had been incubated with autologous serum were well washed and mixed with the appropriate reagent for 1 h at 40. They were then centrifuged and washed three times in the cold with RPMI 1640/5% iFCS before rosetting. w = Weak rosettes.
30
Anne B. Wilson, P. J. Lachmann & R. R. A. Coombs
The adherence of Eg.pig, however, was quite different and was affected neither by anti-C3 nor by anti-Ig, although it was partially inhibited by anti-rabbit whole serum. Unfortunately, no antisera to other purified components of rabbit complement were available to extend these inhibition tests, apart from anti-C6 (raised in C6-deficient rabbits) which had no effect on rosette-formation. 2.Effect of temperature on complement activation by lymphocytes and thymocytes The experiments with EGTA and EDTA indicated that both the classical and the alternative complement pathways were involved. In those tests, lymphoid cells were incubated with serum at 370 and then cooled and washed at 40, and it was not possible to know at which temperature complement activation occurred. Aliquots of lymphoid cells were, therefore, rotated in serum for 1 h either at 40 with subsequent washing at 40 or 370, or at 370 followed by washing at 40 or 37. The results of experiments on blood and thymus are given in Table 3. Other tests on lymph node lymphocytes (not reported) showed similar reactivity to those from blood. The temperature of incubation and washing had little effect on the numbers of lymphocytes reacting with anti-C3, although thymocytes gave a stronger reaction after incubation at 40. Complement
activation, therefore, took place even at low temperatures, and lymphoid cells incubated and washed at 40 always gave the maximum number of rosettes with Ehuman. This was probably because the immune adherence receptor is for C3b, most of which would be converted to C3bi or C3d during the 1 h incubation at 370. In short-term incubation experiments at 370 (not shown), immune adherence reactions were positive after only 1 min, and over 70% of lymphocytes reacted with anti-C3. The reaction of lymphoid cells with Eg.pig was also dependent on contact with serum at low temperatures; the strongest reactions being obtained on lymphocytes and thymocytes incubated and washed at 40. The reactivity was removed when lymphoid cells were warmed at 370 in the presence of serum, although in other tests (not shown) incubation of positive lymphocytes in serum-free medium did not affect their reaction with Eg.pig.
3. The in vitro uptake of Ig by lymphocytes and thymocytes
In some experiments, lymphocytes and thymocytes were tested by DARR for surface Ig both before and after incubation in serum. Tests with reagents specific
for y-Fc and ,u-Fc indicated that most normal Ig-bearing rabbit lymphocytes in blood and lymph nodes
Table 3. Incubation of blood lymphocytes and thymocytes in autologous serum: effect of temperature
Temperature (0)
Percentage of lymphoid cells reacting with erythrocytes
Incubation Wash*
Ox carrying Human Nase-treated Ox carrying anti-C3 (immune adherence) guinea-pig anti-Ig
Origin of lymphoid cells
Bloodt
Thymus
4 4 4 37 37 4 37 37 Untreated 4 4 4 37 4 37 37 37 Untreated
60 66 62 58 3 34 33 24 16 0
37 2
13 14 0 29 0 10 1 0
78 0 25 2 1 61 0 14 0 0
NT NT NI NT NT 73 2 35 8 0
* Where cells were washed at a different temperature from that of the incubation, they were equilibrated at the second temperature for 5 min before centrifuging. t Lymphocytes isolated from ACD blood. NT, Not tested.
31
Complement activation by rabbit lymphocytes IgM, but IgG was detected on only a minority (0-2%) of untreated cells and no evidence was found for the uptake of IgG during in vitro treatment with serum at either 40 or 37°. An increase in total Ig-bearing lymphocytes was found in some experiments on lymph node lymphocytes. One test showed a rise in Ig-positive lymphocytes of from 70 to 80% after incubation with serum at 370 and washing at 40, although the same cells washed at 370 showed no increase in Ig-bearing lymphocytes. Another experiment carried out entirely at 40 produced an increase in Ig-bearing lymphocytes from 63 to 84% in a cell suspension containing 42% T lymphocarry
cytes.
Much clearer evidence for the in vitro uptake of Ig obtained in tests with thymocytes, since untreated suspensions had a much lower 'background' of 0-3% cells with detectable surface Ig. Preliminary experiments had shown that thymocytes will pick up Ig from autologous serum (Table 1), and when tested at different temperatures, this was found to be a cold-dependent phenomenon (Table 3). Immunoglobulin was deposited on thymocytes at 40, but could be removed by washing at 37°. Further investigations showed that at least some of this Ig was IgM (e.g. in one experiment, 76% of thymocytes became Ig-positive and 25% reacted with antibody to u-Fc), but as with lymphocytes, no evidence was found for the acquisition of IgG. All rabbits tested were found to have circulating, was
cold auto-agglutinins, and the properties of the Ig acquired by lymphoid cells indicate that it may be complement-fixing, cold auto-antibody similar to those described in pathological conditions in man (Shumak, Rachkewich & Greaves, 1975; Pruzanski, Farid, Keystone, Armstrong & Greaves, 1975). 4. Effect of EGTA on complement activation at 40 and 370 The probable link between cold auto-antibodies and activation of the classical complement pathway was investigated by examining the inhibitory effect of EGTA at 40 and 370. Experiments on blood lymphocytes and thymocytes (Table 4) showed that complement activation at 40 was strongly inhibited by EGTA, whilst activation at 370 was relatively little affected. Also, EGTA had no effect on the uptake of Ig by thymocytes at 4°. It was thus apparent that complement activation at 40 was principally by the classical pathway, and was probably initiated by the interaction of (IgM?) cold auto-antibodies with lymphocyte or thymocyte cellmembrane antigens. At 370, however, complement was apparently activated by the alternative pathway.
5. Test to find whether peripheral lymphocytes carrying C3 were T or B cells Lymphocytes isolated from ACD blood were incu-
Table 4. Effect of EGTA on the uptake of C3 by peripheral blood lymphocytes and thymocytes (also uptake of Ig on thymocytes) incubated in autologous serum at 40 and 370
Percentage of/cells reacting with erythrocytes Origin of Temperature (0) Ox carrying Human Ox carrying lymphoid anti-C3 (immune adherence) cells Incubation Wash EGTA/Mg2 + anti-Ig
Blood*
4
4
37
37
Absent Present Absent Present
Thymus
Untreated 4
4
37
37
Untreated
Absent Present Absent Present -
70 17 74 80 2 70 14 47 39 2
*Lymphocytes separated from EDTA-blood
36 0 4 1 0 72 1 0 0 0
NT NT NT NT NT 52 57 1 2 2
32
Anne B. Wilson, P. J. Lachmann & R. R. A. Coombs
bated in serum at either 4 or 37°. They were then tested by mixed rosette forming reactions, using fluoresceinconjugated, papain-treated Erabbit as a T-cell marker (Wilson et al, 1975). Lymphocytes not reacting with the Erabbit were regarded as B cells since few 'null' cells are found in the rabbit. After incubation in serum at 40, between 90% and 100% of the B lymphocytes reacted with anti-C3 and were positive for immune adherence. The same proportion of B cells also formed rosettes with Eg.pig. The percentages of T lymphocytes reacting, however, were much lower; 60% combining with anti-C3, 17% with Ehuman and 37% with Eg.pig. The C3 was thus bound preferentially to B lymphocytes. A similar result was obtained at 370 when C3 was detected on 97% of B cells and on 70% of T cells.
DISCUSSION
Complement activation occurred when rabbit blood or lymph node lymphocytes or thymocytes were incubated in autologous serum. The mechanism of activation varied with the temperature of incubation, proceeding by the classical pathway at 4° and by the alternative pathway at 37°. Activated C3 deposited on the surface of lymphoid cells was detected by rosetteformation with E., carrying antibodies to C3 and by immune adherence of Ehuman. In suspensions containing both T and B lymphocytes, C3 was preferentially bound to B lymphocytes and there was blocking of cell-surface receptors for C3b and C3d. The presence of these receptors was not essential for the uptake of C3 since it was also detected on thymocytes and some peripheral T cells which lacked C3-receptors. The C3 on these T cells was presumably attached by hydrophobic bonds as 'nascent' C3b (McConnell & Lachmann, 1977). There was no evidence for a reduction in cell viability following activation of complement. At 40, complement activation was almost exclusively by the classical pathway, as shown by inhibition with EGTA/Mg2 +. In experiments with thymocytes, C3 activation at low temperatures was accompanied by an uptake of Ig, which included IgM but not IgG. There was also evidence that some peripheral T lymphocytes can acquire surface Ig when incubated with serum at 40. It is possible that the Ig was passively adsorbed by reaction with receptors for IgM such as those shown on T cells of man (McConnel & Hurd, 1976) and mouse (Santana, 1977). The rabbit Ig, however, shares properties such as its reactivity at 40 and
release at 370, with the complement activating, cold auto-antibodies described in man (Pruzanski et al, 1975; Shumak et al, 1975). These human antibodies may react with lymphocyte surface determinants antigenically identical to erythrocyte membrane antigens (Shumak et al, 1975). The finding of cold auto-agglutinins in rabbit sera favours the hypothesis that the reaction of such antibodies with lymphocyte antigens is the trigger for complement activation at 40. In contrast to the reaction at 40, complement activation by rabbit lymphocytes and thymocytes at 37° was not inhibited by EGTA/Mg2 +, and was therefore by the alternative pathway. A similar mechanism of complement fixation has also been described in homologous systems involving human tumour and other cell lines incubated in normal human serum (Okada & Baba, 1974; Budzko et al, 1976; Theofilopoulos & Perrin, 1976; Yefenof et al, 1977). These authors found no evidence of complement activation by normal human lymphocytes, although other workers claim that there is a blocking of C3 receptors when these cells are incubated in normal human serum (Abrahamsohn, Nilsson & Abdou, 1974; Erdei, Fiist, Sarmay, Medgyesi & Gergely, 1977). Neo-antigens from the terminal sequence of complement proteins (C5b-C9) have recently been demonstrated on normal human peripheral blood lymphocytes isolated from defibrinated blood by Ficoll-Hypaque centrifugation (Sundsmo, Kolb & Muller-Eberhard, 1978). As with rabbit blood (Wilson et al, 1978), the complement components on these human lymphocytes were acquired in vitro since they were not present on cells isolated from blood collected in EDTA. Unlike the present findings with rabbit C3, the acquisition of C5b-C9 neo-antigens by human lymphocytes during in vitro incubation with serum was inhibited neither by EDTA nor by heating the serum to 560 (Sundsmo, Curd, Kolb & Muller-Eberhard, 1978). As these human cells were not examined for C3, it is not clear whether the generation of activated terminal complement components is linked in any way with alternative pathway activation such as that described in the present investigation. Many agents are known to trigger the alternative complement pathway. These include carbohydrates such as zymosan, inulin and bacterial endotoxins, certain types of antibodies and C3-nephritic factor. C3b itself can activate the alternative pathway by means of the feedback cycle in which the C3 convertase, C3b-Bb, is generated. This process is normally regulated by the action of conglutinogen activation factor
Complement activation by rabbit lymphocytes (C3b inactivator) (KAF) and B1 H which destroy the C3b-Bb complex. Recent work by Fearon & Austen (1977), however, showed that the membranes of certain cells, in this case rabbit erythrocytes, protect bound C3b and C3b-Bb from the action of KAF and ,I3H. The C3b feedback cycle therefore proceeds more or less uncontrolled and C3 conversion is greatly increased. As mentioned above, trace amounts of activated C3 may be present in rabbit serum. It is conceivable, therefore, that rabbit lymphocytes and thymocytes also have 'protected' surfaces and that cell-membrane binding of small amounts of C3b from the serum is sufficient to trigger the rapid and extensive activation of C3 observed in the present investigations. The nature of the reaction between serum-incubated lymphoid cells and Eg.pig remains undetermined. Strong rosette-formation with Eg.pig was produced by lymphocytes and thymocytes incubated in serum at 40, but not at 37°. Rosettes were not formed after incubation in serum containing EGTA/Mg2 + or EDTA, nor when the serum had been heat-inactivated. This indicated that the receptors on Eg.pig may react with one or more components associated with the classical, but not with the alternative, pathway of complement activation (i.e. activated C 1, C4 or C2). Further characterization of the factor(s) responsible for this affinity reaction was not possible in the present study as no purified components of rabbit complement were available. The present findings with rabbit lymphocytes are important in two respects. The first of these has been largely dealt with in an earlier report (Wilson et al, 1978) and involves the practical aspects of isolating lymphocytes, especially from blood. The activation of complement, leading to blocking of receptors for C3, and the uptake of Ig at low temperatures must be avoided in order to retain, as far as possible, the in vivo characteristics and reactivity of the purified lymphocytes. For this reason, it is essential to take blood into an anticoagulant such as citrate which also inhibits complement activation (EDTA kills rabbit T cells), and to avoid cooling the lymphocytes while in contact with serum. The second point for consideration must be the possible in vivo activation of the alternative complement pathway by lymphoid cells. Although in the present study on normal animals only a small percentage (up to 3%) of untreated lymphocytes or thymocytes had detectable surface C3, it appears that only slight changes in cell properties might be necessary to enable them to trigger complement activation. For
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example, the infection of human acute myeloid leukemia cells with Moloney Sarcoma virus enables them to activate complement by the alternative pathway (Okada & Baba, 1974), and enhanced activation has been demonstrated with lymphoblastoid cell lines infected with EB virus (McConnell & Lachmann, 1977). Changes in lymphocytes brought about in vivo by infectious agents or by other alterations in their environment might therefore be sufficient to initiate complement activation.
ACKNOWLEDGMENTS We wish to thank Mrs A. Fieldes for able technical assistance, and the Medical Research Council for financial support. We are also grateful to Dr A. Feinstein for the gift of sheep anti-rabbit ,i-Fc and y-Fc reagents.
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