Clin. exp. Immunol. (1976) 26, 457-462.
Studies on the physico-chemical characteristics of polymorph migration stimulator R. D. STEVENSON & AILEEN C. GRAY University Departments of Medicine & Pathology, Western Infirmary, Glasgow
(Received 28 April 1976)
SUMMARY
Polymorph migration stimulator is a supernatant factor produced by the interaction between glucocorticosteroids and human blood monocytes in culture. Studies on the physical characteristics of this factor show that it is soluble and stable at high and low temperatures. Its activity is reduced by acid and alkali treatment and destroyed by the proteolytic enzyme protease. Experiments involving dialysis, ultrafiltration and Sephadex G-100 gel filtration indicate that the molecular weight of the polymorph migration stimulator is between 12,000 and 15,000. It is suggested that this factor may mediate the anti-inflammatory effects of glucocorticosteroids on phagocytic cells. INTRODUCTION Human blood monocytes, spleen cells and bone marrow cells react with corticosteroids in vitro to produce a supernatant factor which stimulates polymorph migration, using the capillary tube method. The production of this factor, polymorph migration stimulator (PMS), involves an active biological process and occurs at steroid concentrations which are comparable with physiological and pharmacological levels. The amount of PMS generated by different steroids correlates with their anti-inflammatory, glucocorticoid activity (Stevenson, 1973, 1974 and 1976a). These in vitro observations are supported by studies showing that the migration of polymorphs from patients receiving steroid therapy is enhanced in vitro and that this effect is dependent on an interaction between steroids and mononuclear leucocvtes in vivo (Stevenson, 1976b). It has been suggested that this factor may mediate the effects of glucocorticosteroids on the kinetic behaviour of granulocytic cells in the bone marrow and in areas of inflammation. The experiments described in this paper were undertaken to study the physical characteristics of PMS.
MATERIALS AND METHODS Hydrocortisone. Hydrocortisone (Merck, Sharpe & Dohme) was made up as a 1% solution in 95% ethyl alcohol (Heilman, 1972). Further dilutions were made in Eagle's minimal essential medium (MEM). The standard concentration used was 10 jg/ml of medium. Production ofPMS. Previous experiments have shown that lymphocytes do not affect the production ofPMS from steroidtreated monocytes (Stevenson, 1974) and therefore, for ease of preparation in this study, mixed mononuclear leucocytes (MML) and not purified monocytes were cultured with hydrocortisone to produce PMS. The details of the MML culture technique have previously been described (Stevenson, 1974). In experiments which required large volumes of culture supernatants, MML were cultured in glass universal containers in volumes of 6 ml containing 8 x 106 cells. Dialysis and ultrafiltration. Dialysis was carried out using 8/32-in Visking tubing. Supernatants were ultrafiltered through collodion bags (Sartorius) using suction flasks. The pore size of the collodion bags was 8 pm which retained substances of molecular weight greater than 12,400. Gelfiltration of culture supernatants. Culture supernatants (50 ml) were lyophilized and resuspended in 6 ml of distilled Correspondence: Dr R. D. Stevenson, University Department of Medicine, Western Infirmary, Glasgow GIl 6NT.
457
R. D. Stevenson L Aileen C. Gray
458
water. The concentrated samples, after clarification by centrifugation were applied to a Sephadex G-100 column, 2-5 x 100 cm, equilibrated with phosphate-buffered saline (0-01 M phosphate, pH 7-3) with a flow rate of 22 ml/hr. The eluate was collected in 7-ml volumes which were pooled into five fractions on the basis of the chromatogram (OD at 280 nm). After concentration using carbowax, the samples were dialysed against MEM overnight and the final volume of each was adjusted to 12 ml. Before the migration assay, the samples were sterilized by millipore filters. Enzyme treatment of PMS. Enzymatic digestion of PMS was attempted using protease and ribonuclease insolubilized on agarose (Miles Laboratories). The enzyme-agarose preparations were washed with MEM and 3-ml bed volumes were mixed with 3-ml aliquots of culture supernatants. The incubation mixtures were transferred to 25-ml conical flasks and the pH adjusted to 7 0 for ribonuclease and 8-0 for protease. Controls consisted of culture supernatants mixed with agarose alone. Samples were then incubated for 2 hr at 37'C in a shaking water-bath, after which the mixtures were centrifuged, the supernatants decanted and the pH values restored to 7-4. Measurement of PMS activity. The effect of PMS was measured using the polymorph migration technique previously described in detail (Stevenson, 1973). Migration chambers were filled with supernatants from experimental leucocyte cultures and normal human mixed leucocytes were used as a source of migrating polymorphs. Percentage stimulation of migration was calculated as: Larea of migration in steroid-treated culture supernatant 1 J ° area of migration in control culture supernatant
RESULTS The two Figs and Tables 1-6 illustrate typical experiments from each group; each experiment was repeated at least three times. TABLE 1. Effect of ultracentrifugation on the migration-stimulating activity of hydrocortisone-treated MML culture supernatants
Migration area (± s.e.m.) Migration medium Control 100,000 g for 1 hr.
MML culture supernatant MEM MML culture supernatant
Control
Percentage stimulation of migration Hydrocortisone (+ s.e.m.)
78+ 1-73
108+ 3-08
38+ 1-37
69+ 2-83 76+2-20
79+ 3-83 109+264
14+ 0.89 43+ 1.62
Solubility In previous experiments, PMS was assayed using the crude supernatants of steroid-treated MML cultures. It was therefore possible that subcellular fractions, derived from damaged cells, might be responsible for the stimulation of migration. To determine whether PMS was soluble or particulate, control and hydrocortisone-treated culture supernatants were centrifuged at 100,000 g for 1 hr at 20°C and the supernatants assayed for migration-stimulating activity. In experiments, using this system, controls of MEM with and without hydrocortisone are included, since hydrocortisone alone marginally stimulates polymorph migration. This effect occurs because of the reaction between hydrocortisone and the monocytes in the migrating cell population, which produces a migration-stimulating effect on the remaining polymorphs (Stevenson, 1973). The results in Table 1 show that migration-stimulating activity was retained in the particle-free supernatant from the steroid-treated culture. Temperature stability There was no loss of PMS activity when stored at - 20°C, 4°C or room temperature for 1 week. The effect of heat on PMS was studied by incubating control and hydrocortisone-treated culture supernatants in a water-bath for 1 hr at 60°C and 90°C. Table 2 shows that migration-stimulating activity was stable at 60°C but was destroyed by heating at 90°C.
Polymorplh migration stimulator
459
TABLE 2. Effect of temperature on the migration-stimulating activity of hydrocortisonetreated MML culture supernatants
Control
Hydrocortisone
Percentage stimulation of migration (± s.e.m.)
60+ 2-70
86+ 1P04
43+ 2-00
66+ 3-02 61+ 1P43
73+ 1-36 89+ 1-08
11+0*54 46+ 1-21
72+1P38
69+ 1.19
-4+O 10
Migration area
(± s.e.m.) Temperature (OC)
Migration medium
20
MML culture supernatant MEM MML culture supernatant MML culture supernatant
60 90
TABLE 3. Effect of dialysis on the migration-stimulating activity of hydrocortisone-treated MML culture supernatants
Migration Migration medium
area
(± s.e.m.) Control
Dialysed
(a) (b) (a) (b) (a) (b) (c)
Control culture supernatant Hydrocortisone-treated supernatant MEM MEM + hydrocortisone Control culture supernatant Hydrocortisone-treated supernatant Hydrocortisone-treated supernatant reconstituted with hydrocortisone*
68+ 2-18 100+ 0 99 62+1.15 65+0i55 61+ 2-16 74+i64 75+ 2-18
Percentage stimulation of migration (+ s.e.m.)
47+ 1*58 5+0 10 21+ 0-88 23+ 1P05
* Hydrocortisone (10 ug/ml) added to steroid-treated supernatant after dialysis.
Dialysis Control and hydrocortisone-treated culture supernatants were dialysed against phosphate-buffered saline for 24 hr at 4°C with three changes of buffer, followed by overnight dialysis against MEM. Table 3 shows that after dialysis, significant migration-stimulating activity remained, although the effect was greatly reduced. To determine whether the loss of hydrocortisone alone could have resulted in the reduction of PMS activity, the steroid-treated supernatant was reconstituted with hydrocortisone after dialysis. As shown, the diminished PMS effect was not restored by the addition of hydrocortisone.
Ultrafiltration Since there was partial loss of migration-stimulating activity in the dialysis experiments, ultrafiltration studies were carried out in which both the filtrates and retentates were assayed for PMS activity. Ultrafiltration of serum-containing medium results in concentration of the serum proteins, and therefore in these experiments, supernatants were prepared from MML cultured without foetal bovine serum. The absence of serum does not affect the steroid-monocyte reaction, although serum is necessary for the expression of PMS activity (Stevenson, 1976a). Control and hydrocortisone-treated culture supernatants were ultrafiltered until the volume of the
R. D. Stevenson & Aileen C. Gray
460
TABLE 4. Effect of ultrafiltration on the nmigration-stimulating activity of hydrocortisonetreated MML culture supernatants
Control
Hydrocortisone
Percentage stimulation of migration (± s.e.m.)
66+ 386
91 +029
38+ 223
65+1-31 66+ 1.15 61+191
74±3-17 77+2-23 92+422
14±0*66 17±0-57
Migration area (i s.e.m.) Migration medium
Unfiltered
MML culture supernatant MEM Filtrate Retentate
MML culture supernatant
51 + 283
I2Or0oo0 c
.0
z .21 E
801-
0
c 0
60H E
w 0'
cm 40H 02 w
20h
2
0
4
6
8
10
12
pH
FIG. 1. Effect of pH
on
the migration-stimulating activity of hydrocortisone-treated MML culture super-
natants.
2'0 Cytochrome C
E
co
o
1.0
N
a 0
Elution volume (ml) E/////
I
IL
....................... ,,,,\X\\
Ez l m
Froct ions
FIG. 2. Sephadex G-100 fractionation of concentrated MML culture supernatants.
Polymorph migration stimulator
461
TABLE 5. Effect on migration of hydrocortisone-treated MML culture supernatants fractionated on Sephadex G-100
Migration area (± s.e.m.)
Migration medium G-100 fractions
Control
Hydrocortisone
I II III IV V
61+O*61 55+ 1*17 46+ 1*40 57+ 0-21 62+ 1-28
59+ 1-83 48+2 40 48+2-32 92+4-42 84+4*76
Percentage stimulation of migration (± s.e.m.)
-3+0-10 -13+0*71 4+0.23 61+ 2*94 35+2-11
filtrate was equal to the volume of the retentate. Foetal bovine serum was then added at a concentration of 10% and the effect on migration measured. Table 4 shows that the migration-stimulating activity was retained by the filter and that the effect was increased due to the two-fold concentration. In contrast, migration-stimulating activity in the filtrate of the steroid-treated supernatant was no greater than that due to hydrocortisone alone.
Effect of pH HCl and NaOH were added to control and steroid-treated culture supernatants to produce a range of pH from 2-11. The supernatants were then incubated for 1 hr at 370C, following which the pH of the samples was restored to 7*4. Fig. 1 shows that migration-stimulating activity had been diminished at both acid and alkaline pH values with more marked loss of activity at low pH. Effect on migration offractionated culture supernatants Gel filtration on Sephadex G-100 of concentrated culture supernatants containing 10%/o serum produced the characteristic elution profile (Yoshida, Sonozaki and Cohen, 1973). Figure 2 shows a typical chromatogram obtained when the control and steroid-treated culture supernatants were fractionated. Fraction I contained the exclusion peak and fraction II the albumin peak. Fraction III consisted of samples between fractions II and IV and did not contain detectable protein. Fractions IV and V included substances of approximately 12,000 mol. wt. Cytochrome C, a marker of mol. wt 12,400 was eluted with fraction IV. After concentration as described, the fractions derived from each culture supernatant were assayed for migration-stimulating activity. Table 5 shows that only fractions IV and V of the steroidtreated supernatant stimulated polymorph migration. Enzymatic Digestion Control and steroid-treated culturp supernatants were incubated with the enzyme preparations as described. Table 6 shows that migration-stimulating activity was destroyed by incubation with protease but was unaffected by ribonuclease. TABLE 6. Effect of protease and ribonuclease on the migration-stimulating activity of hydrocortisonetreated MML culture supernatants
Migration area (+ s.e.m.) Migration medium MML culture supernatant
Enzyme pre-incubation
Agarose Protease-agarose
Ribonuclease-agarose
Control
Hydrocortisone
40+ 1-38 46+ 2.09 34+0 87
66+ 2-58 45+ 1.15 55+ 2-06
Percentage stimulation of migration (± s.e.m.)
65+ 3*39 - 2+ 0-10
62+ 2-81
462
R. D. Stevenson & Aileen C. Gray
DISCUSSION The results of this study show that PMS is a soluble factor which is stable at - 20'C, 4VC and at 600C but destroyed by heating at 900C. Its activity was reduced by incubating at alkaline pH and more so at acid pH. There was considerable loss of migration-stimulating activity during prolonged dialysis, but in ultrafiltration studies, PMS was retained by a filter which had a retention efficiency of 100%/o for cytochrome C (mol. wt 12,400). Gel filtration of culture supernatants on Sephadex G-100 showed that migration-stimulating activity was localized in the fractions of approximately 12,000-15,000 mol. wt, which is consistent with the results of the experiments using semi-permeable membranes. The activity of PMS was completely destroyed by the proteolytic enzyme protease, insolubilized on agarose, and was unaffected by ribonuclease or by agarose alone. Previous studies have shown that inhibition of protein synthesis during culture of steroid-treated monocytes prevented the production of PMS (Stevenson, 1976a). Migration-stimulating activity must therefore reside in a peptide moiety, actively synthesized under the influence of corticosteroids. Immune complexes and lymphokines inhibit in vitro polymorph migration (Packalen & WVasserman, 1971; Rocklin, 1974). This may represent the localization of polymorphs in inflammatory areas in vivo. It might therefore be suggested that stimulation of polymorph migration in vitro represents a mechanism by which the aggregation of polymorphs would be prevented. In support of this postulate, steroid administration is known to inhibit the inflammatory exudation of polymorphs (Bishop et al., 1968). Further studies are in progress in an attempt to discover whether PMS is responsible for the antiinflammatory effects of corticosteroids on polymorphonuclear leucocytes. We should like to thank Professor G. M. Wilson and Professor J. R. Anderson for their help and encouragement and Mr William Monaghan for excellent technical assistance.
REFERENCES BISHOP, C.R., ATHENS, J.W., BoGGs, D.R., WARNER, H.R., STEVENSON, R.D. (1973) Hydrocortisone and the migration of human leucocytes: an indirect effect mediated by CARTWRIGHT, G.E. & WINTROBE, M.M. (1968) Leukomononuclear cells. Clin. exp. Immunol. 14, 417. kinetic Studies. XIII. A non-steady-state kinetic evaluation of the mechanism of cortisone-induced granulocyto- STEVENSON, R.D. (1974) Polymorph migration stimulator: a new factor produced by hydrocortisone-treated monosis. J. cin. Invest. 47, 249. cytes. Clin. exp. Immunol. 17, 601. HEILMAN, D.H. (1972) Failure of hydrocortisone to inhibit blastogenesis by pokeweed mitogen in human leucocyte STEVENSON, R.D. (1976a) Studies on the production and action of polymorph migration stimulator. Clin. exp. cultvres. Clin. exp. Immunol. 11, 393. Immunol. 27, 527. PACKALUN, T. & WASSERMAN, J. (1971) Inhibition of migration of normal guinea-pig blood leukocytes by homologous STEVENSON, R.D. (1976b) Effect of steroid therapy on in vitro polymorph migration. Clin. exp. Immunol. 23, 285. immune y2-globulin in the presence of specific antigen. YOSHIDA, T., SONOZAKI, H. & COHEN, S. (1973) The Int. Arch. Allergy, 41, 790. production of migration inhibition factor by B and T ROCKLIN, R.E. (1974) Products of activated lymphocytes: cells of the guinea pig. ]. exp. Med. 138, 784. leukocyte inhibitory factor (LIF) distinct from migration inhibitory factor (MIF). J. Immunol. 112, 1461.
Studies on the physico-chemical characteristics of polymorph migration stimulator R. D. STEVENSON & AILEEN C. GRAY University Departments of Medicine & Pathology, Western Infirmary, Glasgow
(Received 28 April 1976)
SUMMARY
Polymorph migration stimulator is a supernatant factor produced by the interaction between glucocorticosteroids and human blood monocytes in culture. Studies on the physical characteristics of this factor show that it is soluble and stable at high and low temperatures. Its activity is reduced by acid and alkali treatment and destroyed by the proteolytic enzyme protease. Experiments involving dialysis, ultrafiltration and Sephadex G-100 gel filtration indicate that the molecular weight of the polymorph migration stimulator is between 12,000 and 15,000. It is suggested that this factor may mediate the anti-inflammatory effects of glucocorticosteroids on phagocytic cells. INTRODUCTION Human blood monocytes, spleen cells and bone marrow cells react with corticosteroids in vitro to produce a supernatant factor which stimulates polymorph migration, using the capillary tube method. The production of this factor, polymorph migration stimulator (PMS), involves an active biological process and occurs at steroid concentrations which are comparable with physiological and pharmacological levels. The amount of PMS generated by different steroids correlates with their anti-inflammatory, glucocorticoid activity (Stevenson, 1973, 1974 and 1976a). These in vitro observations are supported by studies showing that the migration of polymorphs from patients receiving steroid therapy is enhanced in vitro and that this effect is dependent on an interaction between steroids and mononuclear leucocvtes in vivo (Stevenson, 1976b). It has been suggested that this factor may mediate the effects of glucocorticosteroids on the kinetic behaviour of granulocytic cells in the bone marrow and in areas of inflammation. The experiments described in this paper were undertaken to study the physical characteristics of PMS.
MATERIALS AND METHODS Hydrocortisone. Hydrocortisone (Merck, Sharpe & Dohme) was made up as a 1% solution in 95% ethyl alcohol (Heilman, 1972). Further dilutions were made in Eagle's minimal essential medium (MEM). The standard concentration used was 10 jg/ml of medium. Production ofPMS. Previous experiments have shown that lymphocytes do not affect the production ofPMS from steroidtreated monocytes (Stevenson, 1974) and therefore, for ease of preparation in this study, mixed mononuclear leucocytes (MML) and not purified monocytes were cultured with hydrocortisone to produce PMS. The details of the MML culture technique have previously been described (Stevenson, 1974). In experiments which required large volumes of culture supernatants, MML were cultured in glass universal containers in volumes of 6 ml containing 8 x 106 cells. Dialysis and ultrafiltration. Dialysis was carried out using 8/32-in Visking tubing. Supernatants were ultrafiltered through collodion bags (Sartorius) using suction flasks. The pore size of the collodion bags was 8 pm which retained substances of molecular weight greater than 12,400. Gelfiltration of culture supernatants. Culture supernatants (50 ml) were lyophilized and resuspended in 6 ml of distilled Correspondence: Dr R. D. Stevenson, University Department of Medicine, Western Infirmary, Glasgow GIl 6NT.
457
R. D. Stevenson L Aileen C. Gray
458
water. The concentrated samples, after clarification by centrifugation were applied to a Sephadex G-100 column, 2-5 x 100 cm, equilibrated with phosphate-buffered saline (0-01 M phosphate, pH 7-3) with a flow rate of 22 ml/hr. The eluate was collected in 7-ml volumes which were pooled into five fractions on the basis of the chromatogram (OD at 280 nm). After concentration using carbowax, the samples were dialysed against MEM overnight and the final volume of each was adjusted to 12 ml. Before the migration assay, the samples were sterilized by millipore filters. Enzyme treatment of PMS. Enzymatic digestion of PMS was attempted using protease and ribonuclease insolubilized on agarose (Miles Laboratories). The enzyme-agarose preparations were washed with MEM and 3-ml bed volumes were mixed with 3-ml aliquots of culture supernatants. The incubation mixtures were transferred to 25-ml conical flasks and the pH adjusted to 7 0 for ribonuclease and 8-0 for protease. Controls consisted of culture supernatants mixed with agarose alone. Samples were then incubated for 2 hr at 37'C in a shaking water-bath, after which the mixtures were centrifuged, the supernatants decanted and the pH values restored to 7-4. Measurement of PMS activity. The effect of PMS was measured using the polymorph migration technique previously described in detail (Stevenson, 1973). Migration chambers were filled with supernatants from experimental leucocyte cultures and normal human mixed leucocytes were used as a source of migrating polymorphs. Percentage stimulation of migration was calculated as: Larea of migration in steroid-treated culture supernatant 1 J ° area of migration in control culture supernatant
RESULTS The two Figs and Tables 1-6 illustrate typical experiments from each group; each experiment was repeated at least three times. TABLE 1. Effect of ultracentrifugation on the migration-stimulating activity of hydrocortisone-treated MML culture supernatants
Migration area (± s.e.m.) Migration medium Control 100,000 g for 1 hr.
MML culture supernatant MEM MML culture supernatant
Control
Percentage stimulation of migration Hydrocortisone (+ s.e.m.)
78+ 1-73
108+ 3-08
38+ 1-37
69+ 2-83 76+2-20
79+ 3-83 109+264
14+ 0.89 43+ 1.62
Solubility In previous experiments, PMS was assayed using the crude supernatants of steroid-treated MML cultures. It was therefore possible that subcellular fractions, derived from damaged cells, might be responsible for the stimulation of migration. To determine whether PMS was soluble or particulate, control and hydrocortisone-treated culture supernatants were centrifuged at 100,000 g for 1 hr at 20°C and the supernatants assayed for migration-stimulating activity. In experiments, using this system, controls of MEM with and without hydrocortisone are included, since hydrocortisone alone marginally stimulates polymorph migration. This effect occurs because of the reaction between hydrocortisone and the monocytes in the migrating cell population, which produces a migration-stimulating effect on the remaining polymorphs (Stevenson, 1973). The results in Table 1 show that migration-stimulating activity was retained in the particle-free supernatant from the steroid-treated culture. Temperature stability There was no loss of PMS activity when stored at - 20°C, 4°C or room temperature for 1 week. The effect of heat on PMS was studied by incubating control and hydrocortisone-treated culture supernatants in a water-bath for 1 hr at 60°C and 90°C. Table 2 shows that migration-stimulating activity was stable at 60°C but was destroyed by heating at 90°C.
Polymorplh migration stimulator
459
TABLE 2. Effect of temperature on the migration-stimulating activity of hydrocortisonetreated MML culture supernatants
Control
Hydrocortisone
Percentage stimulation of migration (± s.e.m.)
60+ 2-70
86+ 1P04
43+ 2-00
66+ 3-02 61+ 1P43
73+ 1-36 89+ 1-08
11+0*54 46+ 1-21
72+1P38
69+ 1.19
-4+O 10
Migration area
(± s.e.m.) Temperature (OC)
Migration medium
20
MML culture supernatant MEM MML culture supernatant MML culture supernatant
60 90
TABLE 3. Effect of dialysis on the migration-stimulating activity of hydrocortisone-treated MML culture supernatants
Migration Migration medium
area
(± s.e.m.) Control
Dialysed
(a) (b) (a) (b) (a) (b) (c)
Control culture supernatant Hydrocortisone-treated supernatant MEM MEM + hydrocortisone Control culture supernatant Hydrocortisone-treated supernatant Hydrocortisone-treated supernatant reconstituted with hydrocortisone*
68+ 2-18 100+ 0 99 62+1.15 65+0i55 61+ 2-16 74+i64 75+ 2-18
Percentage stimulation of migration (+ s.e.m.)
47+ 1*58 5+0 10 21+ 0-88 23+ 1P05
* Hydrocortisone (10 ug/ml) added to steroid-treated supernatant after dialysis.
Dialysis Control and hydrocortisone-treated culture supernatants were dialysed against phosphate-buffered saline for 24 hr at 4°C with three changes of buffer, followed by overnight dialysis against MEM. Table 3 shows that after dialysis, significant migration-stimulating activity remained, although the effect was greatly reduced. To determine whether the loss of hydrocortisone alone could have resulted in the reduction of PMS activity, the steroid-treated supernatant was reconstituted with hydrocortisone after dialysis. As shown, the diminished PMS effect was not restored by the addition of hydrocortisone.
Ultrafiltration Since there was partial loss of migration-stimulating activity in the dialysis experiments, ultrafiltration studies were carried out in which both the filtrates and retentates were assayed for PMS activity. Ultrafiltration of serum-containing medium results in concentration of the serum proteins, and therefore in these experiments, supernatants were prepared from MML cultured without foetal bovine serum. The absence of serum does not affect the steroid-monocyte reaction, although serum is necessary for the expression of PMS activity (Stevenson, 1976a). Control and hydrocortisone-treated culture supernatants were ultrafiltered until the volume of the
R. D. Stevenson & Aileen C. Gray
460
TABLE 4. Effect of ultrafiltration on the nmigration-stimulating activity of hydrocortisonetreated MML culture supernatants
Control
Hydrocortisone
Percentage stimulation of migration (± s.e.m.)
66+ 386
91 +029
38+ 223
65+1-31 66+ 1.15 61+191
74±3-17 77+2-23 92+422
14±0*66 17±0-57
Migration area (i s.e.m.) Migration medium
Unfiltered
MML culture supernatant MEM Filtrate Retentate
MML culture supernatant
51 + 283
I2Or0oo0 c
.0
z .21 E
801-
0
c 0
60H E
w 0'
cm 40H 02 w
20h
2
0
4
6
8
10
12
pH
FIG. 1. Effect of pH
on
the migration-stimulating activity of hydrocortisone-treated MML culture super-
natants.
2'0 Cytochrome C
E
co
o
1.0
N
a 0
Elution volume (ml) E/////
I
IL
....................... ,,,,\X\\
Ez l m
Froct ions
FIG. 2. Sephadex G-100 fractionation of concentrated MML culture supernatants.
Polymorph migration stimulator
461
TABLE 5. Effect on migration of hydrocortisone-treated MML culture supernatants fractionated on Sephadex G-100
Migration area (± s.e.m.)
Migration medium G-100 fractions
Control
Hydrocortisone
I II III IV V
61+O*61 55+ 1*17 46+ 1*40 57+ 0-21 62+ 1-28
59+ 1-83 48+2 40 48+2-32 92+4-42 84+4*76
Percentage stimulation of migration (± s.e.m.)
-3+0-10 -13+0*71 4+0.23 61+ 2*94 35+2-11
filtrate was equal to the volume of the retentate. Foetal bovine serum was then added at a concentration of 10% and the effect on migration measured. Table 4 shows that the migration-stimulating activity was retained by the filter and that the effect was increased due to the two-fold concentration. In contrast, migration-stimulating activity in the filtrate of the steroid-treated supernatant was no greater than that due to hydrocortisone alone.
Effect of pH HCl and NaOH were added to control and steroid-treated culture supernatants to produce a range of pH from 2-11. The supernatants were then incubated for 1 hr at 370C, following which the pH of the samples was restored to 7*4. Fig. 1 shows that migration-stimulating activity had been diminished at both acid and alkaline pH values with more marked loss of activity at low pH. Effect on migration offractionated culture supernatants Gel filtration on Sephadex G-100 of concentrated culture supernatants containing 10%/o serum produced the characteristic elution profile (Yoshida, Sonozaki and Cohen, 1973). Figure 2 shows a typical chromatogram obtained when the control and steroid-treated culture supernatants were fractionated. Fraction I contained the exclusion peak and fraction II the albumin peak. Fraction III consisted of samples between fractions II and IV and did not contain detectable protein. Fractions IV and V included substances of approximately 12,000 mol. wt. Cytochrome C, a marker of mol. wt 12,400 was eluted with fraction IV. After concentration as described, the fractions derived from each culture supernatant were assayed for migration-stimulating activity. Table 5 shows that only fractions IV and V of the steroidtreated supernatant stimulated polymorph migration. Enzymatic Digestion Control and steroid-treated culturp supernatants were incubated with the enzyme preparations as described. Table 6 shows that migration-stimulating activity was destroyed by incubation with protease but was unaffected by ribonuclease. TABLE 6. Effect of protease and ribonuclease on the migration-stimulating activity of hydrocortisonetreated MML culture supernatants
Migration area (+ s.e.m.) Migration medium MML culture supernatant
Enzyme pre-incubation
Agarose Protease-agarose
Ribonuclease-agarose
Control
Hydrocortisone
40+ 1-38 46+ 2.09 34+0 87
66+ 2-58 45+ 1.15 55+ 2-06
Percentage stimulation of migration (± s.e.m.)
65+ 3*39 - 2+ 0-10
62+ 2-81
462
R. D. Stevenson & Aileen C. Gray
DISCUSSION The results of this study show that PMS is a soluble factor which is stable at - 20'C, 4VC and at 600C but destroyed by heating at 900C. Its activity was reduced by incubating at alkaline pH and more so at acid pH. There was considerable loss of migration-stimulating activity during prolonged dialysis, but in ultrafiltration studies, PMS was retained by a filter which had a retention efficiency of 100%/o for cytochrome C (mol. wt 12,400). Gel filtration of culture supernatants on Sephadex G-100 showed that migration-stimulating activity was localized in the fractions of approximately 12,000-15,000 mol. wt, which is consistent with the results of the experiments using semi-permeable membranes. The activity of PMS was completely destroyed by the proteolytic enzyme protease, insolubilized on agarose, and was unaffected by ribonuclease or by agarose alone. Previous studies have shown that inhibition of protein synthesis during culture of steroid-treated monocytes prevented the production of PMS (Stevenson, 1976a). Migration-stimulating activity must therefore reside in a peptide moiety, actively synthesized under the influence of corticosteroids. Immune complexes and lymphokines inhibit in vitro polymorph migration (Packalen & WVasserman, 1971; Rocklin, 1974). This may represent the localization of polymorphs in inflammatory areas in vivo. It might therefore be suggested that stimulation of polymorph migration in vitro represents a mechanism by which the aggregation of polymorphs would be prevented. In support of this postulate, steroid administration is known to inhibit the inflammatory exudation of polymorphs (Bishop et al., 1968). Further studies are in progress in an attempt to discover whether PMS is responsible for the antiinflammatory effects of corticosteroids on polymorphonuclear leucocytes. We should like to thank Professor G. M. Wilson and Professor J. R. Anderson for their help and encouragement and Mr William Monaghan for excellent technical assistance.
REFERENCES BISHOP, C.R., ATHENS, J.W., BoGGs, D.R., WARNER, H.R., STEVENSON, R.D. (1973) Hydrocortisone and the migration of human leucocytes: an indirect effect mediated by CARTWRIGHT, G.E. & WINTROBE, M.M. (1968) Leukomononuclear cells. Clin. exp. Immunol. 14, 417. kinetic Studies. XIII. A non-steady-state kinetic evaluation of the mechanism of cortisone-induced granulocyto- STEVENSON, R.D. (1974) Polymorph migration stimulator: a new factor produced by hydrocortisone-treated monosis. J. cin. Invest. 47, 249. cytes. Clin. exp. Immunol. 17, 601. HEILMAN, D.H. (1972) Failure of hydrocortisone to inhibit blastogenesis by pokeweed mitogen in human leucocyte STEVENSON, R.D. (1976a) Studies on the production and action of polymorph migration stimulator. Clin. exp. cultvres. Clin. exp. Immunol. 11, 393. Immunol. 27, 527. PACKALUN, T. & WASSERMAN, J. (1971) Inhibition of migration of normal guinea-pig blood leukocytes by homologous STEVENSON, R.D. (1976b) Effect of steroid therapy on in vitro polymorph migration. Clin. exp. Immunol. 23, 285. immune y2-globulin in the presence of specific antigen. YOSHIDA, T., SONOZAKI, H. & COHEN, S. (1973) The Int. Arch. Allergy, 41, 790. production of migration inhibition factor by B and T ROCKLIN, R.E. (1974) Products of activated lymphocytes: cells of the guinea pig. ]. exp. Med. 138, 784. leukocyte inhibitory factor (LIF) distinct from migration inhibitory factor (MIF). J. Immunol. 112, 1461.
