Immunology 1977 32 623
Histamine release from human leucocytes A SERUM FACTOR NECESSARY FOR THE INDUCTION OF HISTAMINE RELEASE AND DESENSITIZATION BY PROTEIN A
B.-A. PETERSSON & G. STALENHEIM Department of Medical and Physiological Chemistry, University of Uppsala, The Biomedical Center, Uppsala, Sweden
Received 17 June 1976; accepted for publication 15 October 1976
ing the leucocytes to release histamine on protein A treatment. Beside being necessary for protein A-induced histamine release, the factor (or factors) is also
Summary. The ability of human leucocytes to release histamine on protein A treatment is lost when the cells are washed repeatedly. It is, however, possible to restore the sensitivity to protein A treatment by incubating the leucocytes in serum. Treatment of the cells with purified IgG does not restore the activity. The material responsible for the resensitization is eluted both in the second and the third protein peak when serum is chromatographed on Sephadex G-200, indicating the possible existence of several active factors. Material with low immunoglobulin content, but with retained capacity to resensitize leucocytes to release histamine on protein A treatment, was obtained by repeated chromatography of peak III material on the Sephadex G-200 column. Furthermore, material from the second and third peaks from Sephadex G-200 deprived of their IgG by passage through a protein A Sepharose or a DEAE-cellulose column had the same capacity to resensitize the leucocytes as unseparated material. When serum was separated by Pevikon block electrophoresis, most of the activity was detected in the a and 8 regions but only little in the y region. The serum fractionations indicate that neither IgG nor the other immunoglobulins are the factor(s) responsible for resensitiz-
essential for protein A-induced desensitization of human leucocytes. INTRODUCTION Protein A from Staphylococcus aureus induces histamine release from human leucocytes. It has been suggested that this reaction is mediated by cell surface attached IgG molecules (Martin & White, 1969, Petersson & Stalenheim, 1975). If the cells are washed several times they lose their capacity to release histamine on protein A treatment while the histamine release induced with optimum amounts of anti-IgE is unaltered. Histamine release from repeatedly washed leucocytes can, however, be triggered by protein A in complex with IgG or with the Fc part of IgG (Petersson, 1975). One proposed explanation for the decrease in protein A reactivity is that IgG is lost during the repeated washings of the leucocytes. In this report, the capacity of serum and different serum fractions to resensitize the leucocytes to release histamine on protein A treatment has been investigated.
Correspondence: Dr B.-A. Petersson, Department of Medical and Physiological Chemistry, The Biomedical Center, Box 575, S-751 23 Uppsala, Sweden.
623
624
B.-A Petersson & G. Stalenheim MATERIALS AND METHODS
Protein A and protein A Sepharose Protein A was purified from lysostaphin-digested bacteria by affinity chromatography on IgG Sepharose; protein A Sepharose was prepared as earlier described (Sjdquist, Meloun & Hjelm, 1972; Hjelm, Hjelm & Sj6quist, 1972).
Human IgG This was isolated from sera by chromatography on DEAE cellulose followed by chromatography on a Sephadex G-200 column (Sj6quist & Stalenheim, 1969) in Tris-buffered saline containing KCI (Tris 0 025 M, NaCl 0 120 M and KCI 0 005 M). In most of the experiments the IgG used was isolated from sera of protein A positive donors, i.e. from donors whose leucocytes release histamine on protein A treatment. In some experiments IgG and leucocytes were isolated from the same donor. Immunoelectrophoresis experiments did not reveal the presence of contaminants in the IgG preparations. In some experiments IgG from pooled serum (Cohn fraction II Kabi AB, Stockholm) was used. Antisera to IgE These were raised in rabbits, and IgG from these antisera was prepared as published (Petersson & Stalenheim, 1975). This rabbit IgG preparation will be called anti-IgE throughout this report.
Pepsin digestion of immunoglobulin Pepsin-digested IgG from anti-IgE sera was passed through a protein A-Sepharose column to remove material containing intact Fc (Petersson & Sta'lenheim, 1975). The unadsorbed material was subsequently run through a Sephadex G-150 column and the main peak was collected. This material will subsequently be called F(ab')2 anti-IgE. IgG-preparation from rabbit antisera against human IgG An IgG preparation from rabbit antiserum against human IgG (heavy chain specific) was purchased from Dakopatts, Copenhagen. Throughout this report this rabbit IgG preparation will be called anti-IgG. Before use, a 1: 5 dilution of anti-IgG was dialysed against Tris-buffered saline containing KC1 for 72 h at 4°. Separation of serum on Sephadex G-200 Human serum was run on a Sephadex G-200 column
in Tris-buffered saline containing KCI. The column was eluted at the rate of 16 5 ml/h and each fraction contained 5 5 ml. In some experiments, fractions (usually every third) were concentrated and their capacity to resensitize leucocytes was tested. In other experiments, the separated material was pooled according to the three peaks obtained. Peak II (immunoglobulin-containing peak) and Peak III (albumin-containing peak) were further processed. Material low in immunoglobulin content was obtained by repeated chromatography of the peak III material on Sephadex G-200. Separation ofpeak II andpeak III material on protein A-Sepharose Peak II or peak III material was also run on a protein A-Sepharose column (15 ml). Material not bound to the column was eluted with Tris-buffered saline containing KCI. Bound material, eluted with 0-1 M glycine-HCI, pH 2-5, was pooled and dialysed against Tris-buffered saline containing KCI. The separated material was concentrated to the volume of the starting material.
Separation of peak II material on DEAE-cellulose column Peak II material dialysed against 0 0175 M phosphate buffer pH 6 3 was applied on a DEAE-cellulose column in the same buffer. After IgG had been collected from the column with the starting buffer, bound material was eluted with 0 0175 M phosphate buffer pH 6 3 containing 1 0 M NaCI. The separated materials were concentrated to the volume of the starting material and dialysed against Tris-buffered saline containing KCl before use in the histamine release experiments. Preparative block electrophoresis This was run according to Muller-Eberhard (1960). Pevikon 870 (Kema Nord, Stockholm, Sweden) was equilibrated with barbital buffer pH 8 6, , = 0 005, and the electrophoresis run at 200 V for 24 h. The block was cut into 1-cm segments, eluted, and the protein content was measured according to Lowry, Rosebrough, Farr & Randall (1951). Quantitative determinations of immunoglobulins IgG and IgA determinations were performed on concentrated samples with the single radial immunodiffusion method. The IgE content was deter-
625
Histamine release from human leucocytes
mined by radioimmunological technique according to the method of Ceska & Lundkvist (1972). Isolation of leucocytes Leucocyte suspensions were isolated from human blood according to Lichtenstein & Osler (1964). Ethylene-diamine tetraacetic acid (EDTA) was used as anticoagulant and the cells were washed with Tris-A (Tris-buffered saline containing KCl and albumin; Lichtenstein & Osler, 1964) unless otherwise stated. The leucocytes were washed according to one of two procedures. The first method includes one washing at 40, while leucocytes prepared according to the other were washed three times. The latter procedure also includes incubation of the cells at 370 for 20 min after the second washing. Throughout this report, cells washed three times will be called 'repeatedly washed leucocytes' (Petersson, 1975). Cell donors were selected in order to get leucocyte preparations releasing more than 30 per cent histamine on treatment with protein A after a single washing. Histamine release The histamine release experiments were performed in a 20-ml sample volume containing 06-10 x 107 leucocytes per ml. A two-step procedure was used to test the capacity of serum or serum fractions to reconstitute the ability of the leucocytes to release histamine on protein A treatment. In the first step, cell samples were incubated in 10 ml of serum or serum fractions. Buffer controls were included. After 40 min incubation at 370 the cells were spun down and washed in Tris-A at 4°. In the second step, the samples were tested with a fixed amount of protein A (1 yg/ml) in 2 0 ml Tris-ACM (Tris-A containing Mg2 + and Ca2 +) for 40 min at 37°. The histamine release during the second step was determined. This procedure will be called 'to resensitize leucocytes' throughout this report. The total histamine content of the cells was determined in two samples of cells after being boiled for 3 min. Histamine assay Histamine was biologically assayed using segments of guinea-pig ileum as published (Cochrane & Muller-Eberhard, 1968; Petersson & Sta'lenheim, 1975). Human IgG (Cohn fraction II, Kabi AB, Stockholm) was added in excess to protein A and anti-IgG containing samples (Petersson & Stalenheim, 1975). B
RESULTS Effect of heparin and Tyrode solution on protein Ainduced histamine release In one type of experiment half of the blood was poured into a mixture of dextran and EDTA and the other half into a mixture of dextran and heparin. After a single washing of the leucocytes protein A (1 ,ug/ml) released the same amount of histamine from the cells isolated in the presence of EDTA or heparin. From repeatedly washed cells, no histamine was released in any of the cases. In another set of experiments, one portion of the leucocytes was washed with Tyrode solution during the preparation (Ohazaki, Ilea, Ohazaki, Reisman & Arbesman, 1975) and the other portion was washed with Tris-A. After one or three washings the Tyrode washed cells were treated with protein A (1 pg/fml) in Tyrode solution and the Tris-A washed cells with protein A (1 pg/ml) in Tris-ACM. Histamine release from once-washed leucocytes was slightly higher if the leucocytes had been treated with Tyrode solution than if the cells were processed according to the original procedure (Lichtenstein & Osler, 1964). No histamine was released after repeated washings in any of the cases. Treatment of repeatedly washed leucocytes with IgG or serum Expt 1 in Table 1 shows the effect of treating repeatedly washed leucocytes with 1 0 ml IgG solution (6 mg/ml) or with 1 0 ml serum diluted 1:2 for 40 min at 37°. A control with buffer was also included. After one washing, each sample was incubated with protein A (1 pg/ml) in Tris-ACM for 40 min at 370 and the histamine released in the second step was determined. Leucocytes preincubated with buffer or IgG liberated no histamine at all on protein A treatment while samples pretreated with serum released 44 per cent of their histamine content. The effect of IgG was tested in ten additional experiments with IgG concentrations varying between 4-7 and 6 8 mg per ml, but in a few experiments lower concentrations (down to 1 mg/ml) were tested. In no case did protein A release histamine from buffer pretreated cells, while serum pretreatment resulted in histamine release of 36-64 per cent. In ten of the experiments protein A induced no histamine release from IgG-pretreated cells. In the remaining experiment the release was 7 per cent.
B.-A. Petersson & G. Stdlenheim
626
Table 1. Protein A-induced histamine release from resensitized human leucocytes
Additive in step 1
Expt Histamine release in step 2 (%)
Exp. No. 1
2
3
4
5
Pretreatment at 370 for 40 min Buffer IgG 6 mg/ml Serum 1: 2 Buffer Serum 1:2 Dialysed serum Serum treated at 560 for 30 min Buffer Serum 1:2 Peak III material* Buffer Serum 1:2 Peak III material* Buffer Serum 1:2 Peak III material*
Table 2. Anti-IgE and anti-IgG induced histamine release from human leucocytes. Specificity of the releasing agents
Test with protein A (1 jug/ml) 0 0 44 0 60 57
60 0 40 48 0 49 52 0 59 56
* This peak III material contains 20 pg IgG per ml and less than 2 jug IgA and 2 ng IgE per ml.
Effect of washings on anti-IgG induced histamine release Preliminary experiments showed that a 1: 200 dilution of anti-IgG gave maximum histamine release from most of the leucocyte preparations tested, and this concentration of anti-IgG has been used throughout. To test the specificity of anti-IgG, samples diluted 1:100 were mixed with 1 or 1O pg of IgE or 10 pg of IgG in a vol. of 1 ml. Buffer controls (Tris-ACM) were also included. After incubation at 370 for 30 min, one ml of cell suspension was added to each sample which was then incubated for an additional 40 min at 37° and the liberated histamine was determined. For comparison, anti-IgE (3 ug/ml) was preincubated in the same way with IgE, 1 pg/ml, IgG, 10 pg/ml or buffer. Table 2 shows that preincubation of anti-IgG with IgG completely abolished the capacity of anti-IgG to release histamine. Pretreatment of anti-IgG with IgE did not significantly decrease its histamine releasing capacity. IgE completely inhibited anti-IgE induced histamine release, while treatment with IgG had no effect. To test the effect of washings on anti-IgG in-
no.
Releasing agent
Anti-IgG 1:200+ buffer Anti-IgG 1:200+0 5 ,pg IgE/mI Anti-IgG 1: 200+ 50 pug IgG/ml Anti-IgE 1-5 ,pg/ml+ buffer Anti-IgE 1-5 pg/ml+0 5 pg IgE/ml Anti-IgE 1-5 ,g/ml+ 5.0 pg IgG/ml 2 Anti-IgG 1:200+ buffer Anti-IgG 1: 200+ 50 ,pg IgE/ml Anti-IgG 1: 200+ 5 0 jug IgG/ml Anti-IgE 1-5 pg/ml+ buffer Anti-IgE 1-5 ug/ml+0.5 pg IgE/ml Anti-IgE 1-5 ug/ml+ 50 pg IgG/ml 3 Anti-IgG 1:200+ buffer Anti-IgG 1: 200+ 5 pg IgE/ml Anti-IgG 1: 200+ 5 plg IgG/ml 1
Histamine release(%) 24 21 0 52 0 56 32 31 0 60 0 62 39 38 0
duced release cell samples were washed once or repeatedly before the addition of anti-IgG (1:200) in Tris-ACM. For comparison the effect of washings on anti-IgE or protein A triggered release was also tested. The experiments in Table 3 show that antiIgG and anti-IgE initiated release was not decreased as an effect of washings. The protein A-induced release on the other hand decreased from high values after one washing down to low values after repeated washings. The effect of dilution, dialysis or heat treatment on the capacity of serum to re-constitute the ability of the leucocytes to release histamine on protein A treatment Samples of repeatedly washed leucocytes were incubated with 1-ml portions of undiluted and diluted autologous serum. A control with Tris-A was included. After 40 min at 370 the cells were spun down and washed once with 2 0 ml Tris-A at 4°. In the second step each sample was incubated with protein A, 1 pg/ml, in Tris-ACM for 40 min at 370, and liberated histamine was measured. Fig. 1 shows the results obtained with three different leucocyte populations. In most experiments, the 1:2 serum dilution gave maximal re-constitution. Serum diluted 1: 20 had no capacity to resensitize leucocytes in any of the experiments performed. Samples incubated with buffer in step one released no histamine at all
627
Histamine release from human leucocytes Table 3. Anti-IgE, anti-IgG and protein A-induced histamine release from once or repeatedly washed human leucocytes
Releasing agent
Expt no.
Anti-IgE 1 5,pg/ml Anti-IgG 1:200 Protein A 1 pg/ml Anti-IgE 1 5 pg/ml Anti-IgG 1:200 Protein A 1 pg/ml F(ab')2anti-IgE 25 ,ug/ml Anti-lgG 1:200 Protein A 1 ug/ml
I
2
3
Histamine release
Histamine release after
after one washing
repeated washings*
(%)
(%)
60 32 35 52 24 37 46 15 37
56 39 0 46 25 0 47 20 8
* The leucocytes were washed three times and incubated for 20 min at the second and third washing.
370
between the
when treated with protein A in the second step. Furthermore, no spontaneous release was obtained on buffer treatment in the second step from samples incubated with serum in the first step. In the following experiments of this report, the 1:2 serum dilution has been consistently used. In five experiments the capacity of native, dialysed and heattreated serum to resensitize leucocytes was studied. Serum (2 ml) was dialysed during 48 h at 40 against 5 x 500 ml Tris-buffered saline containing KCI. Heat treatment was performed at 560 for 30 min. From samples pretreated with native serum (1: 2) the protein A induced release varied between 44 and 89 per cent. At most dialysis resulted in a 5 per cent and heat treatment in a 9 per cent decrease of the capacity of serum to resensitize leucocytes. In some of the experiments dialysed or heat-treated serum gave a somewhat better resensitization than native serum. In additional experiments serum was treated at 56° for 90 or 180 min and in these experiments the stimulating effect varied. In some experiments there was no inactivation of the serum, while in others all reconstituting capacity was lost.
-c E
.r_
Q-
1/8
1/4
Humon
serum
1/2 (dilutions)
1/1
Figure 1. Histamine release induced by protein A from leucocytes resensitized with diluted serum. The different curves represent experiments with cells from different donors.
Effect of washings on resensitized leucocytes Samples of repeatedly washed leucocytes were incubated with serum diluted 1:2. After 40 min incubation at 370 the samples were washed once, twice, or three times, and incubated in the second step with protein A, 1 ,ug/ml, in Tris-ACM for 40 min at 37°. The experiments in Table 4 show that the
B.-A. Petersson & G. Stdlenheim
628
Table 4. Protein A-induced histamine release from extensively washed and resensitized human leucocytes. Effect of washings after the resensitization Additive in step 1 Expt no.
Pretreatment at 370 for 40 min
Number of washings after step 1*
Histamine release in step 2 (Y.)
Buffer Serum Serum Serum Buffer Serum Serum Serum Buffer Serum Serum Serum
1 1 2 3 1 1 2 3 1 1 2 3
0 64 46
I
2
3
Test with protein A (1 pg/ml)
9 0 44 42 27 0 39 20 0
Serum was used diluted 1:2.
*Samples washed three times were incubated at 370for 20 min between the second and third washing.
protein A-induced release decreased again with increasing number of washings.
Separation of serum on Sephadex G-200 Human serum (5 ml) was run on a Sephadex G-200 column (2 5 x 90 cm) in Tris-buffered saline containing KCL. Fractions (usually every third) were concentrated five times and their capacity to resensitize leucocytes was tested. In Fig. 2, the solid line shows the optical density and the dotted line represents the capacity of the fractions to resensitize the leucocytes. One activity peak was detected in protein peak II and another in protein peak III. No activity was detected in peak I. When peak II or peak III material was rechromatographed on the same Sephadex G-200 column, the sensitizing material was eluted in the same position as in the original chromatography. No additional activity peak was obtained in any case. Peak III material with very low immunoglobulin content was obtained by repeated chromatography on Sephadex G-200. Four millilitres of peak III material were obtained from 7 ml of human serum. Expts 3-5 in Table 1 show that this peak III material has the same capacity as native serum (1:2) to resensitize leucocytes. Leucocytes preincubated with buffer released no histamine at all. This peak III material contained
20 pg IgG and less than 2 pg IgA and 2 ng IgE per ml. Peak II and III material from serum separated on Sephadex G-200 were run separately on a protein A-Sepharose column and the capacity of the bound and unbound material to resensitize the leucocytes was tested. Unfractionated peak II and III material as well as serum were also used. Table 5 shows the results of these experiments. Peak II and III material not bound to protein A-Sepharose resensitized the cells to roughly the same extent as unseparated peak II and III material. Bound material had no or only a low capacity to resensitize the cells. Peak III material which passed through protein A-Sepharose contained less than 10 ,g IgG, 2 ,ug IgA and 2 ng IgE per ml. The passage of peak II material through protein A-Sepharose removed 93-96 per cent of the IgG. Peak II material was also deprived of IgG by passage through a DEAE-cellulose column. In this case the active material was bound to the column while IgG was eluted with the starting buffer. The experiments in Table 5 show that peak II material bound to the DEAE-cellulose column resensitizes the leucocytes to the same extent as unseparated peak II material. The material eluted with the starting buffer had no or only low capacity to resensitize leucocytes. Passage of peak II material through the
629
Histamine release from human leucocytes
4). The albumin and a-globulin pools did not show any precipitin line in the y region, whereas contamination of the ,8-globulin pool with y-globulin could not be excluded.
28
80
2-0
C: c
0o00
37°.
60
z w
o
-e Ln
D0
E
40 P
1.0
Z W
20
20
30
40
50
60
Protein A-induced desensitization A three-step procedure was used to study the protein A-induced desensitization of leucocytes pretreated with serum (1:2) or buffer. In the first step, repeatedly washed leucocytes were incubated with After one serum (1:2) or buffer for 40 min at washing, the samples were incubated in the second step with protein A, 1 pug/ml, in Tris-AM (Tris-A containing Mg2 + but no Ca2+) for 40 min at 37°. Buffer controls were also included. After the second step the samples were washed twice and tested for desensitization by adding an optimum amount of F(ab')2 anti-IgE (2 5 #ug/ml) in Tris-ACM and incubating for 40 min at 370 (third step). Liberated histamine in the second and third steps was measured. Table 6 shows that pretreatment of cells with serum enhances the desensitizing activity of protein A yielding a desensitization of 75-100 per cent in comparison with 10-28 per cent desensitization of buffer pretreated cells.
70
Fraction number
Figure 2. Histamine release induced by protein A from leucocytes resensitized with serum fractions chromatographed on a Sephadex G-200 column; (O C) indicates the optical density at 280 nm and (a -* -* - *) the capacity of the tested fractions to resensitize the leucocytes to release histamine on protein A (I pg/mI) treatment. Fraction volume was 5-5 ml.
DEAE-cellulose column removed 94 per cent of IgG from the active material. Separation of serum on Pevikon block electrophoresis Human serum (10 ml) was fractionated by Pevikon block electrophoresis and the material was pooled as indicated in Fig. 3. Each pool was dialysed against KCI-containing Tris-buffered saline and concentrated to 10 ml. One-millilitre portions of each pool were used to test their capacity to resensitize the leucocytes. The curve in Fig. 3 shows the optical density of the eluate and the columns represent the capacity of each pool to resensitize the leucocytes. Most of the active material was found in the a and the ,6 fractions and very little in the y region. Immunoelectrophoresis was run on the concentrated pools using antiserum to whole human serum (Fig.
DISCUSSION The most plausible explanation for the observation that protein A-induced histamine release is reduced on washing of leucocytes is that loosely attached IgG is eluted from the cell surface (Petersson, 1975). Consequently, it was attempted to reattach IgG onto the cells in vitro, and whole human serum was actually found to efficiently restore the protein A elicited histamine release of the cells (Table 1, Fig. 1). However, after incubating the cells for 40 min at 370 with purified IgG at concentrations varying from 1-6 mg/ml, no activity was regained (Table 1). The histamine release observed on challenging serum pretreated cells with protein A is thus unlikely to be the effect of protein A-IgG complexes formed with soluble IgG remaining after the washings. Moreover the amount of IgG calculated to remain in the fluid phase is far too low to give enough complex with protein A to be responsible for the histamine release observed (Petersson, 1975). As with the activity of 'native' cells, the activity of resensitized cells is readily diminished by three
B.-A. Petersson & G. Stdlenheim
630
Table 5. Protein A-induced histamine release from resensitized human leucocytes
Additive in step 1
IgG concentration (mg/ml)
Expt no.
Pretreatment at 370 for 40 min
I
Buffer Serum* Peak III material Peak III material not bound to protein A Sepharoset Peak III material bound to protein A Sepharose Peak II material Peak II material not bound to protein A Sepharose Peak II material bound to protein A Sepharose Buffer Serum Peak II material Peak II material not bound to protein A Sepharose Peak II material bound to protein A Sepharose Peak II material not bound to DEAE-cellulose Peak II material bound to DEAE-cellulose Buffer Serum Peak II material Peak II material not bound to DEAE-cellulose Peak II material bound to DEAE-cellulose
2
3
*
Histamine release in step 2 (0) Test with protein A (I pg/ml)
0-14 < 0 01 Not tested 50 0-2 3-7 6-3 0 45 4-8 56 04 6-3 56 04
° 63 59 56 0 66 54 0 0 49 54 44 8 12 53 0 40 42 0 38
When serum was used for resensitization it was diluted 1:2.
t This peak III material contains less than 2 pg IgA and 2 ng IgE per ml.
washings (Table 4). This is compatible with the concept of some factor being eluted from the cells. The fact that anti-IgG readily releases histamine from extensively washed cells does not, of course, exclude IgG as the factor eluted. The washings could remove part of the cell-bound IgG leaving enough for anti-IgG but not enough for protein A elicited release. That anti-IgG induced release is not diminished as an effect of washings, is in agreement with experiments performed by Lichtenstein (personal communication). Since, generally, washing does not diminish the reactivity of the cells to anti-IgE or anaphylatoxin the factor eluted is either not necessary for the histamine release by these agents, or is needed in lesser amounts than those required by protein A to trigger the cells. In order to characterize the serum factor responsible for the resensitization of cells, serum was fractionated on a Sephadex G-200 column (Fig. 2). Activity was recovered from the second and third but not from the first peak. The material recovered in rechromatographed peak III was found to contain 20 ug of IgG and less than 2 pug IgA and 2 ng IgE per ml but it still had a high capacity, even in com-
parison with unfractionated serum, to resensitize leucocytes. To evaluate further the involvement of IgG, peak II and peak III materials were passed over a protein A-Sepharose column. The active material from both preparations went straight through the column without binding to protein A. Almost all the IgG was bound and active peak III material, passed through the protein A-Sepharose column, contained less than 10 pg IgG per ml. The IgG absorbed from the two preparations could be recovered from the column by lowering the pH of the eluting buffer. When peak II material was fractionated on a DEAE-cellulose column IgG went straight through while the material with resensitizing capacity was bound to the column. The active material was eluted from the column with a buffer containing a high salt concentration. In Pevikon block electrophoresis, the most active material migrated with the a- and fl-globulins (Fig. 3). Some activity was recovered in the albumin peak but only little was found in the y-globulin fraction. These fractionations indicate that IgG could not be the serum factor responsible for resensitization of cells to protein A-induced histamine release. Furthermore,
Histamine release from human leucocytes °0r +
I-
-i
-I
F
0-8
E o 0-6 0-
(s c as
D0
Jo 04 _
i 0-2
K1I
14
18
K,' 13 '--Y
22 26 30 Fraction number
34
38
Figure 3. Histamine release induced by protein A from leucocytes resensitized with pools from serum fractionated by Pevikon block electrophoresis. The anode is to the left and the arrow shows the application line. (o) indicates the optical density at 540 nm, and the material was pooled as shown in the upper part of the figure. The protein concentrations of the pools were: Pool 18-22, 33-2 mg/ml; pool 23-25 4-7 mg/ml, pool 26-29 6-8 mg/ml, pool 30-32 5-2 mg/ml and pool 33-36 5 0 mg/ml. The bars represent the capacity of each pool to reconstitute the ability of the leucocytes to release histamine on protein A (1 pug/ml) treatment.
our results make it unlikely that any other immunoglobulin could be the responsible factor. There could be more than one serum factor with the activity discussed. Alternatively, one factor could exist in a polymeric state or associated with other serum components. The appearance of two peaks in the Sephadex G-200 chromatogram does not seem to be due to an equilibrium between the active material in peaks II and III, since rechromatography of either of them gave essentially homogenous activity peaks with the same elution characteristics as the starting material. It has been reported that serum components have enhancing activity on allergen and anti-IgE induced histamine release (Lichtenstein & Osler, 1966; Ishizaka, Ishizaka, Johansson & Bennich, 1969; Grant & Lichtenstein, 1973). One of the serum
631
factors found by Lichtenstein and co-workers has sufficiently low molecular size to pass dialysis membranes and another is macromolecular and excluded from Biogel P 300 (Lichtenstein, 1968). A third enhancing factor described in the same report could be eluted from leucocytes. Material capable of restoring the activity of cells to release histamine on protein-A treatment is found not to pass through dialysis membranes when serum is dialysed against a small volume of buffer. Nor does the activity decrease in serum after prolonged dialysis against large volumes of buffer. Thus the factor under study is not identical with the low mol. wt factor studied by Lichtenstein. If the lipoprotein described by the same author was responsible for some of the activity observed by us, some activity should have appeared in the first peak of the Sephadex G-200 chromatogram. Cells extracted according to Lichtenstein (1968) do not release histamine on protein A treatment but the extract, even if concentrated four times, has no capacity to restore the activity of repeatedly washed cells. Since heating (560, 30 min) does not diminish the resensitizing activity of serum, haemolytic complement is not likely to be responsible. This is in accordance with what has been found for allergen and anti-IgE-induced histamine release (Ishizaka et al., 1969; Grant et al., 1973). Immunoglobulin-mediated histamine release involves a mechanism which is activated even in the absence of calcium ions. Prolonged treatment in calcium-free medium induces a state of unresponsivness in the leucocytes (Lichtenstein, 1971). Earlier investigations have also shown that histamine release mediated by IgE and IgG at least partially takes place by the same biochemical mechanism (Petersson, 1975; Petersson & Stailenheim 1975). Anaphylatoxin, on the other hand, induces histamine release and desensitization by a different mechanism (Petersson, Nilsson & StAlenheim, 1975). Table 6 shows that the serum factor (or factors) necessary for protein A-induced histamine release is needed even for the desensitization induced by protein A in the absence of calcium ions. It could be concluded that serum contains materials which interact with basophils to make them liable to release histamine on challenge with protein A. This activity does not seem to be a property of IgG or any other immunoglobulin. Since activity is found in two peaks in Sephadex G-200 chromatography more than one factor could be involved. The activity is associated with high mol. wt material and
632
B.-A. Petersson & G. Stdlenheim
Figure 4. Immunoelectrophoresis of serum and the five pools obtained, when serum was fractionated by Pevikon block electrophoresis. The electrophoresis was run 2 h and the anti-serum used was against human serum proteins. The anode is to the right and the samples were from top to bottom: serum, pool 18-22, pool 23-25, pool 26-29. pool 30-32 and pool 33-36. Table 6. Protein A-induced desensitization of human leucocytes. Effect of serum pretreatment
Additive in step 2 Histamine release in step 3 (%) Expt no.
Treatment Additive in in the absence of step 1 Ca2+
1
Serum Serum Buffer Buffer
2
Serum Serum Buffer Buffer
3
Serum Serum Buffer Buffer
Buffer Protein A 1 pg/ml Buffer Protein A 1 ug/ml Buffer Protein A 1 pg/ml Buffer Protein A I pg/ml Buffer Protein A 1 pg/ml Buffer Protein A 1 pg/ml
Histamine release in step 2 (0/)
Test with F(ab')2 anti-IgE (2-5 ug/ml)
0 0 0 0
55 0 50 45
0 0 0 0
46 0 43 31
0 0 0 0
92 23 89 68
Repeatedly washed leucocytes were used. Control samples were treated with buffer or serum (1: 2) in the first step and with protein A (1 pg/ml) in Tris-ACM in the second step. In experiment 1 the histamine release was 3 per cent from buffer-treated and 44 per cent from serum-treated leucocytes. The corresponding figures for experiment 2 was 7 and 40 per cent and for experiment 3 15 and 88 per cent.
Histamine release from human leucocytes moves as a- and fl-globulins in electrophoresis. Furthermore, the activity does not seem to be associated with the factors previously described by Lichtenstein (1968).
ACKNOWLEDGMENTS The authors wish to thank Dr S. G. 0. Johansson for performing the IgE and IgA determinations. The excellent technical assistance of Miss B. Kruse and the secretarial work of Mrs M. Gustafson is also gratefully acknowledged. This investigation was supported by grants from the Swedish Medical Research Council (13X-04763 and 13X-02518), Livforsakringsbolagens namnd for Medicinsk forskning, IF :s Stiftelse for Farmaceutisk forskning and Ernst och Martha Janssons foundation. REFERENCES CESKA M. & LUNDKVIST U. (1972) A new and simple radioimmunoassay method for the determination of IgE. Immunochemistry, 9, 1021. COCHRANE C.G. & MULLER-EBERHARD H.J. (1968) The derivation of two distinct anaphylatoxin activities from the third and fifth components of human complement. J. exp. Med. 127, 371. GRANT J.A. & LICHTENSTEIN L.M. (1973) The role of complement in human immediate hypersensitivity: Evidence against involvement of the alternate pathway of complement activation. J. Immunol. 111, 733. HJELM H., HJELM K. & SJOQUIST J. (1972) Protein A from Staphylococcus aureus. Its isolation by affinity chromatography and its use as an immunosorbent for isolation of immunoglobulins. FEBS Letters, 28, 73. ISHIZAKA T., ISHIZAKA K., JOHANSSON S.G.O. & BENNICH H. (1969) Histamine release from human leukocytes by anti-yE antibodies. J. Immunol. 102. 884.
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LICHTENSTEIN L.M. (1968) Mechanism of allergic histamine release from human leukocytes, Biochemistry of the Acute Allergic Reactions (ed. by K.F. Austen and E.L. Becker), p. 153. Blackwell Scientific Publications, Oxford. LICHTENSTEIN L.M. (1971) The immediate allergic response. In vitro separation of antigen activation, decay and histamine release. J. Immunol. 107, 1122. LICHTENSTEIN L.M. & OSLER A.G. (1964) Studies on the mechanisms of hypersensitivity phenomena. J. exp. Med. 120, 507. LICHTENSTEIN L.M. & OSLER A.G. (1966) Studies on the mechanism of hypersensitivity phenomena. Xl. The effect of normal human serum on the release of histamine from human leukocytes by ragweed pollen antigen. J. Immunol. 96, 159. LOWRY O.H., ROSEBROUGH N.J., FARR A.L. & RANDALL R.J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265. MARTIN R.R. & WHITE A. (1969) The in vitro release of leukocyte histamine by staphylococcal antigens. J. Immunol. 102, 437. MULLER-EBERHARD H.J. (1960) A new supporting medium for preparative electrophoresis. Scand. J. dlin. Lab. Invest. 12, 33. OHAZAKI T., ILEA V., OHAZAKi A., REISMAN R.E. & ARBESMAN C.E. (1975) The effect of Tris-buffered media and Tyrode physiologic saline solution on the antigenic release of histamine from human leukocytes. J. Allergy, Clin. Immunol. 56, 27. PETERSSON B.-A. (1975) Induction of histamine release and desensitization in human leukocytes. IgG-mediated histamine release. Scand. J. Immunol. 4, 777. PETERSSON B.-A., NILSSON A. & STXLENHEIM G. (1975) Induction of histamine release and desensitization in human leukocytes. Effect of anaphylotoxin. J. Immunol. 114, 1581. PETERSSON B.-A. & STALENHEIM G. (1975) Induction of histamine release and desensitization in human leukocytes. Effect of anti-IgE or protein A from Staphylococcus aureus. Scand. J. Immunol. 4, 103. SJOQUIST J., MELOUN B. & HJELM H. (1972) Protein A isolated from Staphylococcus aureus after digestion with lysostaphin. Europ. J. Biochem. 29, 572. SJoQUIST J. & STALENHEIM G. (1969) Protein A from Staphylococcus aureus. IX. Complement fixing activity of protein A-lgG complexes. J. Immunol. 103, 467.
Histamine release from human leucocytes A SERUM FACTOR NECESSARY FOR THE INDUCTION OF HISTAMINE RELEASE AND DESENSITIZATION BY PROTEIN A
B.-A. PETERSSON & G. STALENHEIM Department of Medical and Physiological Chemistry, University of Uppsala, The Biomedical Center, Uppsala, Sweden
Received 17 June 1976; accepted for publication 15 October 1976
ing the leucocytes to release histamine on protein A treatment. Beside being necessary for protein A-induced histamine release, the factor (or factors) is also
Summary. The ability of human leucocytes to release histamine on protein A treatment is lost when the cells are washed repeatedly. It is, however, possible to restore the sensitivity to protein A treatment by incubating the leucocytes in serum. Treatment of the cells with purified IgG does not restore the activity. The material responsible for the resensitization is eluted both in the second and the third protein peak when serum is chromatographed on Sephadex G-200, indicating the possible existence of several active factors. Material with low immunoglobulin content, but with retained capacity to resensitize leucocytes to release histamine on protein A treatment, was obtained by repeated chromatography of peak III material on the Sephadex G-200 column. Furthermore, material from the second and third peaks from Sephadex G-200 deprived of their IgG by passage through a protein A Sepharose or a DEAE-cellulose column had the same capacity to resensitize the leucocytes as unseparated material. When serum was separated by Pevikon block electrophoresis, most of the activity was detected in the a and 8 regions but only little in the y region. The serum fractionations indicate that neither IgG nor the other immunoglobulins are the factor(s) responsible for resensitiz-
essential for protein A-induced desensitization of human leucocytes. INTRODUCTION Protein A from Staphylococcus aureus induces histamine release from human leucocytes. It has been suggested that this reaction is mediated by cell surface attached IgG molecules (Martin & White, 1969, Petersson & Stalenheim, 1975). If the cells are washed several times they lose their capacity to release histamine on protein A treatment while the histamine release induced with optimum amounts of anti-IgE is unaltered. Histamine release from repeatedly washed leucocytes can, however, be triggered by protein A in complex with IgG or with the Fc part of IgG (Petersson, 1975). One proposed explanation for the decrease in protein A reactivity is that IgG is lost during the repeated washings of the leucocytes. In this report, the capacity of serum and different serum fractions to resensitize the leucocytes to release histamine on protein A treatment has been investigated.
Correspondence: Dr B.-A. Petersson, Department of Medical and Physiological Chemistry, The Biomedical Center, Box 575, S-751 23 Uppsala, Sweden.
623
624
B.-A Petersson & G. Stalenheim MATERIALS AND METHODS
Protein A and protein A Sepharose Protein A was purified from lysostaphin-digested bacteria by affinity chromatography on IgG Sepharose; protein A Sepharose was prepared as earlier described (Sjdquist, Meloun & Hjelm, 1972; Hjelm, Hjelm & Sj6quist, 1972).
Human IgG This was isolated from sera by chromatography on DEAE cellulose followed by chromatography on a Sephadex G-200 column (Sj6quist & Stalenheim, 1969) in Tris-buffered saline containing KCI (Tris 0 025 M, NaCl 0 120 M and KCI 0 005 M). In most of the experiments the IgG used was isolated from sera of protein A positive donors, i.e. from donors whose leucocytes release histamine on protein A treatment. In some experiments IgG and leucocytes were isolated from the same donor. Immunoelectrophoresis experiments did not reveal the presence of contaminants in the IgG preparations. In some experiments IgG from pooled serum (Cohn fraction II Kabi AB, Stockholm) was used. Antisera to IgE These were raised in rabbits, and IgG from these antisera was prepared as published (Petersson & Stalenheim, 1975). This rabbit IgG preparation will be called anti-IgE throughout this report.
Pepsin digestion of immunoglobulin Pepsin-digested IgG from anti-IgE sera was passed through a protein A-Sepharose column to remove material containing intact Fc (Petersson & Sta'lenheim, 1975). The unadsorbed material was subsequently run through a Sephadex G-150 column and the main peak was collected. This material will subsequently be called F(ab')2 anti-IgE. IgG-preparation from rabbit antisera against human IgG An IgG preparation from rabbit antiserum against human IgG (heavy chain specific) was purchased from Dakopatts, Copenhagen. Throughout this report this rabbit IgG preparation will be called anti-IgG. Before use, a 1: 5 dilution of anti-IgG was dialysed against Tris-buffered saline containing KC1 for 72 h at 4°. Separation of serum on Sephadex G-200 Human serum was run on a Sephadex G-200 column
in Tris-buffered saline containing KCI. The column was eluted at the rate of 16 5 ml/h and each fraction contained 5 5 ml. In some experiments, fractions (usually every third) were concentrated and their capacity to resensitize leucocytes was tested. In other experiments, the separated material was pooled according to the three peaks obtained. Peak II (immunoglobulin-containing peak) and Peak III (albumin-containing peak) were further processed. Material low in immunoglobulin content was obtained by repeated chromatography of the peak III material on Sephadex G-200. Separation ofpeak II andpeak III material on protein A-Sepharose Peak II or peak III material was also run on a protein A-Sepharose column (15 ml). Material not bound to the column was eluted with Tris-buffered saline containing KCI. Bound material, eluted with 0-1 M glycine-HCI, pH 2-5, was pooled and dialysed against Tris-buffered saline containing KCI. The separated material was concentrated to the volume of the starting material.
Separation of peak II material on DEAE-cellulose column Peak II material dialysed against 0 0175 M phosphate buffer pH 6 3 was applied on a DEAE-cellulose column in the same buffer. After IgG had been collected from the column with the starting buffer, bound material was eluted with 0 0175 M phosphate buffer pH 6 3 containing 1 0 M NaCI. The separated materials were concentrated to the volume of the starting material and dialysed against Tris-buffered saline containing KCl before use in the histamine release experiments. Preparative block electrophoresis This was run according to Muller-Eberhard (1960). Pevikon 870 (Kema Nord, Stockholm, Sweden) was equilibrated with barbital buffer pH 8 6, , = 0 005, and the electrophoresis run at 200 V for 24 h. The block was cut into 1-cm segments, eluted, and the protein content was measured according to Lowry, Rosebrough, Farr & Randall (1951). Quantitative determinations of immunoglobulins IgG and IgA determinations were performed on concentrated samples with the single radial immunodiffusion method. The IgE content was deter-
625
Histamine release from human leucocytes
mined by radioimmunological technique according to the method of Ceska & Lundkvist (1972). Isolation of leucocytes Leucocyte suspensions were isolated from human blood according to Lichtenstein & Osler (1964). Ethylene-diamine tetraacetic acid (EDTA) was used as anticoagulant and the cells were washed with Tris-A (Tris-buffered saline containing KCl and albumin; Lichtenstein & Osler, 1964) unless otherwise stated. The leucocytes were washed according to one of two procedures. The first method includes one washing at 40, while leucocytes prepared according to the other were washed three times. The latter procedure also includes incubation of the cells at 370 for 20 min after the second washing. Throughout this report, cells washed three times will be called 'repeatedly washed leucocytes' (Petersson, 1975). Cell donors were selected in order to get leucocyte preparations releasing more than 30 per cent histamine on treatment with protein A after a single washing. Histamine release The histamine release experiments were performed in a 20-ml sample volume containing 06-10 x 107 leucocytes per ml. A two-step procedure was used to test the capacity of serum or serum fractions to reconstitute the ability of the leucocytes to release histamine on protein A treatment. In the first step, cell samples were incubated in 10 ml of serum or serum fractions. Buffer controls were included. After 40 min incubation at 370 the cells were spun down and washed in Tris-A at 4°. In the second step, the samples were tested with a fixed amount of protein A (1 yg/ml) in 2 0 ml Tris-ACM (Tris-A containing Mg2 + and Ca2 +) for 40 min at 37°. The histamine release during the second step was determined. This procedure will be called 'to resensitize leucocytes' throughout this report. The total histamine content of the cells was determined in two samples of cells after being boiled for 3 min. Histamine assay Histamine was biologically assayed using segments of guinea-pig ileum as published (Cochrane & Muller-Eberhard, 1968; Petersson & Sta'lenheim, 1975). Human IgG (Cohn fraction II, Kabi AB, Stockholm) was added in excess to protein A and anti-IgG containing samples (Petersson & Stalenheim, 1975). B
RESULTS Effect of heparin and Tyrode solution on protein Ainduced histamine release In one type of experiment half of the blood was poured into a mixture of dextran and EDTA and the other half into a mixture of dextran and heparin. After a single washing of the leucocytes protein A (1 ,ug/ml) released the same amount of histamine from the cells isolated in the presence of EDTA or heparin. From repeatedly washed cells, no histamine was released in any of the cases. In another set of experiments, one portion of the leucocytes was washed with Tyrode solution during the preparation (Ohazaki, Ilea, Ohazaki, Reisman & Arbesman, 1975) and the other portion was washed with Tris-A. After one or three washings the Tyrode washed cells were treated with protein A (1 pg/fml) in Tyrode solution and the Tris-A washed cells with protein A (1 pg/ml) in Tris-ACM. Histamine release from once-washed leucocytes was slightly higher if the leucocytes had been treated with Tyrode solution than if the cells were processed according to the original procedure (Lichtenstein & Osler, 1964). No histamine was released after repeated washings in any of the cases. Treatment of repeatedly washed leucocytes with IgG or serum Expt 1 in Table 1 shows the effect of treating repeatedly washed leucocytes with 1 0 ml IgG solution (6 mg/ml) or with 1 0 ml serum diluted 1:2 for 40 min at 37°. A control with buffer was also included. After one washing, each sample was incubated with protein A (1 pg/ml) in Tris-ACM for 40 min at 370 and the histamine released in the second step was determined. Leucocytes preincubated with buffer or IgG liberated no histamine at all on protein A treatment while samples pretreated with serum released 44 per cent of their histamine content. The effect of IgG was tested in ten additional experiments with IgG concentrations varying between 4-7 and 6 8 mg per ml, but in a few experiments lower concentrations (down to 1 mg/ml) were tested. In no case did protein A release histamine from buffer pretreated cells, while serum pretreatment resulted in histamine release of 36-64 per cent. In ten of the experiments protein A induced no histamine release from IgG-pretreated cells. In the remaining experiment the release was 7 per cent.
B.-A. Petersson & G. Stdlenheim
626
Table 1. Protein A-induced histamine release from resensitized human leucocytes
Additive in step 1
Expt Histamine release in step 2 (%)
Exp. No. 1
2
3
4
5
Pretreatment at 370 for 40 min Buffer IgG 6 mg/ml Serum 1: 2 Buffer Serum 1:2 Dialysed serum Serum treated at 560 for 30 min Buffer Serum 1:2 Peak III material* Buffer Serum 1:2 Peak III material* Buffer Serum 1:2 Peak III material*
Table 2. Anti-IgE and anti-IgG induced histamine release from human leucocytes. Specificity of the releasing agents
Test with protein A (1 jug/ml) 0 0 44 0 60 57
60 0 40 48 0 49 52 0 59 56
* This peak III material contains 20 pg IgG per ml and less than 2 jug IgA and 2 ng IgE per ml.
Effect of washings on anti-IgG induced histamine release Preliminary experiments showed that a 1: 200 dilution of anti-IgG gave maximum histamine release from most of the leucocyte preparations tested, and this concentration of anti-IgG has been used throughout. To test the specificity of anti-IgG, samples diluted 1:100 were mixed with 1 or 1O pg of IgE or 10 pg of IgG in a vol. of 1 ml. Buffer controls (Tris-ACM) were also included. After incubation at 370 for 30 min, one ml of cell suspension was added to each sample which was then incubated for an additional 40 min at 37° and the liberated histamine was determined. For comparison, anti-IgE (3 ug/ml) was preincubated in the same way with IgE, 1 pg/ml, IgG, 10 pg/ml or buffer. Table 2 shows that preincubation of anti-IgG with IgG completely abolished the capacity of anti-IgG to release histamine. Pretreatment of anti-IgG with IgE did not significantly decrease its histamine releasing capacity. IgE completely inhibited anti-IgE induced histamine release, while treatment with IgG had no effect. To test the effect of washings on anti-IgG in-
no.
Releasing agent
Anti-IgG 1:200+ buffer Anti-IgG 1:200+0 5 ,pg IgE/mI Anti-IgG 1: 200+ 50 pug IgG/ml Anti-IgE 1-5 ,pg/ml+ buffer Anti-IgE 1-5 pg/ml+0 5 pg IgE/ml Anti-IgE 1-5 ,g/ml+ 5.0 pg IgG/ml 2 Anti-IgG 1:200+ buffer Anti-IgG 1: 200+ 50 ,pg IgE/ml Anti-IgG 1: 200+ 5 0 jug IgG/ml Anti-IgE 1-5 pg/ml+ buffer Anti-IgE 1-5 ug/ml+0.5 pg IgE/ml Anti-IgE 1-5 ug/ml+ 50 pg IgG/ml 3 Anti-IgG 1:200+ buffer Anti-IgG 1: 200+ 5 pg IgE/ml Anti-IgG 1: 200+ 5 plg IgG/ml 1
Histamine release(%) 24 21 0 52 0 56 32 31 0 60 0 62 39 38 0
duced release cell samples were washed once or repeatedly before the addition of anti-IgG (1:200) in Tris-ACM. For comparison the effect of washings on anti-IgE or protein A triggered release was also tested. The experiments in Table 3 show that antiIgG and anti-IgE initiated release was not decreased as an effect of washings. The protein A-induced release on the other hand decreased from high values after one washing down to low values after repeated washings. The effect of dilution, dialysis or heat treatment on the capacity of serum to re-constitute the ability of the leucocytes to release histamine on protein A treatment Samples of repeatedly washed leucocytes were incubated with 1-ml portions of undiluted and diluted autologous serum. A control with Tris-A was included. After 40 min at 370 the cells were spun down and washed once with 2 0 ml Tris-A at 4°. In the second step each sample was incubated with protein A, 1 pg/ml, in Tris-ACM for 40 min at 370, and liberated histamine was measured. Fig. 1 shows the results obtained with three different leucocyte populations. In most experiments, the 1:2 serum dilution gave maximal re-constitution. Serum diluted 1: 20 had no capacity to resensitize leucocytes in any of the experiments performed. Samples incubated with buffer in step one released no histamine at all
627
Histamine release from human leucocytes Table 3. Anti-IgE, anti-IgG and protein A-induced histamine release from once or repeatedly washed human leucocytes
Releasing agent
Expt no.
Anti-IgE 1 5,pg/ml Anti-IgG 1:200 Protein A 1 pg/ml Anti-IgE 1 5 pg/ml Anti-IgG 1:200 Protein A 1 pg/ml F(ab')2anti-IgE 25 ,ug/ml Anti-lgG 1:200 Protein A 1 ug/ml
I
2
3
Histamine release
Histamine release after
after one washing
repeated washings*
(%)
(%)
60 32 35 52 24 37 46 15 37
56 39 0 46 25 0 47 20 8
* The leucocytes were washed three times and incubated for 20 min at the second and third washing.
370
between the
when treated with protein A in the second step. Furthermore, no spontaneous release was obtained on buffer treatment in the second step from samples incubated with serum in the first step. In the following experiments of this report, the 1:2 serum dilution has been consistently used. In five experiments the capacity of native, dialysed and heattreated serum to resensitize leucocytes was studied. Serum (2 ml) was dialysed during 48 h at 40 against 5 x 500 ml Tris-buffered saline containing KCI. Heat treatment was performed at 560 for 30 min. From samples pretreated with native serum (1: 2) the protein A induced release varied between 44 and 89 per cent. At most dialysis resulted in a 5 per cent and heat treatment in a 9 per cent decrease of the capacity of serum to resensitize leucocytes. In some of the experiments dialysed or heat-treated serum gave a somewhat better resensitization than native serum. In additional experiments serum was treated at 56° for 90 or 180 min and in these experiments the stimulating effect varied. In some experiments there was no inactivation of the serum, while in others all reconstituting capacity was lost.
-c E
.r_
Q-
1/8
1/4
Humon
serum
1/2 (dilutions)
1/1
Figure 1. Histamine release induced by protein A from leucocytes resensitized with diluted serum. The different curves represent experiments with cells from different donors.
Effect of washings on resensitized leucocytes Samples of repeatedly washed leucocytes were incubated with serum diluted 1:2. After 40 min incubation at 370 the samples were washed once, twice, or three times, and incubated in the second step with protein A, 1 ,ug/ml, in Tris-ACM for 40 min at 37°. The experiments in Table 4 show that the
B.-A. Petersson & G. Stdlenheim
628
Table 4. Protein A-induced histamine release from extensively washed and resensitized human leucocytes. Effect of washings after the resensitization Additive in step 1 Expt no.
Pretreatment at 370 for 40 min
Number of washings after step 1*
Histamine release in step 2 (Y.)
Buffer Serum Serum Serum Buffer Serum Serum Serum Buffer Serum Serum Serum
1 1 2 3 1 1 2 3 1 1 2 3
0 64 46
I
2
3
Test with protein A (1 pg/ml)
9 0 44 42 27 0 39 20 0
Serum was used diluted 1:2.
*Samples washed three times were incubated at 370for 20 min between the second and third washing.
protein A-induced release decreased again with increasing number of washings.
Separation of serum on Sephadex G-200 Human serum (5 ml) was run on a Sephadex G-200 column (2 5 x 90 cm) in Tris-buffered saline containing KCL. Fractions (usually every third) were concentrated five times and their capacity to resensitize leucocytes was tested. In Fig. 2, the solid line shows the optical density and the dotted line represents the capacity of the fractions to resensitize the leucocytes. One activity peak was detected in protein peak II and another in protein peak III. No activity was detected in peak I. When peak II or peak III material was rechromatographed on the same Sephadex G-200 column, the sensitizing material was eluted in the same position as in the original chromatography. No additional activity peak was obtained in any case. Peak III material with very low immunoglobulin content was obtained by repeated chromatography on Sephadex G-200. Four millilitres of peak III material were obtained from 7 ml of human serum. Expts 3-5 in Table 1 show that this peak III material has the same capacity as native serum (1:2) to resensitize leucocytes. Leucocytes preincubated with buffer released no histamine at all. This peak III material contained
20 pg IgG and less than 2 pg IgA and 2 ng IgE per ml. Peak II and III material from serum separated on Sephadex G-200 were run separately on a protein A-Sepharose column and the capacity of the bound and unbound material to resensitize the leucocytes was tested. Unfractionated peak II and III material as well as serum were also used. Table 5 shows the results of these experiments. Peak II and III material not bound to protein A-Sepharose resensitized the cells to roughly the same extent as unseparated peak II and III material. Bound material had no or only a low capacity to resensitize the cells. Peak III material which passed through protein A-Sepharose contained less than 10 ,g IgG, 2 ,ug IgA and 2 ng IgE per ml. The passage of peak II material through protein A-Sepharose removed 93-96 per cent of the IgG. Peak II material was also deprived of IgG by passage through a DEAE-cellulose column. In this case the active material was bound to the column while IgG was eluted with the starting buffer. The experiments in Table 5 show that peak II material bound to the DEAE-cellulose column resensitizes the leucocytes to the same extent as unseparated peak II material. The material eluted with the starting buffer had no or only low capacity to resensitize leucocytes. Passage of peak II material through the
629
Histamine release from human leucocytes
4). The albumin and a-globulin pools did not show any precipitin line in the y region, whereas contamination of the ,8-globulin pool with y-globulin could not be excluded.
28
80
2-0
C: c
0o00
37°.
60
z w
o
-e Ln
D0
E
40 P
1.0
Z W
20
20
30
40
50
60
Protein A-induced desensitization A three-step procedure was used to study the protein A-induced desensitization of leucocytes pretreated with serum (1:2) or buffer. In the first step, repeatedly washed leucocytes were incubated with After one serum (1:2) or buffer for 40 min at washing, the samples were incubated in the second step with protein A, 1 pug/ml, in Tris-AM (Tris-A containing Mg2 + but no Ca2+) for 40 min at 37°. Buffer controls were also included. After the second step the samples were washed twice and tested for desensitization by adding an optimum amount of F(ab')2 anti-IgE (2 5 #ug/ml) in Tris-ACM and incubating for 40 min at 370 (third step). Liberated histamine in the second and third steps was measured. Table 6 shows that pretreatment of cells with serum enhances the desensitizing activity of protein A yielding a desensitization of 75-100 per cent in comparison with 10-28 per cent desensitization of buffer pretreated cells.
70
Fraction number
Figure 2. Histamine release induced by protein A from leucocytes resensitized with serum fractions chromatographed on a Sephadex G-200 column; (O C) indicates the optical density at 280 nm and (a -* -* - *) the capacity of the tested fractions to resensitize the leucocytes to release histamine on protein A (I pg/mI) treatment. Fraction volume was 5-5 ml.
DEAE-cellulose column removed 94 per cent of IgG from the active material. Separation of serum on Pevikon block electrophoresis Human serum (10 ml) was fractionated by Pevikon block electrophoresis and the material was pooled as indicated in Fig. 3. Each pool was dialysed against KCI-containing Tris-buffered saline and concentrated to 10 ml. One-millilitre portions of each pool were used to test their capacity to resensitize the leucocytes. The curve in Fig. 3 shows the optical density of the eluate and the columns represent the capacity of each pool to resensitize the leucocytes. Most of the active material was found in the a and the ,6 fractions and very little in the y region. Immunoelectrophoresis was run on the concentrated pools using antiserum to whole human serum (Fig.
DISCUSSION The most plausible explanation for the observation that protein A-induced histamine release is reduced on washing of leucocytes is that loosely attached IgG is eluted from the cell surface (Petersson, 1975). Consequently, it was attempted to reattach IgG onto the cells in vitro, and whole human serum was actually found to efficiently restore the protein A elicited histamine release of the cells (Table 1, Fig. 1). However, after incubating the cells for 40 min at 370 with purified IgG at concentrations varying from 1-6 mg/ml, no activity was regained (Table 1). The histamine release observed on challenging serum pretreated cells with protein A is thus unlikely to be the effect of protein A-IgG complexes formed with soluble IgG remaining after the washings. Moreover the amount of IgG calculated to remain in the fluid phase is far too low to give enough complex with protein A to be responsible for the histamine release observed (Petersson, 1975). As with the activity of 'native' cells, the activity of resensitized cells is readily diminished by three
B.-A. Petersson & G. Stdlenheim
630
Table 5. Protein A-induced histamine release from resensitized human leucocytes
Additive in step 1
IgG concentration (mg/ml)
Expt no.
Pretreatment at 370 for 40 min
I
Buffer Serum* Peak III material Peak III material not bound to protein A Sepharoset Peak III material bound to protein A Sepharose Peak II material Peak II material not bound to protein A Sepharose Peak II material bound to protein A Sepharose Buffer Serum Peak II material Peak II material not bound to protein A Sepharose Peak II material bound to protein A Sepharose Peak II material not bound to DEAE-cellulose Peak II material bound to DEAE-cellulose Buffer Serum Peak II material Peak II material not bound to DEAE-cellulose Peak II material bound to DEAE-cellulose
2
3
*
Histamine release in step 2 (0) Test with protein A (I pg/ml)
0-14 < 0 01 Not tested 50 0-2 3-7 6-3 0 45 4-8 56 04 6-3 56 04
° 63 59 56 0 66 54 0 0 49 54 44 8 12 53 0 40 42 0 38
When serum was used for resensitization it was diluted 1:2.
t This peak III material contains less than 2 pg IgA and 2 ng IgE per ml.
washings (Table 4). This is compatible with the concept of some factor being eluted from the cells. The fact that anti-IgG readily releases histamine from extensively washed cells does not, of course, exclude IgG as the factor eluted. The washings could remove part of the cell-bound IgG leaving enough for anti-IgG but not enough for protein A elicited release. That anti-IgG induced release is not diminished as an effect of washings, is in agreement with experiments performed by Lichtenstein (personal communication). Since, generally, washing does not diminish the reactivity of the cells to anti-IgE or anaphylatoxin the factor eluted is either not necessary for the histamine release by these agents, or is needed in lesser amounts than those required by protein A to trigger the cells. In order to characterize the serum factor responsible for the resensitization of cells, serum was fractionated on a Sephadex G-200 column (Fig. 2). Activity was recovered from the second and third but not from the first peak. The material recovered in rechromatographed peak III was found to contain 20 ug of IgG and less than 2 pug IgA and 2 ng IgE per ml but it still had a high capacity, even in com-
parison with unfractionated serum, to resensitize leucocytes. To evaluate further the involvement of IgG, peak II and peak III materials were passed over a protein A-Sepharose column. The active material from both preparations went straight through the column without binding to protein A. Almost all the IgG was bound and active peak III material, passed through the protein A-Sepharose column, contained less than 10 pg IgG per ml. The IgG absorbed from the two preparations could be recovered from the column by lowering the pH of the eluting buffer. When peak II material was fractionated on a DEAE-cellulose column IgG went straight through while the material with resensitizing capacity was bound to the column. The active material was eluted from the column with a buffer containing a high salt concentration. In Pevikon block electrophoresis, the most active material migrated with the a- and fl-globulins (Fig. 3). Some activity was recovered in the albumin peak but only little was found in the y-globulin fraction. These fractionations indicate that IgG could not be the serum factor responsible for resensitization of cells to protein A-induced histamine release. Furthermore,
Histamine release from human leucocytes °0r +
I-
-i
-I
F
0-8
E o 0-6 0-
(s c as
D0
Jo 04 _
i 0-2
K1I
14
18
K,' 13 '--Y
22 26 30 Fraction number
34
38
Figure 3. Histamine release induced by protein A from leucocytes resensitized with pools from serum fractionated by Pevikon block electrophoresis. The anode is to the left and the arrow shows the application line. (o) indicates the optical density at 540 nm, and the material was pooled as shown in the upper part of the figure. The protein concentrations of the pools were: Pool 18-22, 33-2 mg/ml; pool 23-25 4-7 mg/ml, pool 26-29 6-8 mg/ml, pool 30-32 5-2 mg/ml and pool 33-36 5 0 mg/ml. The bars represent the capacity of each pool to reconstitute the ability of the leucocytes to release histamine on protein A (1 pug/ml) treatment.
our results make it unlikely that any other immunoglobulin could be the responsible factor. There could be more than one serum factor with the activity discussed. Alternatively, one factor could exist in a polymeric state or associated with other serum components. The appearance of two peaks in the Sephadex G-200 chromatogram does not seem to be due to an equilibrium between the active material in peaks II and III, since rechromatography of either of them gave essentially homogenous activity peaks with the same elution characteristics as the starting material. It has been reported that serum components have enhancing activity on allergen and anti-IgE induced histamine release (Lichtenstein & Osler, 1966; Ishizaka, Ishizaka, Johansson & Bennich, 1969; Grant & Lichtenstein, 1973). One of the serum
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factors found by Lichtenstein and co-workers has sufficiently low molecular size to pass dialysis membranes and another is macromolecular and excluded from Biogel P 300 (Lichtenstein, 1968). A third enhancing factor described in the same report could be eluted from leucocytes. Material capable of restoring the activity of cells to release histamine on protein-A treatment is found not to pass through dialysis membranes when serum is dialysed against a small volume of buffer. Nor does the activity decrease in serum after prolonged dialysis against large volumes of buffer. Thus the factor under study is not identical with the low mol. wt factor studied by Lichtenstein. If the lipoprotein described by the same author was responsible for some of the activity observed by us, some activity should have appeared in the first peak of the Sephadex G-200 chromatogram. Cells extracted according to Lichtenstein (1968) do not release histamine on protein A treatment but the extract, even if concentrated four times, has no capacity to restore the activity of repeatedly washed cells. Since heating (560, 30 min) does not diminish the resensitizing activity of serum, haemolytic complement is not likely to be responsible. This is in accordance with what has been found for allergen and anti-IgE-induced histamine release (Ishizaka et al., 1969; Grant et al., 1973). Immunoglobulin-mediated histamine release involves a mechanism which is activated even in the absence of calcium ions. Prolonged treatment in calcium-free medium induces a state of unresponsivness in the leucocytes (Lichtenstein, 1971). Earlier investigations have also shown that histamine release mediated by IgE and IgG at least partially takes place by the same biochemical mechanism (Petersson, 1975; Petersson & Stailenheim 1975). Anaphylatoxin, on the other hand, induces histamine release and desensitization by a different mechanism (Petersson, Nilsson & StAlenheim, 1975). Table 6 shows that the serum factor (or factors) necessary for protein A-induced histamine release is needed even for the desensitization induced by protein A in the absence of calcium ions. It could be concluded that serum contains materials which interact with basophils to make them liable to release histamine on challenge with protein A. This activity does not seem to be a property of IgG or any other immunoglobulin. Since activity is found in two peaks in Sephadex G-200 chromatography more than one factor could be involved. The activity is associated with high mol. wt material and
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B.-A. Petersson & G. Stdlenheim
Figure 4. Immunoelectrophoresis of serum and the five pools obtained, when serum was fractionated by Pevikon block electrophoresis. The electrophoresis was run 2 h and the anti-serum used was against human serum proteins. The anode is to the right and the samples were from top to bottom: serum, pool 18-22, pool 23-25, pool 26-29. pool 30-32 and pool 33-36. Table 6. Protein A-induced desensitization of human leucocytes. Effect of serum pretreatment
Additive in step 2 Histamine release in step 3 (%) Expt no.
Treatment Additive in in the absence of step 1 Ca2+
1
Serum Serum Buffer Buffer
2
Serum Serum Buffer Buffer
3
Serum Serum Buffer Buffer
Buffer Protein A 1 pg/ml Buffer Protein A 1 ug/ml Buffer Protein A 1 pg/ml Buffer Protein A I pg/ml Buffer Protein A 1 pg/ml Buffer Protein A 1 pg/ml
Histamine release in step 2 (0/)
Test with F(ab')2 anti-IgE (2-5 ug/ml)
0 0 0 0
55 0 50 45
0 0 0 0
46 0 43 31
0 0 0 0
92 23 89 68
Repeatedly washed leucocytes were used. Control samples were treated with buffer or serum (1: 2) in the first step and with protein A (1 pg/ml) in Tris-ACM in the second step. In experiment 1 the histamine release was 3 per cent from buffer-treated and 44 per cent from serum-treated leucocytes. The corresponding figures for experiment 2 was 7 and 40 per cent and for experiment 3 15 and 88 per cent.
Histamine release from human leucocytes moves as a- and fl-globulins in electrophoresis. Furthermore, the activity does not seem to be associated with the factors previously described by Lichtenstein (1968).
ACKNOWLEDGMENTS The authors wish to thank Dr S. G. 0. Johansson for performing the IgE and IgA determinations. The excellent technical assistance of Miss B. Kruse and the secretarial work of Mrs M. Gustafson is also gratefully acknowledged. This investigation was supported by grants from the Swedish Medical Research Council (13X-04763 and 13X-02518), Livforsakringsbolagens namnd for Medicinsk forskning, IF :s Stiftelse for Farmaceutisk forskning and Ernst och Martha Janssons foundation. REFERENCES CESKA M. & LUNDKVIST U. (1972) A new and simple radioimmunoassay method for the determination of IgE. Immunochemistry, 9, 1021. COCHRANE C.G. & MULLER-EBERHARD H.J. (1968) The derivation of two distinct anaphylatoxin activities from the third and fifth components of human complement. J. exp. Med. 127, 371. GRANT J.A. & LICHTENSTEIN L.M. (1973) The role of complement in human immediate hypersensitivity: Evidence against involvement of the alternate pathway of complement activation. J. Immunol. 111, 733. HJELM H., HJELM K. & SJOQUIST J. (1972) Protein A from Staphylococcus aureus. Its isolation by affinity chromatography and its use as an immunosorbent for isolation of immunoglobulins. FEBS Letters, 28, 73. ISHIZAKA T., ISHIZAKA K., JOHANSSON S.G.O. & BENNICH H. (1969) Histamine release from human leukocytes by anti-yE antibodies. J. Immunol. 102. 884.
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LICHTENSTEIN L.M. (1968) Mechanism of allergic histamine release from human leukocytes, Biochemistry of the Acute Allergic Reactions (ed. by K.F. Austen and E.L. Becker), p. 153. Blackwell Scientific Publications, Oxford. LICHTENSTEIN L.M. (1971) The immediate allergic response. In vitro separation of antigen activation, decay and histamine release. J. Immunol. 107, 1122. LICHTENSTEIN L.M. & OSLER A.G. (1964) Studies on the mechanisms of hypersensitivity phenomena. J. exp. Med. 120, 507. LICHTENSTEIN L.M. & OSLER A.G. (1966) Studies on the mechanism of hypersensitivity phenomena. Xl. The effect of normal human serum on the release of histamine from human leukocytes by ragweed pollen antigen. J. Immunol. 96, 159. LOWRY O.H., ROSEBROUGH N.J., FARR A.L. & RANDALL R.J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265. MARTIN R.R. & WHITE A. (1969) The in vitro release of leukocyte histamine by staphylococcal antigens. J. Immunol. 102, 437. MULLER-EBERHARD H.J. (1960) A new supporting medium for preparative electrophoresis. Scand. J. dlin. Lab. Invest. 12, 33. OHAZAKI T., ILEA V., OHAZAKi A., REISMAN R.E. & ARBESMAN C.E. (1975) The effect of Tris-buffered media and Tyrode physiologic saline solution on the antigenic release of histamine from human leukocytes. J. Allergy, Clin. Immunol. 56, 27. PETERSSON B.-A. (1975) Induction of histamine release and desensitization in human leukocytes. IgG-mediated histamine release. Scand. J. Immunol. 4, 777. PETERSSON B.-A., NILSSON A. & STXLENHEIM G. (1975) Induction of histamine release and desensitization in human leukocytes. Effect of anaphylotoxin. J. Immunol. 114, 1581. PETERSSON B.-A. & STALENHEIM G. (1975) Induction of histamine release and desensitization in human leukocytes. Effect of anti-IgE or protein A from Staphylococcus aureus. Scand. J. Immunol. 4, 103. SJOQUIST J., MELOUN B. & HJELM H. (1972) Protein A isolated from Staphylococcus aureus after digestion with lysostaphin. Europ. J. Biochem. 29, 572. SJoQUIST J. & STALENHEIM G. (1969) Protein A from Staphylococcus aureus. IX. Complement fixing activity of protein A-lgG complexes. J. Immunol. 103, 467.
