In Vitro and In Vivo Production of Chemotactic Inhibitors by Tumor Cells Marion C. Cohen, PhD, John P. Brozna, BS, and Peter A. Ward, MD
The ascites fluid or peritoneal washings of DBA/2 mice bearing the P815 mastocytoma have been found to contain a chemotactic factor inactivator (CFI) which inactivates the bacterial chemotactic factor as well as the chemotactic activitv associated with the C3 and C5 fragments when assayed on rabbit neutrophils. The amount of CFI is proportional to the number of tumor cells in the peritoneal exudate. The inactivator is also found in tumor cell hemogenates as well as in culture fluid from tumor cells growing in vitro. The activity is heat-labile but is not affected by protease inhibitors. Its molecular weight is greater than 50,000 daltons, based on Sephadex chromatography and sucrose density gradient ultracentrifugation studies. In C57BL/6 mice, which reject the mastocytoma, CFI levels decrease in proportion to the decreasing numbers of tumor cells. (Am j Pathol 94:603-614, 1979)
IN CLINICAL SETTINGS and in experimental models, there is abundant evidence that inflammatory responses as well as immune reactions may be defective in the presence of neoplastic disease (reviewved in Reference 1). Since mechanisms for tumor killing are known that involve neutrophils 2 and macrophages 34 as wvell as lymphocytes,5 defective inflammatory- responses could favor the tumor at the expense of the host. The mechanism of such subversion of defense mechanisms is not fullunderstood. Howvever, in recent years, a number of soluble inhibitory factors have been found in body fluids of tumor-bearing animals and humans. 8 Of special interest in relation to the studies described here is the report that tw o rat tumor lines are capable of generating and releasing a factor that can inhibit the response of neutrophils to chemotactic agents.7 Such an inhibitor, if effective in vivo, could obviously prevent the accumulation of such inflammatory cells at sites of neoplastiC disease. The present study extends these observations to another species, the mouse, and compares the chemotactic inhibitor in a strain capable of rejecting the tumor with that found in a strain Xwhich cannot reject the tumor cells. The temporal relationship of the factor to the presence of tumor cells and inflammatory cells is explored. From the Department of Connecticut.
Pathology. University
of Connecticut Health Center. Farmington.
Supported bh- Grants XI-09651 and CX-192S6 from the National Institutes of Health. Accepted for publication November 13. 1975. Address reprint requests to Peter A. Ward. NID. Department of Pathology. U niv ersitx of Connecticut Health Center. Farmington. CT 060;32 603 0002-9440/79/0308-0603$01.00 (C American Association of Pathologists
604
COHEN ET AL
American Journal of Pathology
Materials and Methods Chemotactic Assays
Chemotactic assays were performed in vitro in modified Boyden chambers containing 0.65-,i micropore filters (Millipore Corp.). Assays were carried out using rabbit neutrophils obtained by intraperitoneal injection of glycogen as described previously.9 Leukocytes were suspended in Hanks' balanced salt solution (HBSS) containing 0.1% bovine serum albumin. Chemotaxis values represent the average number of migrating leukocytes in five high-power (400X) fields per membrane. Analysis of chemotactic factor inactivator (CFI) activity was accomplished by preincubation of test samples with individual chemotactic factors for 30 minutes at 37 C, followed by assay of residual chemotactic activity. CFI activity is expressed as percent inhibition of a given chemotactic factor. Chemotactic Factors
The bacterial chemotactic factor in culture medium after overnight growth of Escherichia coli was used."0 This factor is heat-stable and resists boiling. The chemotactic factors derived from the third (C3) and fifth (C5) components of complement were also used in some experiments. These were obtained by activation of normal human serum with epsilon-amino-n-caproic acid (EACA) in the presence or absence of zymosan." Animals and Tumor Cells DBA/2 mice, 6 to 8 weeks old, were obtained from West Seneca Laboratories (Buffalo). C57BL/6 mice, 6 to 8 weeks old, were obtained from The Jackson Laboratory (Bar Harbor,
Me). The P815 mastocytoma has been maintained in our laboratory in ascites form in DBA/2 mice by serial intraperitoneal passage of 0.1 ml ascites fluid at 6- to 8-day intervals. Under these conditions, the tumor cells grow progressively as nonhemorrhagic tumors and kill all inoculated mice in 15 to 18 days.
Preparation of Murine Tumor Cell-Derived CFI The peritoneal cavity of each mouse injected with P815 mastocytoma was washed with 1.0 ml HBSS, and the amount of fluid recovered was measured and then subjected to centrifugation at 4 C at 1000 rpm (200g) for 10 minutes. The supernatants were removed and stored at -70 C. Before use, they were cleared by centrifugation at 4 C at 3000 rpm (1800g) for 30 minutes. The cell pellet was suspended in normal mouse serum (NMS); smears were made and stained with Wright's stain. Differential counts were made using an oil immersion lens (IOOX). For larger amounts of chemotactic inactivator, 4- or 5-day-old peritoneal exudates of mastocytoma cells were prepared as described above. Protease Inhibitors
The effect of enzyme inhibitors on tumor-associated chemotactic inhibitor was studied by incubating 100 ,ul of inhibitor preparations with 100 ,l of peritoneal washings for 20 minutes at room temperature and then assaying for inactivation of chemotaxis. The following inhibitors were prepared in HBSS: soybean trypsin inhibitor (SBTI) (1 mg/ml); epsilon-amino-n-caproic acid (EACA) (70 mg/ml); lima bean trypsin inhibitor (LBTI) (1 mg/ml). Two milligrams of elastase inhibitor (Me-O-Suc-Ala-Pro-Val CHCI2) was added to 0.1 ml dimethylsulfoxide (DMSO) and dissolved by incubating at 37 C for 30 minutes, followed by the addition of 0.9 ml HBSS. This inhibitor was used in a final concentration of 0.1 mM. All data presented in the following sections were reproduced in at least three successive experiments. What is printed is characteristic of the overall results.
605
PRODUCTION OF CHEMOTACTIC INHIBITORS
Vol. 94, No. 3 March 1979
Table 1-Inverse Relationship Between Presence of CFI and Neutrophil Accumulation
CFI activity
peritoneal
No. of cells injection
recovered (X 104)
1 2 3 4 7
8.2 37.0 68.8 162.5 -
Days following
No. of tumor cells in exudate (X 10')
Neutrophils in exudate
in washings* (% inactivation
Total (X 106)
% of total cells
of bacterial chemotactic factor)
4
49 27 16 8 0
55 15 80 80 80
3 16 66 163
10 11 13
-
-
* In 50-gl peritoneal washings and assayed using 50 Al bacterial factor. Each number represents pooled washings from 2 mice. In these, as in all other experiments, reaction mixtures were assayed for percent inactivation of chemotactic activity using modified Boyden chambers, as described in the text.
Results Time Course of Appearance of Chemotactic lnhbtory Acivity
We first determined that ascites fluid from DBA/2 mice implanted 7 days previously with 1 X 106 mastocytoma cells demonstrated inhibitory activity for the bacterial chemotactic factor. Twenty-five microliters of cell-free ascites fluid and 50 yl of the bacterial chemotactic factor were incubated at 37 C for 30 minutes and then assaved for residual chemotactic activitv using rabbit neutrophils as the target cells. Under these conditions, approximately 50% inhibition of chemotaxis was observed. We next studied the time course of this reaction in relation to the presence of tumor cells and inflammatorv cells in the peritoneal cavity. Nlice were injected intraperitoneally with 1 X 106 tumor cells. At 1, 2, 3, 4, and 7 days, 2 mice were killed and the peritoneal cavity of each was washed with 1.0 ml HBSS; the washings from each pair were combined. The cells were separated from the suspension by centrifugation and resuspended in normal mouse serum (NMS); differential counts were done of smears stained with Wright's stain. The peritoneal washings from each pair were assayed for inhibitor activitv. Chemotactic inhibitor activity in peritoneal washings, as demonstrated by inhibition of chemotactic activity, was found to be lowest at 2 days following tumor injection. As the tumor cells proliferated, the inhibitor reached maximal activity at 3 days and was maintained at this level through Day 7 as cell multiplication continued (Table 1). Peritoneal washings from mice injected with sterile glycogen had no detectable inhibitory activity. Within the first 24 hours following intraperitoneal injection of the
606
American Journal of Pathology
COHEN ET AL
Table 2-CFI Activity in Tumor Cell Homogenates
Inactivation of bacterial factor
(100 JI)*
Inactivation of C5 fragment (25 gl)*
Quantity of preparation (IAl)
Ascites fluid
Cell homogenatet
Ascites fluid
Cell homogenatet
25 50 100
ND 31.4 74.8
38.7 66.1 74.2
ND ND 60.0
65.8 82.2 79.5
ND = not done. Numbers represent percent inactivation of chemotactic activity. t Mastocytoma cells, 2 x 107/ml, were sonicated on ice three times (15-second intervals) using a Bronwell Blosonic Sonicator. *
tumor cells, there was an extensive neutrophil response. A small number of mononuclear cells was also observed, but the magnitude of this response remained unchanged. The percentage of neutrophils in the peritoneal washings decreased as the number of mastocytoma cells increased; therefore, by 7 days they formed an insignificant part of the total number of cells as assessed by analysis of the cell smears. Thus, with the exception of Day 1, it can be seen that where the CFI level is low, the neutrophil count is high. As the level of the inactivator and the number of tumor cells increase, the percentage of neutrophils decreases, and the number of neutrophils remains essentially unaltered. These results are summarized in Table 1. As will be shown below, the inhibitor activity cannot be related to leukocytic proteases with known CFI activity. Inhibitor Activity in Tumor Cell Extracts
To determine whether the chemotactic inhibitory activity could be found in extracts of viable cells, homogenates of tumor cells were prepared by sonication of cell pellets of 7-day tumor growths. Cells were disrupted using a "Biosonic" probe disruptor. These homogenates were able to inactivate both the bacterial chemotactic factor and the C5derived chemotactic factor, indicating that the inactivator was contained within the tumor cells (Table 2). These data are similar to those recently reported with another tumor of a completely different cytologic type and of different origin.7 Gel Filtration
To determine the molecular weight of the inactivator in the ascitic fluid, four to five peritoneal washings from 8 mice injected with mastocytoma cells were pooled and concentrated three-fold on a PM10 membrane; the retentate was applied to a Sephadex G150 column (2.5 X 50 cm) previously calibrated with bovine serum albumin (BSA) (molecular
Vol. 94, No. 3 March 1979
- .-. .-- . --.-
....
..r%.Iqr"%rl%^
PRODUCTION OF CHEMOTACTIC INHIBIIURS --
bU7
chrcff*C
BSA bocteriol
>
~50 TEXT-FIGURE I-Elution of chemotactic factor inSephadex G 150 after application of ascitic fluid. The eluting solution was phosphate-buffered saline. As the tw-o frames indicate. there was a heterogeneity of inactivator actisity of tu-o chemotactic factors. Positions of the reference standards (BSA and cvtochrome C) are shown bh- the arrous.
25-
activator activity from
o , E 0 I C5 u fvgwt O50z
0
25-
< 0-
Z
E
20
FRACTION
30
I, 40
NUMBER
weight, 67,000) and cvtochrome C (molecular weight, 12,300). Chemotactic inhibitorv activity was determined using bacterial factor and the C( fragment as chemotactic agents. The inactivator appeared in a broad band around the BSA marker, indicating heterogeneity of molecular weight (Text-figure 1). Ultracentrifual Studies
Peritoneal washings of mice injected 4 days earlier with mastocytoma cells were fractionated by sucrose density gradient ultracentrifugation, and each fraction was tested at neutral pH for inhibitory activity against the bacterial chemotactic factor and the C( fragment As shown in Textfigure 2, the inactivator activity sedimented in two zones, ie, near the IgG and albumin (BSA) markers. This pattern suggests that the inactivator is heterogeneous, similar to the CFI found in human serum,12 and confirms molecular weight estimations based on gel filtration chromatography. Heat Lability of Chemotactic Inhibto Activit
In determining if the inhibitor acted directly on the chemotactic factors, 100 ul of the peritoneal washings of mice injected 4 days previously wvith 1 X 106 mastocvtoma cells was found to inhibit chemotactic activity if first incubated with 30 Ml of the CM fragment or 30 Ml of bacterial factor for 30 minutes at 37 C prior to heat inactivation (56 C for 30 minutes) (Table 3). However, prior exposure of the inactivator to 56 C for 30 minutes preceding incubation with the chemotactic factor resulted in loss ot the ability to inhibit chemotaxis (Table 3). These data suggest that the
American Journal of Pathology
COHEN ET AL
608
IgG >
cytochrome c
BSA
bocterial factor 50-
inTEXT-FICURE 2-Distribution of chemotactic factor activity (for two chemotactic factors) in fractions from sucrose density gradient ultracentrifugation.
|
|
25-
U 25-
activator
iS O
>I:
Approximately 0.2 ml ascitic fluid was applied to the top of the gradient. Conditions for ultracentrifugation
o-
C5
U O
o-
fragment
included a 7.5 to 35% sucrose gradient, 0.05 M phosphate buffer, pH 7.4, and centrifugal force of 100,OOOg for 16 hours.
\
Z
0 25-
The ascites fluid or peritoneal washings of DBA/2 mice bearing the P815 mastocytoma have been found to contain a chemotactic factor inactivator (CFI) which inactivates the bacterial chemotactic factor as well as the chemotactic activitv associated with the C3 and C5 fragments when assayed on rabbit neutrophils. The amount of CFI is proportional to the number of tumor cells in the peritoneal exudate. The inactivator is also found in tumor cell hemogenates as well as in culture fluid from tumor cells growing in vitro. The activity is heat-labile but is not affected by protease inhibitors. Its molecular weight is greater than 50,000 daltons, based on Sephadex chromatography and sucrose density gradient ultracentrifugation studies. In C57BL/6 mice, which reject the mastocytoma, CFI levels decrease in proportion to the decreasing numbers of tumor cells. (Am j Pathol 94:603-614, 1979)
IN CLINICAL SETTINGS and in experimental models, there is abundant evidence that inflammatory responses as well as immune reactions may be defective in the presence of neoplastic disease (reviewved in Reference 1). Since mechanisms for tumor killing are known that involve neutrophils 2 and macrophages 34 as wvell as lymphocytes,5 defective inflammatory- responses could favor the tumor at the expense of the host. The mechanism of such subversion of defense mechanisms is not fullunderstood. Howvever, in recent years, a number of soluble inhibitory factors have been found in body fluids of tumor-bearing animals and humans. 8 Of special interest in relation to the studies described here is the report that tw o rat tumor lines are capable of generating and releasing a factor that can inhibit the response of neutrophils to chemotactic agents.7 Such an inhibitor, if effective in vivo, could obviously prevent the accumulation of such inflammatory cells at sites of neoplastiC disease. The present study extends these observations to another species, the mouse, and compares the chemotactic inhibitor in a strain capable of rejecting the tumor with that found in a strain Xwhich cannot reject the tumor cells. The temporal relationship of the factor to the presence of tumor cells and inflammatory cells is explored. From the Department of Connecticut.
Pathology. University
of Connecticut Health Center. Farmington.
Supported bh- Grants XI-09651 and CX-192S6 from the National Institutes of Health. Accepted for publication November 13. 1975. Address reprint requests to Peter A. Ward. NID. Department of Pathology. U niv ersitx of Connecticut Health Center. Farmington. CT 060;32 603 0002-9440/79/0308-0603$01.00 (C American Association of Pathologists
604
COHEN ET AL
American Journal of Pathology
Materials and Methods Chemotactic Assays
Chemotactic assays were performed in vitro in modified Boyden chambers containing 0.65-,i micropore filters (Millipore Corp.). Assays were carried out using rabbit neutrophils obtained by intraperitoneal injection of glycogen as described previously.9 Leukocytes were suspended in Hanks' balanced salt solution (HBSS) containing 0.1% bovine serum albumin. Chemotaxis values represent the average number of migrating leukocytes in five high-power (400X) fields per membrane. Analysis of chemotactic factor inactivator (CFI) activity was accomplished by preincubation of test samples with individual chemotactic factors for 30 minutes at 37 C, followed by assay of residual chemotactic activity. CFI activity is expressed as percent inhibition of a given chemotactic factor. Chemotactic Factors
The bacterial chemotactic factor in culture medium after overnight growth of Escherichia coli was used."0 This factor is heat-stable and resists boiling. The chemotactic factors derived from the third (C3) and fifth (C5) components of complement were also used in some experiments. These were obtained by activation of normal human serum with epsilon-amino-n-caproic acid (EACA) in the presence or absence of zymosan." Animals and Tumor Cells DBA/2 mice, 6 to 8 weeks old, were obtained from West Seneca Laboratories (Buffalo). C57BL/6 mice, 6 to 8 weeks old, were obtained from The Jackson Laboratory (Bar Harbor,
Me). The P815 mastocytoma has been maintained in our laboratory in ascites form in DBA/2 mice by serial intraperitoneal passage of 0.1 ml ascites fluid at 6- to 8-day intervals. Under these conditions, the tumor cells grow progressively as nonhemorrhagic tumors and kill all inoculated mice in 15 to 18 days.
Preparation of Murine Tumor Cell-Derived CFI The peritoneal cavity of each mouse injected with P815 mastocytoma was washed with 1.0 ml HBSS, and the amount of fluid recovered was measured and then subjected to centrifugation at 4 C at 1000 rpm (200g) for 10 minutes. The supernatants were removed and stored at -70 C. Before use, they were cleared by centrifugation at 4 C at 3000 rpm (1800g) for 30 minutes. The cell pellet was suspended in normal mouse serum (NMS); smears were made and stained with Wright's stain. Differential counts were made using an oil immersion lens (IOOX). For larger amounts of chemotactic inactivator, 4- or 5-day-old peritoneal exudates of mastocytoma cells were prepared as described above. Protease Inhibitors
The effect of enzyme inhibitors on tumor-associated chemotactic inhibitor was studied by incubating 100 ,ul of inhibitor preparations with 100 ,l of peritoneal washings for 20 minutes at room temperature and then assaying for inactivation of chemotaxis. The following inhibitors were prepared in HBSS: soybean trypsin inhibitor (SBTI) (1 mg/ml); epsilon-amino-n-caproic acid (EACA) (70 mg/ml); lima bean trypsin inhibitor (LBTI) (1 mg/ml). Two milligrams of elastase inhibitor (Me-O-Suc-Ala-Pro-Val CHCI2) was added to 0.1 ml dimethylsulfoxide (DMSO) and dissolved by incubating at 37 C for 30 minutes, followed by the addition of 0.9 ml HBSS. This inhibitor was used in a final concentration of 0.1 mM. All data presented in the following sections were reproduced in at least three successive experiments. What is printed is characteristic of the overall results.
605
PRODUCTION OF CHEMOTACTIC INHIBITORS
Vol. 94, No. 3 March 1979
Table 1-Inverse Relationship Between Presence of CFI and Neutrophil Accumulation
CFI activity
peritoneal
No. of cells injection
recovered (X 104)
1 2 3 4 7
8.2 37.0 68.8 162.5 -
Days following
No. of tumor cells in exudate (X 10')
Neutrophils in exudate
in washings* (% inactivation
Total (X 106)
% of total cells
of bacterial chemotactic factor)
4
49 27 16 8 0
55 15 80 80 80
3 16 66 163
10 11 13
-
-
* In 50-gl peritoneal washings and assayed using 50 Al bacterial factor. Each number represents pooled washings from 2 mice. In these, as in all other experiments, reaction mixtures were assayed for percent inactivation of chemotactic activity using modified Boyden chambers, as described in the text.
Results Time Course of Appearance of Chemotactic lnhbtory Acivity
We first determined that ascites fluid from DBA/2 mice implanted 7 days previously with 1 X 106 mastocytoma cells demonstrated inhibitory activity for the bacterial chemotactic factor. Twenty-five microliters of cell-free ascites fluid and 50 yl of the bacterial chemotactic factor were incubated at 37 C for 30 minutes and then assaved for residual chemotactic activitv using rabbit neutrophils as the target cells. Under these conditions, approximately 50% inhibition of chemotaxis was observed. We next studied the time course of this reaction in relation to the presence of tumor cells and inflammatorv cells in the peritoneal cavity. Nlice were injected intraperitoneally with 1 X 106 tumor cells. At 1, 2, 3, 4, and 7 days, 2 mice were killed and the peritoneal cavity of each was washed with 1.0 ml HBSS; the washings from each pair were combined. The cells were separated from the suspension by centrifugation and resuspended in normal mouse serum (NMS); differential counts were done of smears stained with Wright's stain. The peritoneal washings from each pair were assayed for inhibitor activitv. Chemotactic inhibitor activity in peritoneal washings, as demonstrated by inhibition of chemotactic activity, was found to be lowest at 2 days following tumor injection. As the tumor cells proliferated, the inhibitor reached maximal activity at 3 days and was maintained at this level through Day 7 as cell multiplication continued (Table 1). Peritoneal washings from mice injected with sterile glycogen had no detectable inhibitory activity. Within the first 24 hours following intraperitoneal injection of the
606
American Journal of Pathology
COHEN ET AL
Table 2-CFI Activity in Tumor Cell Homogenates
Inactivation of bacterial factor
(100 JI)*
Inactivation of C5 fragment (25 gl)*
Quantity of preparation (IAl)
Ascites fluid
Cell homogenatet
Ascites fluid
Cell homogenatet
25 50 100
ND 31.4 74.8
38.7 66.1 74.2
ND ND 60.0
65.8 82.2 79.5
ND = not done. Numbers represent percent inactivation of chemotactic activity. t Mastocytoma cells, 2 x 107/ml, were sonicated on ice three times (15-second intervals) using a Bronwell Blosonic Sonicator. *
tumor cells, there was an extensive neutrophil response. A small number of mononuclear cells was also observed, but the magnitude of this response remained unchanged. The percentage of neutrophils in the peritoneal washings decreased as the number of mastocytoma cells increased; therefore, by 7 days they formed an insignificant part of the total number of cells as assessed by analysis of the cell smears. Thus, with the exception of Day 1, it can be seen that where the CFI level is low, the neutrophil count is high. As the level of the inactivator and the number of tumor cells increase, the percentage of neutrophils decreases, and the number of neutrophils remains essentially unaltered. These results are summarized in Table 1. As will be shown below, the inhibitor activity cannot be related to leukocytic proteases with known CFI activity. Inhibitor Activity in Tumor Cell Extracts
To determine whether the chemotactic inhibitory activity could be found in extracts of viable cells, homogenates of tumor cells were prepared by sonication of cell pellets of 7-day tumor growths. Cells were disrupted using a "Biosonic" probe disruptor. These homogenates were able to inactivate both the bacterial chemotactic factor and the C5derived chemotactic factor, indicating that the inactivator was contained within the tumor cells (Table 2). These data are similar to those recently reported with another tumor of a completely different cytologic type and of different origin.7 Gel Filtration
To determine the molecular weight of the inactivator in the ascitic fluid, four to five peritoneal washings from 8 mice injected with mastocytoma cells were pooled and concentrated three-fold on a PM10 membrane; the retentate was applied to a Sephadex G150 column (2.5 X 50 cm) previously calibrated with bovine serum albumin (BSA) (molecular
Vol. 94, No. 3 March 1979
- .-. .-- . --.-
....
..r%.Iqr"%rl%^
PRODUCTION OF CHEMOTACTIC INHIBIIURS --
bU7
chrcff*C
BSA bocteriol
>
~50 TEXT-FIGURE I-Elution of chemotactic factor inSephadex G 150 after application of ascitic fluid. The eluting solution was phosphate-buffered saline. As the tw-o frames indicate. there was a heterogeneity of inactivator actisity of tu-o chemotactic factors. Positions of the reference standards (BSA and cvtochrome C) are shown bh- the arrous.
25-
activator activity from
o , E 0 I C5 u fvgwt O50z
0
25-
< 0-
Z
E
20
FRACTION
30
I, 40
NUMBER
weight, 67,000) and cvtochrome C (molecular weight, 12,300). Chemotactic inhibitorv activity was determined using bacterial factor and the C( fragment as chemotactic agents. The inactivator appeared in a broad band around the BSA marker, indicating heterogeneity of molecular weight (Text-figure 1). Ultracentrifual Studies
Peritoneal washings of mice injected 4 days earlier with mastocytoma cells were fractionated by sucrose density gradient ultracentrifugation, and each fraction was tested at neutral pH for inhibitory activity against the bacterial chemotactic factor and the C( fragment As shown in Textfigure 2, the inactivator activity sedimented in two zones, ie, near the IgG and albumin (BSA) markers. This pattern suggests that the inactivator is heterogeneous, similar to the CFI found in human serum,12 and confirms molecular weight estimations based on gel filtration chromatography. Heat Lability of Chemotactic Inhibto Activit
In determining if the inhibitor acted directly on the chemotactic factors, 100 ul of the peritoneal washings of mice injected 4 days previously wvith 1 X 106 mastocvtoma cells was found to inhibit chemotactic activity if first incubated with 30 Ml of the CM fragment or 30 Ml of bacterial factor for 30 minutes at 37 C prior to heat inactivation (56 C for 30 minutes) (Table 3). However, prior exposure of the inactivator to 56 C for 30 minutes preceding incubation with the chemotactic factor resulted in loss ot the ability to inhibit chemotaxis (Table 3). These data suggest that the
American Journal of Pathology
COHEN ET AL
608
IgG >
cytochrome c
BSA
bocterial factor 50-
inTEXT-FICURE 2-Distribution of chemotactic factor activity (for two chemotactic factors) in fractions from sucrose density gradient ultracentrifugation.
|
|
25-
U 25-
activator
iS O
>I:
Approximately 0.2 ml ascitic fluid was applied to the top of the gradient. Conditions for ultracentrifugation
o-
C5
U O
o-
fragment
included a 7.5 to 35% sucrose gradient, 0.05 M phosphate buffer, pH 7.4, and centrifugal force of 100,OOOg for 16 hours.
\
Z
0 25-
