Production of Chemotactic Factor for Lymphocytes by Neutral SH-Dependent Protease of Rabbit PMN Leukocytes from Immunoglobulins, Especially lgMl** YASUNORI
In inflammation, PMN leukocyte emigration is followed by lymphocyte emigration. Two neutral proteases were isolated from lysosomal fraction of rabbit PMN leukocytes and purified by chromatography. The SH-dependent protease converted in vitro a naturally occurring IgM and specific antibody IgM to a chemotactic factor for lymphocytes; its molecular size was assumed to be around 14,000. Lymphocytes were collected from the thoracic duct lymph of rats. The chemotactic generation was induced by a short treatment with small amount of the enzyme, but the chemotactic factor produced was inactivated by a prolonged digestion with the enzyme. The chemotactic generation by the enzyme of rabbit IgG was apparently less marked. On the other hand, the SH-independent protease was ineffective for such chemotactic generation, sug,gesting different enzymatic characteristics of these proteases.
INTRODUCTION As previously described, we (H.H.) have isolated a chemotactic factor (leukoegresin) specific for neutrophilic PMN leukocytes from the inflamed sites and then purified by chromatography (l-3). Leukoegresin satisfied several criteria to make it acceptable as a natural mediator of inflammatory leukotaxis, i.e., local availability, parallelity between amount and time-course of the reaction, and morphological resemblance of the reaction (1, 4, 5). The substance shared common antigenic sites with serum IgG (6)) and was produced in zrifvo (7, 8) and in viva ( 1, 4) from IgG of rabbit and man by a neutral SH-dependent protease from inflammatory tissue. The molecular size of the chemotactic factor was approximately 140,000, suggesting a minor structural change of the IgG molecule (9). However, the peptides released by the enzyme from the IgG molecule were ineffective for PMN leukocytes. As is well-known, PMN leukocyte emigration is followed by lymphocyte emigration in inflammation. We have recently found two neutral proteases SH-dependent and SH-independent in the lysosomal fraction of rabbit PMN leukocytes, and effectively purified by chromatography ( 10, 11) . The present communication deals with in vitro production of a chemotactic factor for lymphocytes by the PMN SH-dependent protease from rabbit immunoglobulins, especially IgM. 1 This work was supported by grants from the Mitsubishi Foundation, Tokyo, the Malignant Rheumatoid Arthritis Study Group, and The Society for Metabolism Research, Tokyo. 2 This is No. 1 of Studies of Lymphocyte Chemotaxis in Inflammation. 100 0 1975 by Academic Press, Inc. of reproduction in any form reserved.
The capacity to promote lymphocyte migration was measured in vitro by a modification (6) of Boyden’s method ( 12) using Millipore filters (SSWPO 1300, pore size 3 pm ; Millipore Filter Co., Bedford), and a metal chamber containing two l-ml compartments. Test samples, in 0.067 M phosphate buffer (pH 7.4)) were placed in the lower compartment; the filter was placed on it, and lymphocytes, collected from the thoracic duct lymph of male Donryu rats (20&250 g) and suspended in Eagle’s MEM containing 2% human serum albumin (pH 7.4) at a concentration of 4 x lo8 cells/ml, were poured into the upper compartment. was A “clear vinyl tube,” 1.0 mm diameter (Dural Plastics Co., Australia) inserted into the thoracic duct under the diaphragm according to the method of Bollman, Chanin, and Grindlay (13). F rom 12-15 hr after the operation, the thoracic lymph, which spontaneously dropped into Eagle’s MEM containing heparin (500 units/ml) and 2% human serum albumin (pH 7.4)) was collected in the cold for 3 hr, and immediately centrifuged at 400 rpm for 10 min for collection of lymphocytes. The- chambers were incubated for 3 hr at 37” C using a 5% CO, atmosphere. The presence of COz in the environment seemed to be a requirement for lymphocyte migration. The cells that had migrated through the filter were stained with hematoxylin and counted over 20 microscopic fields (10 x 40) randomly selected. Identification of the migrated cells was performed without any trouble, because the majority (over 95%) of suspended cells was of small lymphocyte. Isolation
PMN leukocytes of male albino rabbits (2.8-3.0 kg) were collected from the peritoneal exudates produced by injection of glycogen (14). The isolation of two neutral proteases from PMN leukocyte was performed according to the previous methods (10, 11) . After homogenization in 0.34 M sucrose solution, the celIs ( lo8 cells/ml) were extracted with 0.01 M citric acid (pH 2.8) in the cold for 4 hr essentially according to the method of Cohn and Hirsch (15)) and centrifuged at 12,000 rpm. for 20 min. The supernatant fluid was fractionated with ammonium sulfate at 70% saturation. After desalting through a Sephadex G-50 column, the protein fraction was eluted through a DEAE-Sephadex A-50 column (1 X 18 cm) by concentration stepwise changes of eluting buffers with the same pH (7.4) as follows: 0.02 M phosphate buffer, 0.067 M phosphate buffer, 0.067 M plus 0.2 M NaCl, and finally 1.0 M NaCl. The protein fraction eluted in 0.02 M phosphate buffer contained two neutral proteases, and was further eluted through a CM-Sephadex C-50 column (1 x 18 cm) by increasing salt gradient (from 0.02 M phosphate buffer to 0.02 M plus 0.5 1M NaCl) and then in 1.0 M NaCl. The neutral SH-dependent protease was concentrated in the fraction eluted in 0.02 M phosphate buffer ; its molecular size was about 14,000, and the enzyme induced marked inflammatory reactions on experimental injection (11). The neutral SH-independent protease eluted in 1.0 M NaCl induced no inflammatory reaction on experimental injection. Before use, the SH-dependent protase was activated by adding cysteine to a final concentration of lo-’ M
of Protease Activity
Activity was measured by a modification (16, 17) of the casein digestion method of Kunitz (IS). The pH of the reaction was 7.1 and the ionic strength was 0.25. After incubation at 37” C for 60 min, an equal volume of 6% trichloroacetic acid was added to the reaction mixture and filtered when precipitation was completed. The extinction at 276 nm of the filtrate was measured. The arbitrary unit of proteolytic activity was defined as the amount of enzyme which would cause an increase of 0.001 unit of extinction per minute of digestion (19). Accordingly, one proteolytic unit represents 0.06 absorbance units at 276 nm. Enzyme samples with proteolytic activity of 0.66 or 0.16 units/ml were used. Preparation
Rabbit IgG fraction was separated in the cold from fresh sera of normal rabbits according to the method of Kapusta and Halberstam (20)) using sodium sulfate precipitation and DEAE-cellulose chromatography with 0.01 M phosphate buffer (pH 8.0) ; the isolated IgG was homogeneous as judged by immunoelectrophoresis with goat antiserum against rabbit serum. The IgG fraction was dissolved in 0.067 M phosphate buffer, pH 7.4 at concentration of 200 pg/ml. Rabbit IgM fraction was also separated in the cold from fresh sera of normal rabbits according to a modification of the method of Vaerman, Heremans, and Vaerman (21) by dialysis, Sephadex G-200 chromatography, and block electrophoresis on Pevikon C-870 (Superfosfat Aktiebolag, Stockholm, Sweden) ; the isolated IgM was homogeneous as judged by immunoelectorphoresis with goat antiserum against rabbit serum. The IgM fraction was dissolved in 0.067 M phosphate buffer, pH 7.4 at concentration of 200 pg/ml. Preparation of Antibody IgM Against Salmonella typhimurium This was performed essentially according to the method of Kishimoto and Onoue (22). Male albino rabbits (2.5-3.0 kg) were injected iv with 3 mg (wet weight) of heat-killed S. typhimurium, and 2 wk later, a booster injection of 10 mg of the same antigen was given. The animals were bled on the 5th, 7th, 9th, and 11th days after the second injection. A globulin fraction of pooled antisera was precipitated twice with ammonium sulfate at 50% saturation. The precipitate *as dissolved and dialyzed against 0.15 M NaCl buffered at pH 7.2 with phosphate buffer. Heatkilled S. typhimurium (3 x 1OX2cells) was added to 500 ml of the globulin fraction in the presence of 0.01 M EDTA. The mixture was agitated gently at 37” C for 90 min and at 4” C for 18 hr. The cells were separated and washed with phosphate-buffered saline of pH 7.2 until the optic density of .the supernatant ’ fluid was less than 0.05 at 280 nm. The antibodies absorbed were eluted with 0.1 M sodium acetate (pH 3.9) at 37” C for 90 min. The eluate was neutralized, concentrated, and dialyzed against 0.2 M Tris-HCl buffer (PH. 8.0). For separation of IgM and IgG antibodies, the eluate was subjected to gel filtration on Sephadex G-ZOO equlibrated with 0.3 A4 NaCI-O.05 M Tris-phosphate buffer (pH 8.0). Gel filtration was carried out with a 2.5 X lOO-cm upward-flow column (Pharmacia, Uppsala, Sweden). After rechromatography, the IgM antibody preparations were further purified by block electrophoresis through a column of Pevikon C-870 equilibrated with 0.05 M Verona1 buffer (pH 8.6) at a current of
1.5 mA for 48 hr (23). The isolated antibody IgM was homogeneous as judged by immunoelectrophoresis with goat antiserum against rabbit serum. The antibody IgM was dialyzed against 0.067 M phosphate buffer (pH 7.4) for 16 hr, and stored at 4” C after passage through Millipore filter (0.45 pm). Estimation
of Molecular Size of a Chewzoiactic Factor for Lymphocytes
This was performed by gel filtration on Sephadex G-200 (24). Glucagon (3,500, MW; Sigma, St. Louis, MO), cytochrome c (12,400, MW; Sigma, St. Louis, MO) and bovine serum albumin (67,000 in monomer MW ; Armour, Kankakee, Ic) were used as standard substances. RESULTS Generation of Chenzotactic Activity Dependent Protease
from Rabbit Imnzunoglobulins
Equal volumes (0.5 ml) of rabbit IgM or IgG (200 pg/ml in 0.067 111phosphate buffer, pH 7.4) and the SH-dependent protease or SH-independent protease solution (0.16 and 0.66 units/ml in 0.067 1M phosphate buffer, pH 7.4) were mixed and incubated at 37” C for 3 hr. As summarized in Table 1, rabbit IgM had no chemotactic activity on lymphocytes, but it became strongly chemotactic after treatment with the SH-dependent protease (0.08 and 0.33 units) in the presence of cysteine ( 1O-3 M) ; and the TABLE
In Vitro CHEMOTACTIC
GENERATION OF RABBIT IMMUNOGLOBULIN SH-DEPENDENT PROTEASE FROM PMN LEUKOCYTES’
Samples in lower compartment Rabbit immunoglobulins
kM I& Buffere Buffer
100 100 100 100 100 100 100 100 100 100
Proteases (proteolytic units)
Cysteine (final concn)
0.33c O.O& Buffer Buffer 0.33c 0.33c 0.ot-v Buffer Buffer 0.33d 0.33d 0.33d Buffer
lo+ M 1O-3 M 1O-3 M Buffer 10-s M lo+ M lo+ M lo-* M Buffer Buffer Buffer Buffer Buffer
y The mixture of immunoglobulin and protease (0.5 ml) placed in the lowere compartment for assay of chemotactic * Mean values of five assays. c Proteolytic units of SH-dependent protease from P-MN unit/ml. d Proteolytic units of SH-independent protease from P-MN * 0.067 M phosphate buffer (pH 7.4).
No. of lymphocytes migrated*
240 166 45 41 40 156 69 27 25 58 60 39 42
was incubated at 37’C for 3 hr and potency. See text. leukocytes
added were 0.16 or 0.66
added were 0.66 unit/ml.
CHEMOTACTIC POTENCY OF RABBIT IgM AFTER PROLONGED DIGESTION NEUTRAL SH-DEPENDENT PROTEASE FROM PMN LEUKOCYTES”
Samples in lower compartment
Rabbit IgM at 3-hr digestion lo-hr digestion 24-hr digestion Nondigestion Bufferc
No. 0f lymphocytes migratedb
171 90 50 41 38
(LThe mixture of each sample (0.5 ml) was placed in the lower compartment for assay of chemotactic potency. Rabbit IgM (200 pg/ml) had been previously treated with the SH-dependent protease (0.16 unit/ml) in the presence of 10V31Mcysteine. See text. b Mean values of five assays. 0 0.067 M phosphate buffer (pH 7.4).
intensity of chemotactic generation seemed to be associated with the activity of the SH-dependent protease added. The protease itself was ineffective for lymphocytes. By contrast, no or little chemotactic generation of IgM for lymphocytes was observed even after treatment with higher activity of SH-independent protease (0.33 units). The protease itself showed no chemotactic activity. On the other hand, rabbit IgG became chemotactic only when treated with higher activity of the SH-dependent protease (0.33 units), though its chemotactic intensity was less marked. Little chemotactic activity was revealed when treated with lower activity
of the enzyme
chemotactic generation of IgG. Chemotactic Activity of Rabbit IgM at Various Durations
Treated with PMhT SH-Depepldent Pvotease
Rabbit IgM was used in this assay, because of its stronger chemotactic generation by PMN SH-dependent protease. Equal volumes (0.5 ml) of rabbit IgM (ZOO pg/ml) and the cysteine-activated SH-dependent protease (0.16 units/ml) were mixed and incubated at 37” C for 3, 10, and 24 hr, respectively. As summarized in Table 2, in a parallel with prolongation of the enzymatic treatment, the chemotactic activity was decreased; and little or no chemotactic activity was found with the samples after 24-hr digestion. Generation of Chemotactic Activity Dependent Protease
from Rabbit Antibody
In order to verify the chemotactic generation from IgM molecule, specific rabbit antibody IgM against S. typhimurium was used in the same type of experiment as mentioned above. Equal volumes (0.5 ml) of antibody IgM (200 pg/ml in 0.067 M phosphate buffer, pH 7.4) and the cysteine-activated SH-dependent protease (0.16 and 0.66 units/ml in 0.067 M phosphate buffer, pH 7.4) were mixed and incubated at 37” C for 3 hr.
As summarized in Table 3, antibody IgM itself showed no chemotactic effect on lymphocytes, but it became strongly chemotactic after treatment with the activated SH-dependent protease ; and the intensity of the chemotactic generation seemed to be associated with the activity of the enzyme added. These observations indicated that the chemotact? activity for lymphocytes was similarly generated by the enzymatic treatment from naturally occurring IgM as well as from antibody IgM of rabbits. Gel Filtration
of IgM Treated with PMN SH-Dependent Protease
Based on the observations mentioned above, normal serum IgM and antibody IgM, respectively, were treated with the SH-dependent protease in the presence of 1O-3M cysteine at 37” C for 3 hr, and the reaction mixtures, respectively, were eluted through a Sephadex G-ZOO upward-flow column (1.5 X 95 cm) equilibrated with 0.067 M phosphate buffer (pH 7.4). The flow rate was 7 ml/hr and 2.8 ml effluent fraction was collected, respectively. As shown in Fig. la, four chromatographic peaks were obtained when normal serum IgM (2 mg) treated with the SE-I-dependent protease (6.6 units) was eluted. The total yield measured as the absorbancy at 280 nm was about 93% of the applied sample. The first peak contained 16.7%, the second 4.0%, the third 30.6%, and the fourth 43.6%. The chemostatic activity for lymphocytes was predominantly concentrated in the fourth component. Since the fourth peak contained the SH-dependent protease, but the enzyme itself had no chemotactic activity for lymphocytes (Tables 1 and 3), it was reasonable that the chemotactic activity of this peak was due to the presence of a chemotactic factor for lymphocytes. The molecular size of the chemotactic factor was assumed to be around 14,000, because the elution volume of the fourth peak was about 185 ml, that of cytochrome c about 190 ml, that of bovine serum albumin about 126 ml, and that of glucagon about 217 ml. The molecular size of the enzyme reported was about 14,000 ( 19). TABLE
In Vitro CHEMOTACTIC GENERATION OF RABBIT ANTI-S. typhimurium IgM BY NEUTRAL SH-DEPENDENT PROTEASE FROM PMN LEUKOCYTES~ Samples in lower compartment Rabbit antibody IgM k) 100 100 100 100 Bufferc Buffer
SH-dependent (proteolytic 0.33 0.08 Buffer Buffer 0.33 Buffer
Cysteine (final concn) 10-a M lO+ M 10-s M Buffer 1OW M Buffer
No. of lymphocytes migrated6
225 162 64 62 59 51
a The mixture of each sample (0.5 ml) was incubated at 37°C for 3 hr and placed in the lower compartment for assay of chemotactic potency. The proteolytic activity of SH-dependent protease added was 0.06 or 0.66 unit/ml. See text. b Mean values of five assays. c 0.067 M phosphate buffer (pH 7.4).
As seen in Fig. lb, when antibody IgM (1 mg) was treated under the similar enzymatic condition (3.3 units) and eluted, five chromatograpbic peaks were obtained; the first contained about 15.1%, the second about S.S%, the third 10.6%, and the fourth about 8.4%, and the fifth about 42.6%. The total yield was about S470 of proteins at 280 nm in the eluate. The cbemotactic activity ior lymphocytes was revealed in the fifth peak corresponding to the fourth peak in Fig. la. DISCUSSION The previous observations suggested that after the increase in vascular permeability (4, 25), the neutral SH-dependent protease, which was activated in and released from the mesenchymal connective tissue cells by inflammatory stimulation, e.g., antigen-antibody reaction (1 ( 26-2s). first converted the exuded IgG molecule to a cbemotactic factor (leukoegresin) which caused the emigration of PMN leukocytes into the inflammatory lesion (2, 5, i). In view of the observations that PMN emigration is followed by lymphocyte emigration in inflammation, the present results seemed to be reasonable that the neutral SH-dependent protease from PMN leukocytes converted the exuded immul~oglobulil~s, especially IgM, to a chemotactic factor for lymphocytes. The acti\-ation and release of the SH-dependent protease in and from rabbit PMN leukocytes was caused by antigen-antibody complexes ( 10). The observations presented demonstrated that rabbit Igl\l became strongly chemo-
tactic for lymphocytes even when treated with smaller amount of the SH-dependent protease
supported by the use of specific antibody IghI.
On the other hand,
FIG. 1. Distribution of chemotactic activity generated from IgM on Sephadex G-ZOO.Stippled columns show chemotactic potency. la: Norma1 serum IgM (2 mg) was incubated with activated SH-dependent protease (6.6 units) at 37” C for 3 hr, eluted, and then tested for chemotactic activity for lymphocytes. Protein concentrations at 280 nm of each fraction tested were as follows : a, 0.18; b, 0.10; c, 0.12; d, 0.23. Fraction d contained the SH-dependent protease, but the enzyme itself had no chemotactic potency (See Tables 1 and 3). Elution profile of the SH-dependent protease (- - - - - - -) was superimposed after separate elution. lb : Antibody Ighl (1 mg) was similarly incubated with activated SH-dependent protease (3.3 units) and eluted. Protein concentrations at 280 nm of each fraction tested were as follows: a, 0.12; h, 0.06; c, 0.10; d, 0.07; e, 0.07: f, 0.10; g. 0.10: h, 0.10. Fractions f. g, and h contained the SH-dependent protease. Difference in the chromatographic patterns between normal Ighi and antibody IgM after enzymatic treatment was supposed to be related to the previous treatment of the antibody Ighl in an acidic condition during separation of this antibody. I, Bovine serum albumin; ----+, Cytochrome c.
chemotactic generation of rabbit IgG under the same enzymatic conditions was apparently less marked. The reason of the phcnoinenon is obscure at present, but it might be concerned with the structure specificit! of immunoglobulin molecules or with the favorable affinity of the molecule to lymphocytes. At present, the molecular size of the cbemotactic factor, produced from normal serum IgAI and antibody IgRI, was assumed to be similar to that of the SHdependent protease (about 14,000) because both of the chemotactic factor and enzyme were found in the same chromatographic fraction eluted on Sephadex G-200. The chromatographic observations suggested that the chemotactic factor was apparently different from i~~~i~~unoglobulin-derived chemotactic factor or leukoegresin for PAIN leukocytes ; their molecular size was similarly about 140,000, and they were ineffective for lymphocytes (2. 29). Such separation of chemotactically active products of im~~~unoglobuli~~ seemed to be important, because it is supposed that the natural mediators for cell emigration in inflammation may be specific in their action. Leukoegresin was apparently specilic for emigration of neutrophilic PMN leukocytes (29). The problem of isolation of such chemotactic factor for lymphocytes from inflamed sites remains to be studied. A chemotactic activity for lymphocytes was detected in culture fluids from antigen-stimulated lymphoid cells of rats (30). However. it was difficult to compare these two chemotactic factors for lymphocytes at present purification. Such chemotactic generation for lymphocytes by the SH-dependent protease was found to decrease in parallel with a prolongation of the digestion period; and the chemotactic activity was almost abolished on 21-hr digestion. Such loss of chemotactic actkity was assumed to be associated with an advanced degradation of the chemotactic factor by the SH-dependent protease. A similar loss of chemotactic activity by prolonged digestion with the SH-dependent protease has been observed with leukoegresin (9). At the present experiment, lymphocytes were collected from the thoracic lymph of rats under the careful conditions described above. Most of the cells collected were small lymphocytes. Accordingly, we had no trouble in identifying the migrated cells, and obtained reasonable constant results. At the preliminary experiment, the cells from the lymph nodes of rats and rabbits were tested for chemotactic potency, but some difficulties were experienced in the identification or counting of migrated cells. It was of interest to note that no chemotactic generation of immunoglobuli~~ for lymphocytes was induced by treatment \vith the SH-independent protease, indicating the characteristics of these two proteases of PIIT\’ leukocytes. As previously suggested (31), rabbit IgG had no chemotactic actkit! for mononuclear cells collected from oil-induced peritoneal exudates. but it became chemotactic when treated with the SH-independent protease described above. In contrast, the SH-dependent protease did not generate chemostatic activity for momnlclear cells during incubation with IgG molecule when tested at the same level of acti\.ity. These preliminary observations suggested that production of different types of chemotactic factors might be associated with the mode of cleavage of im~~~L1i~oglobulinmolecules bj different proteases.
\Ve are indebted to Dr. T. Kouno in this laboratory for purification of PMN leukocyte proKyushu University Dental School, teases, to Dr. K. Onoue, Department of Biochemistry,
Eukut)ka, for puriticatiou of rahljit antibody IgM, and to Dr. Y. Nawa, in this Medical School for collection of thoraric lymphocytes.
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