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A macrophage surface component related to fibronectin is involved in the response to migration inhibitory factor
CELLULAR
58, 175-187
IMMUNOLOGY
(1981)
A Macrophage
Surface
Involved
in the Response
HEINZ Departments Division
Component
G. REMOLD,
JUDITH
Related
to Migration E. SHAW,
to Fibronectin Inhibitory
May
21. 1980;
accepted
Factor
AND JOHN R. DAVID
of Medicine. Harvard Medical School and Robert B. Brigham of the Affiliated Hospitals Center, Inc.. Boston. Massachusetts Received
Is
June
Hospital, 021 I5
25, 1980
Guinea pig peritoneal exudate cells release a substance upon incubation with hypotonic buffer or 6 M urea which (1) augments the response of normal macrophages to migration inhibitory factor, and (2) restores the response of trypsinized macrophages to migration inhibitory factor. Adsorption of peritoneal exudate cell extract with antibodies to guinea pig plasma fibronectin results in a loss of both the MIF-response-enhancing and restoring activity. These results indicate that both activities are associated with a macrophage surface component serologically related to plasma fibronectin. Furthermore, preincubation of macrophages with plasma fibronectin abrogates the enhancement of the MIF response caused by peritoneal exudate cell extracts. This suggests that the active component and plasma tibronectin compete for a common binding site. It is likely that this substance plays a regulatory role in the macrophage response to migration inhibitory factor.
INTRODUCTION Lymphocyte mediators are soluble effector substances of cellular immunity with a variety of biological activities. They include the closely related factors migration inhibitory factor (MIF)’ and macrophage activating factor (MAF), which both affect the macrophage, resulting in inhibition of macrophage migration and an increased capacity of the cells to kill microorganisms and tumor cells (l-4). In previous studies it has been shown that the response of macrophages to MIF and MAF can be enhanced by pretreatment of the cells with esterase inhibitors or chemicals affecting the cell surface (5, 6) and by macrophage glycolipids (7). In the present study we demonstrate that incubation of guinea pig macrophages with a component obtained by hypotonic treatment from the macrophage surface enhances their response to MIF and restores the ability of trypsinized macrophages to respond to MIF. This component, which is referred to as MIF-response enhancing factor (EF), is obtained from guinea pig peritoneal macrophages by treatment with hypotonic buffer. It is not an esterase inhibitor, and it is absorbed out by antiguinea pig plasma fibronectin-IgG. Fibronectin, the transformation-sensitive gly’ Abbreviations used: IgG, Immunoglobulin G; MIF, migration inhibitory factor; MAF, macrophage activating factor; EF, MIF-response enhancing factor; PEC, peritoneal exudate cells; HBSS, Hanks’ balanced salt solution; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; PMN, polymorphonuclear leukocytes; PBS, phosphate-buffered saline; DFP, diisopropylphosphorofluoridate, MEM-PS, Eagles minimum essential medium containing 100 units/ml penicillin and 100 rg/ml streptomycin; PF, plasma fibronectin. 175
0008-8749/81/030175-13$02.00/O Copyright 0 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.
176
REMOLD,
SHAW, AND DAVID
coprotein of connective tissue cells, has been shown to occur in two different molecular forms (8). Cell surface fibronectin, found on the cell surface of connective tissue cells, has an important function in cell aggregation (9), adhesion (lo), and motility (1 I). This substance is antigenically identical with plasma fibronectin (cold insoluble globulin) present in plasma or serum, but differs from it in several biochemical and functional characteristics ( 12). MATERIALS
AND
METHODS
MEM and HBSS were obtained from Microbiological Associates, Bethesda, Maryland, and sodium caseinate from Eastman Kodak, Rochester, New York. DFP and gelatin from swine skin were bought from Sigma Chemical Company, St. Louis, Missouri. Protein A-Sepharose and fluoresceinated protein A were purchased from Pharmacia Fine Chemicals, Piscataway, New Jersey; bovine fibrinogen and TPCK-trypsin from Worthington Enzymes, Freehold, New Jersey. Na1251 (17 Ci/mg) was obtained from New England Nuclear Corporation, Boston, Massachusetts. Production of MIF. MIF-rich and control lymphocyte supernatants were obtained as previously described (13). Briefly, 2.4 X 10’ guinea pig lymph node lymphocytes/ml were incubated for 24 hr at 37°C with concanavalin A (10 pg/ml) to stimulate MIF production. Concanavalin A was added to a parallel control supernatant after the 24-hr incubation. The cells were removed by centrifugation and the supernatants were lyophilized and filtered over Sephadex G-100 columns. The MIF-containing fractions were pooled as were parallel fractions from the control Sephadex G- 100 column. Fractions were concentrated to l/ 100 the volume of the original supernatant and stored at -70°C. The amounts of MIF mentioned in the Results section refer to this fraction. Assay for MZF. Fractions to be tested were diluted to 2 ml with MEM-PS containing 15% normal guinea pig serum and assayed for MIF activity on normal guinea pig PEC using a capillary tube assay (13). MIF activity (%I) was expressed as the percentage of the inhibition of migration of cells incubated with MIF compared to the migration of the same cells in the control fraction. percentage I = 100 -
average migration of MIF-containing fractions x 100. average migration in control fractions
At least 20% inhibition must be obtained for significant activity (13). To test for MIF-enhancing activity, limiting concentrations of MIF were chosen so that MIF itself produced little or no inhibition of migration. Production of PEC extracts containing EF. PECs were induced in guinea pigs by peritoneal injection of mineral oil (Marco1 52, Exxon Corporation, Everett, Mass.) or 1% sodium caseinate in saline; PECs were harvested 3-4 days later as previously described (14) and washed by pelleting in HBSS. The cells (6 X lo’/ ml) were incubated at 37°C for 60 min during which time they were maintained in suspension. 7.5% NaHCO, solution was added dropwise in order to maintain the pH at 7.4. The supernatants from these cells were tested for EF activity. In order to obtain larger amounts of EF activity, a volume of 10-30 ml PEC (6 X 10’ cells/ml) was incubated in HBSS containing 10m3M DFP for 30 min at 37°C at pH 7.4, and washed. The cells were then incubated in a lo-fold volume
A FIBRONECTIN-LIKE
CELL
SURFACE
COMPONENT
177
of hypotonic buffer (5 mM Tris-HCl, 75 mM sucrose, and 1 mM EDTA, pH 8.2), for 15 min at 4°C which causes the cells to swell but not to lyse (15). Cells were then removed by low-speed centrifugation (250g for 5 min), and 0.25 vol of 20 mM Tris-HCl, 1.5 mM EDTA, and 0.15 M NaCl, pH 7.4, was added to the supernatant which was then centrifuged at 900g for 7 min to precipitate any remaining cells or cellular debris. Cell breakage was monitored by measuring the lactate dehydrogenase activity ( 16) in the extracts and comparing it to the activity of the enzyme in the cells after homogenization. This PEC extract contains 600 +- 200 yg protein/ml (13 determinations). The resulting extracts were stored at -70°C. In some experiments, PEC were incubated with 6 M urea in PBS for 15 min in HBSS at 4°C and processed in the same fashion. Production of EF from lymph node ceils and PMNs. Lymph node cells were obtained from guinea pigs immunized with CFA (13). The lymph nodes were harvested, and the cells were obtained by teasing. More than 95% of the cells were lymphoid cells. PMNs were elicited by intraperitoneal injection of 1% casein. The cells harvested after 12 hr were 99% PMNs. To obtain EF the lymph node cells (7 x lO’/ml) and PMNs (8 X lO’/ml) were subjected to the same hypotonic treatment as described for the PECs. Viability of these cells was greater than 90% after hypotonic treatment. Purification of guinea pig plasma proteins. Plasma fibronectin was prepared from frozen guinea pig plasma as described (17) for human plasma fibronectin with a modification. An additional final step, gelatin-Sepharose affinity chromatography, was employed (18). Plasma fibronectin prepared by this method contained no fibrinogen as judged by immunodiffusion and by its ability to clot, and it showed one precipitation line when tested against rabbit anti-guinea pig serum. In the presence of 2-mercaptoethanol it showed one band on SDS-PAGE with an apparent MW of 220,000. Guinea pig fibrinogen and AT III were prepared as previously described (19, 20). Fibrinogen showed one precipitation line in immunoelectrophoresis using antiguinea pig plasma serum as an antigen. AT III showed one band with an apparent MW of 68,000 on SDS-PAGE under reducing conditions. Production of antisera. Antibodies against guinea plasma fibronectin, guinea pig fibrinogen, and guinea pig AT III were obtained by injecting subcutaneously 175200 pg of the purified proteins in complete Freund’s adjuvant into New Zealand white rabbits. After 14 days the animals were boosted with a second dose of antigen in incomplete Freund’s adjuvant. This procedure was repeated three to four times. The resulting antisera showed a precipitation line in the Ouchterlony gel diffusion assay at a dilution of 1:8 when a concentration of 1.6 mg/ml antigen was used. Preparation of IgG-Sepharose 4B. Aliquots of 5-ml CNBr-activated Sepharose 4B (2 1) were each coupled with 8 mg of normal rabbit IgG or rabbit anti-guinea pig PF IgG purified on protein A-Sepharose columns. The IgG-Sepharose was packed into 5-ml syringes and washed with 0.05 M Tris-HCl, pH 7.2. EF (8.2 ml) was filtered over each IgG-Sepharose column, washed with the same buffer, and eluted with 4 M guanidine HCl, pH 7.3. Aliquots from the void volume and from the guanidine HCl wash were tested for EF activity. Reaction of PEC extracts with antibodies against several guinea pig proteins. Anti-guinea PF IgG, anti-AT III IgG, and anti-fibrinogen IgG were obtained by
178
REMOLD,
SHAW,
AND
DAVID
affinity chromatography of 2.0 ml crude antiserum on a protein A-Sepharose column (1 X 5 cm) (22). IgG was eluted from the column with 0.1 M glycine HCl buffer, pH 3.0, and immediately adjusted to pH 7.0 with NaOH. The eluted IgG was concentrated to 2 ml. PEC extract (2.5 ml) was incubated with 0.5 ml IgG for 60 min at 4°C. As an additional control, IgG was incubated in buffer alone. Ten percent heat-killed suspension (0.5 ml) of Stuphylococcus aureus Cowan I (a gift from Dr. E. Remold-O’Donnell) was then added to the solution, and they were mixed at 4°C for 30 min. The suspensions were then centrifuged at 9OOg for 10 min. No IgG could be detected in the supernatants after treatment with Staphylococcus aureus by gel diffusion. The supernatants were dialyzed against MEMPS and assayed for MIF-enhancing activity. Surface fluorescence staining of peritoneal macrophages with anti-PF serum. Normal rabbit serum ( 100 ~1) or rabbit anti-guinea pig PF serum were incubated with 100 ~1 of a fluorescein-labeled protein A solution (1.1 mg/ml) at 4°C for 30 min, as previously described (23). One hundred microliters from serial dilutions (1:l to 1:64) of the incubation mixture was incubated with 5 X lo6 washed guinea pig peritoneal exudate cells at 4°C for 30 min. The cells were then washed with PBS four times and examined for surface fluorescent staining. Glycolipid extraction. PEC supernatants obtained by hypotonic treatment were extracted using a modification of the method of Esselman et al. (24). In brief, 4 ml of chloroform/methanol (2 v/v) was added to a lyophilized preparation of the PEC supernatants obtained from 6 X 10’ cells. The suspension was sonicated at 4°C for 1 min using a Model 250 ultrasonic cleaning bath (E/MC, Division of Rai Research, Fisher Scientific, Medford, Mass.) and centrifuged. The pellet was reextracted with chloroform:methanol. The combined supernatants were filtered on a medium-porosity sintered glass filter and evaporated to dryness. The glycolipids were dissolved in sterile MEM-PS by sonication for 15-30 set and incorporated into liposomes using the method of Kinsky (25). The liposomes were assayed for enhancement of MIF response (7). Flotation of PEC extracts in salt solutions with increasing densities using a procedure developed for the separation of lipoprotein classes. PEC extracts were centrifuged in NaCl-NaBr solutions of increasing density (26). Sequential fractionations were carried out in solutions with densities of 1.0063 (0.196 A4 NaCl), 1.067 (0.0786 M NaBr), and 1.210 (2.962 M NaBr) g/ml in a SW 50.1 rotor (Beckman Instruments, Palo Alto, Calif.) at 45,000 rpm for 23 hr at 9°C. The resulting pellets and supernatants were dialyzed against MEM-PS and assayed for enhancement of the MIF response. Determination of protein. Protein was measured by the method of Lowry et al. (27). Measurement of pinocytosis. Pinocytosis of PEC was measured according to the method of Steinman and Cohn (28) as applied to guinea pig macrophages (29). RESULTS Enhancement
of the MIF Response of Macrophages
by PEC Extracts
Supernatants obtained after incubation of peritoneal exudate cells in MEM-PS for 1 hr at 37°C were found to markedly enhance the response of fresh PEC to a limiting amount of the lymphocyte mediator MIF. The results of these experi-
A FIBRONECTIN-LIKE
CELL
SURFACE
COMPONENT
179
EXPEUIMEN T NUMBER
FIG. 1. Effect of PEC supernatants on a low dose of MIF. MIF activity is shown on the ordinate; the abscissa shows six experiments. The stippled bars represent the response to a low dose of MIF on normal macrophages. The cross-hatched bars represent the response of macrophages in the presence of 0.5 ml PEC supernatants diluted with 0.5 ml MEM-PS. Note that the presence of PEC supernatants increases the response of macrophages to MIF.
ments are shown in Fig. 1, where it can be seen that optimal increase in the MIF response was found when 0.5 ml of PEC supernatant/ml of assay medium was used. An amount of 1.0 ml was also effective (results not shown), but amounts smaller than 0.5 ml did not result in enhancement of MIF response. More consistent enhancing activity was obtained when casein- or oil-induced PEC were extracted with cold hypotonic buffer (see Materials and Methods). Figure 2 shows six such experiments where 0.35 ml PEC extract caused a marked enhancement of migration inhibition. Similar results were obtained when PEC were extracted with 6 it4 urea in the cold. Urea extract (0.7 ml/ml) containing 350 pg protein enhanced the response of macrophages to a limiting concentration of MIF (2 and 5% I) to 43 and 289’0 I respectively. It should be noted that PEC extracts alone in several concentrations had no MIF-like activity (data not shown).
:-~- -L 0 10 zo M/f
30
-IL 40 50
ACr/vlTY
60 70 /%I)
FIG. 2. Enhancement of the MIF response of macrophages by hypotonic PEC extracts. The abscissa shows MIF activity; the ordinate six experiments. The amounts of PEC extract added per milliliter medium is indicated on the left. Note that in all experiments as little as 0.35 ml PEC extracts/ml medium induced an enhanced response of the macrophage to a low dose of MIF.
180
REMOLD,
SHAW, TABLE
Restoration
of the Response
of Trypsinized
AND
DAVID
1 Macrophages
to MIF
Percentage Experiment 1 2 3 4 5 6 7 R Migration b Amounts
inhibition of trypsinized of PEC extract included
Ob 14 17 0 11 15 3 16
by PEC
Extracts”
inhibition
0.3sb
o.7b
24 22 42
34 23 45 44 52 24 40
macrophages exposed to 150 ~1 MIF per ml medium. in the assay medium in milliliters per milliliter of medium.
Hypotonic treatment of lymph node cells and PMN also released a substance which enhanced the migration inhibition of macrophages. Inhibition in the presence of a limiting concentration of MIF (0 and 13% I) was increased by lymph node cell extracts to 45 and 67% I and by PMN extracts to 59 and 49% I. Restoration of the Response of Trypsin-Treated by PEC Extracts
Macrophages
to MIF
It was shown in earlier studies that macrophages treated with trypsin fail to respond to MIF (30, 31). Since the EF described above augments the response of normal macrophages to MIF, we asked whether it would also restore the response of trypsinized macrophages to MIF. When trypsinized PEC, which were unresponsive to MIF, were incubated with PEC extracts, the response to MIF was consistently restored (Table 1). Specific Absorption of EF Activity Guinea Pig Plasma Fibronectin
from
PEC Extracts
by Antibodies
against
Fibronectin plays an important role in a variety of cellular functions, such as aggregation and motility. Therefore, it was of interest to investigate whether EF activity is associated with a fibronectin-like macrophage surface component. To test this, we incubated PEC extracts containing EF activity with antibodies to PF. PEC extracts were incubated with rabbit anti-guinea pig PF IgG, and, as a control, with normal rabbit IgG and rabbit anti-guinea pig AT III IgG. After incubation, the IgG was removed by addition of heat-killed Staphylococcus aureus Cowan I and subsequent centrifugation. In a second set of controls, anti-guinea pig PF IgG and anti-guinea pig AT III IgG were incubated with PBS alone and then precipitated with Staphylococcus aureus. In each case, the supernatants were dialyzed and tested for migration inhibition enhancing activity. The results of 10 such experiments are summarized in Fig. 3. It can be seen that enhancing activity is abrogated when PEC extracts are absorbed with anti-PF IgG, but is unaltered in all controls.
A FIBRONECTIN-LIKE EC
PRETREATMENT
-
NOW
CELL
SURFACE
COMPONENT
181
~-~-
onf~-ATil
FIG. 3. Effect of anti-PF IgG on the MIF-response enhancing activity of EF. PEC extracts (EF, 0.7 ml/ml medium) or PBS as a control (-) were incubated with medium alone (none), normal rabbit IgG (normal IgG), anti-AT-III IgG (anti-AT III), and anti-PF IgG (anti-PF) for 60 min at 4°C. The solutions were then incubated with heat-killed Staphylococcus aureus and centrifuged. The supernatants were tested for enhancement of MIF response using a limiting concentration of MIF (0.2 ml/ml medium) on normal macrophages. The columns summarize the MIF activity of 10 experiments (abscissa). The standard error is indicated. Note that treatment of PEC extracts with anti-PF IgG abolished the enhancing effect.
Fibrin is also known to be a macrophage surface component (32). It is of note that absorption of PEC extracts with anti-guinea pig fibrinogen-IgG did not remove EF activity: in three experiments the macrophage response to a low amount of MIF (13,0, and 5% I) was augmented by addition of PEC extracts pretreated with anti-fibrinogen IgG to 62, 56, and 53% I, respectively, whereas PEC extracts pretreated with anti-PF IgG lacked the MIF-response enhancing effect (16, 18, and 19% I). We conclude from these experiments that EF serologically cross-reacts with plasma fibronectin, but not with fibrinogen. In a similar set of experiments we tested the effect of anti-PF IgG on the capacity of PEC extracts to restore the response of trypsinized macrophages to MIF. The results, shown in Table 2, indicate that restoration of MIF response to trypsinized macrophages is abolished by incubation of these extracts with anti-PF IgG. In contrast, after treatment with normal rabbit IgG, the MIF response is restored. In other controls normal rabbit IgG or anti PF IgG was incubated in MEM-PS alone and was then precipitated with heat-killed Staphylococcus aureus. The supernatants did not restore the response of macrophages to MIF indicating that abolishment of restoration of the MIF response to trypsinized macrophages is not due to material present in the supernatant after precipitation with Staphylococcus aureus. These data indicate that both activities, MIF enhancing and restoring activity, are associated with the same substance. Since EF can be absorbed out by anti-PF antibodies, we questioned whether purified plasma fibronectin from guinea pig plasma is able to substitute for EF, i.e., is PF able to enhance the response of normal macrophages to MIF. When guinea pig plasma fibronectin was added to PEC at a concentration of 100 or 50 pg/ml medium, no enhancement of the MIF response was seen (experiments not shown). In three additional experiments, plasma fibronectin did not restore the response of trypsinized PEC to MIF (17, 13, and 19% I in the controls versus 2, 17, and 26% I using 100 yg/ml plasma fibronectin and 15, 22, and 10% I using 50 pg/ml plasma fibronectin). As a positive control, 0.7 ml PEC extracts/ml me-
182
REMOLD,
Effect
of Antibody
Treatment
SHAW,
AND
TABLE
2
on the Capacity of PEC Extracts Trypsinized Macrophages to MIF” Percentage
Experiment
PBS + normal rabbit-IgG
I 2 3 4
DAVID
7 4 19 10
EF + normal rabbit-IgG 41 20 32 35
to Restore
the Response
of
inhibition PBS + antiPF-IgG 9 2 4 7
EF + antiPF-IgG 18 11 21 6
a Migration inhibition of trypsinized macrophages in the presence of 150-300 WI/ml MIF and various pretreated PEC extracts. Aliquots (2.5 ml) of PEC extract (EF) were incubated with S-10 @I normal rabbit IgG or with 5-10 rg rabbit anti-guinea pig PF-IgG for 60 min at 4°C. Subsequently heat-killed S~uphylococcus nrrrelcs (0.5 ml of a 10% solution) was added and after 30 min at 4°C the suspensions were centrifuged. The supernatants were tested for their capacity to restore the response of trypsinized macrophages to MIF. In parallel control samples PBS replaced the PEC extracts. Note that the lacking MIF response of trypsinized macrophages (PBS + normal rabbit-IgG) is restored when PEC extracts are added (EF + normal rabbit-IgG). This effect is abrogated when the PEC extracts were absorbed with anti-PF IgG (EF + anti-PF IgG).
dium enhanced the migration inhibition significantly to 41, 42, and 33% I. These results show that although antiplasma fibronectin antibodies cross-react with EF, plasma fibronectin is not capable of augmenting or restoring the migration inhibition of macrophages. It is, therefore, not identical with EF. To further study the relationship between fibronectin and EF, experiments were carried out to see if plasma fibronectin competes with EF for a common binding site. Preincubation of macrophages with plasma fibronectin consistently prevented enhancement of the MIF response due to EF (Table 3). When macrophages were preincubated with 200 pg/ml myoglobin (used as a control) they still responded to EF. This experiment suggests strongly the EF and plasma fibronectin compete for a common binding site on the macrophage. Furthermore, when anti-PF IgG was absorbed out with purified plasma fibronectin, it failed to remove EF activity from PEC extracts, indicating that anti-PF IgG and not a contaminating antibody is responsible for the abolition of EF (three experiments, data not shown). In addition, anti-PF-IgG-PF complexes did not absorb out EF activity making it unlikely that EF binds nonspecifically to immune complexes. Cell Surface Location
of EF
The conditions used to extract EF from the cells were such that cell breakage was minimal. To demonstrate this, the activity of the cytoplasmic enzyme lactate dehydrogenase was measured in PEC extracts. Activity of lactate dehydrogenase in hypotonic PEC extracts was 7.0 f 0.01% (SE) of the activity in the corresponding cell homogenates (eight determinations). These values are similar to those obtained from isotonic macrophage supernatants (data not shown) and suggest that EF activity is derived from the cell surface. In order to establish this point more fully, we investigated whether material which crossreacts serologically with plasma
A FIBRONECTIN-LIKE
CELL TABLE
Abrogation
of EF Activity
by Preincubation
SURFACE 3 of Macrophages Percentage
Ob Experiment I 2 3 4 5 6
Medium 1 0 9 0 0 0
183
COMPONENT
with
Plasma
Fibronectin”
inhibition 3oob
2oob EF
Medium
EF
Medium
EF
35 36 39 39 30 34
1 0 4 0 0 18
17 21 24 7 0 15
18 0
4 9
-
a Macrophages (6 X 10’ cells/ml) were preincubated with 200 pg/ml HBSS and 300 Kg/ml plasma fibronectin for 1 hr at 37°C. They were then assayed for their response to a limiting concentration of MIF in the presence (EF) and absence (medium) of 0.7 ml PEC extract. Note that macrophages preincubated with plasma fibronectin show decreased or no EF-dependent enhancement of MIF response. b Amounts of plasma fibronectin (micrograms per milliliter) used for preincubation.
fibronectin is present on the macrophage cell surface. Oil-induced PEC, consisting of 90% macrophages, were incubated for 30 min at 4°C with rabbit anti-guinea pig-PF IgG complexed with fluorescein-conjugated protein A and then extensively washed (see Materials and Methods). Cells treated in this manner consistently showed fluorescent staining on their surface (Fig. 4.1). The staining pattern, as seen in Fig. 4.1, is patchy, and the patches have a diameter of 0.4-0.8 pm. 34 f 1% of the PEC are stained with anti-PF antiserum (three experiments). On the other hand, cells incubated with normal rabbit serum complexed with fluoresceinconjugated protein A did not show staining of the cell surface (Fig. 4.3). These results are similar to the recent findings of Colvin et al. (33) and demonstrate that fibronectin-containing material is a component of the macrophage cell surface. It should be noted that trypsinized macrophages do not show fluorescent staining by anti-PF IgG, indicating that the structures specific for anti-PF IgG are trypsin sensitive. EF Activity Glycolipids,
is Not Caused by the Action of Esterase Inhibitors, or Lipoproteins
In previous studies, we showed that pretreatment of macrophages with proteinase inhibitors enhances the response of macrophages to a limiting concentration of MIF (5). It was therefore of importance to investigate (a) whether hypotonic PEC extracts contain esterase inhibitors and (b) whether EF activity can be attributed to the action of an esterase inhibitor. A small amount of esterase inhibitors in hypotonic extracts from PEC was found; undiluted PEC extracts when added to ‘251-fibrin plates (34) inhibit the activity of low concentrations of trypsin (0.1 pg/ml), whereas higher trypsin concentrations (30-60 pg/ml) were not inhibited. We were, however, unable to remove the esterase inhibitory activity from the PEC extracts by absorption with anti-PF antibodies (H. G. Remold and J. E. Shaw, manuscript in preparation). We also found that
FIG. 4. Fibronectin-like material on the surface of guinea pig peritoneal macrophages. 1. Cells incubated with rabbit-anti-guinea pig PF serum complexed with fluorescein-conjugated protein A (see Materials and Methods). Note that the cell surface is stained. 2. A cell photographed in the plane of the cell surface. Note the patchy distribution of the fluorescent stain on the cell surface. 3. Cells incubated with normal rabbit serum complexed with fluorescent protein A. No fluorescent surface stain can be seen. X900.
A FIBRONECTIN-LIKE
CELL
SURFACE
COMPONENT
185
esterase inhibitors did not restore the response of trypsinized macrophages to MIF. For instance, trypsin-treated macrophages that did not respond to a large amount of MIF (16% I) did respond when 0.7 ml PEC extract was added per ml medium (65% I). AT III (100 pg/ml), soybean trypsin inhibitor (100 pg/ml), and DFP ( lop4 M), which are potent serine esterase inhibitors, were unable to restore this response (21, 8, and 0% I, respectively). These experiments demonstrate that the action of EF is not that of an esterase inhibitor. Higgins et al. (7) showed that macrophage glycolipids enhance the response of macrophges to MIF. To investigate whether EF activity is due to the action of a glycolipid, PEC extracts were fractionated using an extraction method for glycolipids (see Materials and Methods). No biological activity was recovered in the glycolipid fraction, demonstrating that EF is not a glycolipid (six experiments, not shown). Since lipoproteins have immunoregulatory effects (35), we also investigated whether EF is a lipoprotein. PEC extracts were fractionated using a method developed for the separation of lipoprotein classes (26). In three experiments the response to a limiting amount of MIF (10, 0, and 5% I) was not enhanced when the fractions with a density of 1.0063 (21, 0, and 0% I), 1.067 (15, 0, and 0% I) m 1 were tested. EF activity accumulated in the pellet and 1.210(12,0,andO%I)g/ of the densest fraction (1.210 g/ml), which contains components denser than lipoproteins. The densest fraction enhanced the response to a low concentration of MIF from 10, 0, and 5% I to 70, 23, and 37% I, respectively. These experiments show that EF does not have the characteristics of a lipoprotein. DISCUSSION The experiments described here demonstrate that hypotonic extracts from guinea pig peritoneal exudate cells contain a material which enhances the response of normal macrophages to MIF and which restores the MIF response of trypsinized macrophages. The data also show that antibodies against purified plasma fibronectin absorb out both activities from PEC extracts, suggesting that they are related to each other and are associated with the same substance. This substance is referred to here as MIF-response enhancing factor, or EF. In earlier studies, we found that the response of macrophages to MIF is enhanced by three groups of substances, proteinase inhibitors (5), reagents which alter cell surface components, such as diazotized sulfanilic acid (6), and fucogangliosides from the macrophage cell membrane (36). The EF described here is not an esterase inhibitor, nor a glycolipid, indicating that the action of EF on the macrophage differs from the action of esterase inhibitors or glycolipids. There are several lines of evidence to indicate that EF activity is associated with a substance related to fibronectin. First, EF activity can be absorbed out by antiPF IgG. Second, plasma fibronectin competes with EF for a common binding site on the macrophage, and third, trypsin treatment abolishes the response of macrophages to MIF. Concomitantly, the capacity of the cells to stain with fluorescent anti-PF IgG is also abolished by trypsin. On the other hand, plasma fibronectin cannot substitute for EF; therefore it is not identical with it. Plasma fibronectin does not enhance the response of normal macrophages to MIF, nor does it restore the MIF response of trypsinized mac-
186
REMOLD,
SHAW,
AND
DAVID
rophages. Furthermore, plasma fibronectin is relatively easy to purify, but we were unable to purify EF by conventional methods such as gel filtration, sucrose density gradient centrifugation, or electrophoresis, probably because of its strong tendency to aggregate. For example, affinity chromatography of EF on anti-PF-IgG-sepharose columns was inconclusive, because EF, due to its high affinity to particulate matter, absorbed to normal-IgG-sepharose columns as well as to anti-PF-IgG-sepharose columns. It could be eluted from both resins with 4 M guanidine HCl. It was reported that anti-PF IgG was also found to react with human neutrophil surfaces (37). These cells displayed immunofluorescent staining for fibronectin which, surprisingly, persisted after trypsin treatment. The reported resistance of the fibronectin-like structure to trypsin on the neutrophil strongly indicates that this material is modified, possibly by cross-linking. In addition, a cold insoluble protein with an apparent MW of 125,000 was found on mouse lymphocytes which plays a role in the regulation of the immune response (38). The findings presented in this study extend the importance of fibronectin-like substances to the physiology of the macrophage. Further studies will be necessary to characterize the material associated with the peritoneal macrophage and its role in the mechanism of the MIF-macrophage interaction. It will also be necessary to investigate whether other macrophage functions, such as tumoricidal capacity, are dependent on this cell surface component. At first sight the function of the fibronectin-like substance on macrophages seems to be different from the known role of fibronectins in other cells. One function of fibronectin can be interpreted as enabling cells to respond to messages from other cells through cell to cell contact. For example, addition of cell surface fibronectin to transformed fibroblasts restores their morphology, adhesiveness, and contact inhibition of movement (10). In our experiments, fibronectin enables macrophages to respond to a soluble mediator released by lymphocytes. These substances are messengers allowing the transfer of a signal through the surrounding medium. We see no basic difference between messages delivered by cell to cell contact and messages conveyed by means of lymphokines. An important role for fibronectin in the macrophage response to lymphocyte mediators should not then be too surprising. ACKNOWLEDGMENTS This work a grant-in-aid
was supported by United States Public from the American Heart Association.
Health
Service
Grants
AI121
10 and AI07685
and
REFERENCES I. 2. 3. 4. 5. 6. 7. 8. 9. IO. 11.
David, J. R., Proc. Nat. Acad. Sci. USA 56, 72, 1966. Bloom, B. R., and Bennett, B., Science 153, 80, 1966. Fowles, R. E., Fajardo, I. M., and David, J. R., J. Exp. Med. 138, 952, 1973. Piessens, W. F., Churchill, W. H., and David, J. R., J. Immunol. 114, 293, 1975. Remold, H. G., and Rosenberg, R. D., J. Biol. Chem. 250, 6608, 1975. Remold, H. G., J. Immunol. 118, 1, 1977. Higgins, T. J., Sabatino, A. P., Remold, H. G., and David, J. R., J. Immunol. 121, 880, 1978. Yamada, K. M., and Olden, K., Nature (London) 275, 179, 1978. Yamada, K. M., Yamada, S. S., and Pastan, I., Proc. Nat. Acad. Sci. USA 72, 3158, 1975. Yamada, K. M., Yamada, S. S., and Pastan, I., Proc. Nat. Acad. Sci. USA 73, 1217, 1976. Pouyssegur, T. M., Willingham, J. M., and Pastan, I., Proc. Nat. Acad. Sci. USA 74, 243, 1977.
A FIBRONECTIN-LIKE 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38.
CELL
SURFACE
COMPONENT
187
Yamada, K. M., and Kennedy, D. W., J. Cell Biol. 80, 492, 1979. Remold, H. G., Katz, A. B., Haber, E., and David, J. R., Cell. Immunol. 1, 133, 1970. Nathan, C. F., Karnovsky, M. L., and David, J. R., g. Exp. Med. 133, 1356, 1971. Remold-O’Donnell, E. M., Prep. Biochem. 7, 441, 1977. Udenfriend, S., In “Fluorescence Assay in Biology and Medicine,” Vol. 2, p. 422. Academic Press, New York/London, 1969. Chen, A. B., and Mossesson, M. W., Anal. Biochem. 79, 144, 1977. Engvall, E., and Ruoslahti, E., Intern. J. Cancer 20, 1, 1977. Blomback, B., and Blomback, M., Ark. Kemi. 10, 415, 1956. Heck, L. W., Rosenberg, R. D., and Remold, H. G., Prep. Biochem. 9, 359, 1979. Cuatrecasas, P., J. Biol. Chem. 245, 3059, 1970. Goding, J. W., J. Immunol. Methods 20, 241, 1978. Ades, E. W., Phillips, D. J., Shore, S. L., Gordon, D. S., LaVia, M. F., Black, C. M., and Reimer, C. B., J. Immunol. 117, 2119, 1976. Esselman, W. J., Laine, R. A., and Sweeley, C. C., In “Methods in Enzymology” (V. Ginsburg, Ed.), Vol. 28, p. 140. Academic Press, New York, 1972. Kinsky, S. C., In “Methods in Enzymology” (S. Fleisher and L. Racker, Eds.), Vol. 32, p. 501. Academic Press, New York, 1975. Lindgren, F. T., In “Analysis of Lipids and Lipoproteins” (E. G. Perkins, Ed.), p. 704. American Oil Chemists Society, Champaign, Ill., 1975. Lowry, 0. H., Rosebough, N. J., Farr, A. L., and Randall, R. J., J. Biol. Chem. 193, 265, 1951. Steinman, R. M., and Cohn, Z., J. Cell Biol. 55, 186, 1972. Schubert, R. D., Wong, J. A., and David, J. R., Cell. Immunol. 55, 145, 1980. Bartfeld, H., and Atoynatan, T., Proc. Sot. Exp. Biol. Med. 130, 497, 1969. Remold, H. G., Transplant. Proc. 10, 152, 1972. Colvin, R. B., and Dvorak, H. F., J. Exp. Med. 142, 1377, 1975. Colvin, R. B., Lanigan, J., Clark, R. A., Ebert, T. H., Verderber, E., and Hammond, M. E., Fed. Proc. Abstr. 38, 1408, 1979. Unkeless, J. C., Gordon, S., and Reich, E., J. Exp. Med. 139, 834, 1974. Curtiss, L. K., and Edgington, T. S., J. Immunol. 118, 1966, 1977. Liu, D. Y., Petschek, K. D., Remold, H. G., and David, J. R., 1. Immunof. 124, 2042, 1980. Hoffstein, S. T., J. Cell Biol. 83, 69aCS, 1979. Hauptman, S. P., Kansu, E., and Godfrey, S., J. Immunol. 123, 1007, 1979.
58, 175-187
IMMUNOLOGY
(1981)
A Macrophage
Surface
Involved
in the Response
HEINZ Departments Division
Component
G. REMOLD,
JUDITH
Related
to Migration E. SHAW,
to Fibronectin Inhibitory
May
21. 1980;
accepted
Factor
AND JOHN R. DAVID
of Medicine. Harvard Medical School and Robert B. Brigham of the Affiliated Hospitals Center, Inc.. Boston. Massachusetts Received
Is
June
Hospital, 021 I5
25, 1980
Guinea pig peritoneal exudate cells release a substance upon incubation with hypotonic buffer or 6 M urea which (1) augments the response of normal macrophages to migration inhibitory factor, and (2) restores the response of trypsinized macrophages to migration inhibitory factor. Adsorption of peritoneal exudate cell extract with antibodies to guinea pig plasma fibronectin results in a loss of both the MIF-response-enhancing and restoring activity. These results indicate that both activities are associated with a macrophage surface component serologically related to plasma fibronectin. Furthermore, preincubation of macrophages with plasma fibronectin abrogates the enhancement of the MIF response caused by peritoneal exudate cell extracts. This suggests that the active component and plasma tibronectin compete for a common binding site. It is likely that this substance plays a regulatory role in the macrophage response to migration inhibitory factor.
INTRODUCTION Lymphocyte mediators are soluble effector substances of cellular immunity with a variety of biological activities. They include the closely related factors migration inhibitory factor (MIF)’ and macrophage activating factor (MAF), which both affect the macrophage, resulting in inhibition of macrophage migration and an increased capacity of the cells to kill microorganisms and tumor cells (l-4). In previous studies it has been shown that the response of macrophages to MIF and MAF can be enhanced by pretreatment of the cells with esterase inhibitors or chemicals affecting the cell surface (5, 6) and by macrophage glycolipids (7). In the present study we demonstrate that incubation of guinea pig macrophages with a component obtained by hypotonic treatment from the macrophage surface enhances their response to MIF and restores the ability of trypsinized macrophages to respond to MIF. This component, which is referred to as MIF-response enhancing factor (EF), is obtained from guinea pig peritoneal macrophages by treatment with hypotonic buffer. It is not an esterase inhibitor, and it is absorbed out by antiguinea pig plasma fibronectin-IgG. Fibronectin, the transformation-sensitive gly’ Abbreviations used: IgG, Immunoglobulin G; MIF, migration inhibitory factor; MAF, macrophage activating factor; EF, MIF-response enhancing factor; PEC, peritoneal exudate cells; HBSS, Hanks’ balanced salt solution; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; PMN, polymorphonuclear leukocytes; PBS, phosphate-buffered saline; DFP, diisopropylphosphorofluoridate, MEM-PS, Eagles minimum essential medium containing 100 units/ml penicillin and 100 rg/ml streptomycin; PF, plasma fibronectin. 175
0008-8749/81/030175-13$02.00/O Copyright 0 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.
176
REMOLD,
SHAW, AND DAVID
coprotein of connective tissue cells, has been shown to occur in two different molecular forms (8). Cell surface fibronectin, found on the cell surface of connective tissue cells, has an important function in cell aggregation (9), adhesion (lo), and motility (1 I). This substance is antigenically identical with plasma fibronectin (cold insoluble globulin) present in plasma or serum, but differs from it in several biochemical and functional characteristics ( 12). MATERIALS
AND
METHODS
MEM and HBSS were obtained from Microbiological Associates, Bethesda, Maryland, and sodium caseinate from Eastman Kodak, Rochester, New York. DFP and gelatin from swine skin were bought from Sigma Chemical Company, St. Louis, Missouri. Protein A-Sepharose and fluoresceinated protein A were purchased from Pharmacia Fine Chemicals, Piscataway, New Jersey; bovine fibrinogen and TPCK-trypsin from Worthington Enzymes, Freehold, New Jersey. Na1251 (17 Ci/mg) was obtained from New England Nuclear Corporation, Boston, Massachusetts. Production of MIF. MIF-rich and control lymphocyte supernatants were obtained as previously described (13). Briefly, 2.4 X 10’ guinea pig lymph node lymphocytes/ml were incubated for 24 hr at 37°C with concanavalin A (10 pg/ml) to stimulate MIF production. Concanavalin A was added to a parallel control supernatant after the 24-hr incubation. The cells were removed by centrifugation and the supernatants were lyophilized and filtered over Sephadex G-100 columns. The MIF-containing fractions were pooled as were parallel fractions from the control Sephadex G- 100 column. Fractions were concentrated to l/ 100 the volume of the original supernatant and stored at -70°C. The amounts of MIF mentioned in the Results section refer to this fraction. Assay for MZF. Fractions to be tested were diluted to 2 ml with MEM-PS containing 15% normal guinea pig serum and assayed for MIF activity on normal guinea pig PEC using a capillary tube assay (13). MIF activity (%I) was expressed as the percentage of the inhibition of migration of cells incubated with MIF compared to the migration of the same cells in the control fraction. percentage I = 100 -
average migration of MIF-containing fractions x 100. average migration in control fractions
At least 20% inhibition must be obtained for significant activity (13). To test for MIF-enhancing activity, limiting concentrations of MIF were chosen so that MIF itself produced little or no inhibition of migration. Production of PEC extracts containing EF. PECs were induced in guinea pigs by peritoneal injection of mineral oil (Marco1 52, Exxon Corporation, Everett, Mass.) or 1% sodium caseinate in saline; PECs were harvested 3-4 days later as previously described (14) and washed by pelleting in HBSS. The cells (6 X lo’/ ml) were incubated at 37°C for 60 min during which time they were maintained in suspension. 7.5% NaHCO, solution was added dropwise in order to maintain the pH at 7.4. The supernatants from these cells were tested for EF activity. In order to obtain larger amounts of EF activity, a volume of 10-30 ml PEC (6 X 10’ cells/ml) was incubated in HBSS containing 10m3M DFP for 30 min at 37°C at pH 7.4, and washed. The cells were then incubated in a lo-fold volume
A FIBRONECTIN-LIKE
CELL
SURFACE
COMPONENT
177
of hypotonic buffer (5 mM Tris-HCl, 75 mM sucrose, and 1 mM EDTA, pH 8.2), for 15 min at 4°C which causes the cells to swell but not to lyse (15). Cells were then removed by low-speed centrifugation (250g for 5 min), and 0.25 vol of 20 mM Tris-HCl, 1.5 mM EDTA, and 0.15 M NaCl, pH 7.4, was added to the supernatant which was then centrifuged at 900g for 7 min to precipitate any remaining cells or cellular debris. Cell breakage was monitored by measuring the lactate dehydrogenase activity ( 16) in the extracts and comparing it to the activity of the enzyme in the cells after homogenization. This PEC extract contains 600 +- 200 yg protein/ml (13 determinations). The resulting extracts were stored at -70°C. In some experiments, PEC were incubated with 6 M urea in PBS for 15 min in HBSS at 4°C and processed in the same fashion. Production of EF from lymph node ceils and PMNs. Lymph node cells were obtained from guinea pigs immunized with CFA (13). The lymph nodes were harvested, and the cells were obtained by teasing. More than 95% of the cells were lymphoid cells. PMNs were elicited by intraperitoneal injection of 1% casein. The cells harvested after 12 hr were 99% PMNs. To obtain EF the lymph node cells (7 x lO’/ml) and PMNs (8 X lO’/ml) were subjected to the same hypotonic treatment as described for the PECs. Viability of these cells was greater than 90% after hypotonic treatment. Purification of guinea pig plasma proteins. Plasma fibronectin was prepared from frozen guinea pig plasma as described (17) for human plasma fibronectin with a modification. An additional final step, gelatin-Sepharose affinity chromatography, was employed (18). Plasma fibronectin prepared by this method contained no fibrinogen as judged by immunodiffusion and by its ability to clot, and it showed one precipitation line when tested against rabbit anti-guinea pig serum. In the presence of 2-mercaptoethanol it showed one band on SDS-PAGE with an apparent MW of 220,000. Guinea pig fibrinogen and AT III were prepared as previously described (19, 20). Fibrinogen showed one precipitation line in immunoelectrophoresis using antiguinea pig plasma serum as an antigen. AT III showed one band with an apparent MW of 68,000 on SDS-PAGE under reducing conditions. Production of antisera. Antibodies against guinea plasma fibronectin, guinea pig fibrinogen, and guinea pig AT III were obtained by injecting subcutaneously 175200 pg of the purified proteins in complete Freund’s adjuvant into New Zealand white rabbits. After 14 days the animals were boosted with a second dose of antigen in incomplete Freund’s adjuvant. This procedure was repeated three to four times. The resulting antisera showed a precipitation line in the Ouchterlony gel diffusion assay at a dilution of 1:8 when a concentration of 1.6 mg/ml antigen was used. Preparation of IgG-Sepharose 4B. Aliquots of 5-ml CNBr-activated Sepharose 4B (2 1) were each coupled with 8 mg of normal rabbit IgG or rabbit anti-guinea pig PF IgG purified on protein A-Sepharose columns. The IgG-Sepharose was packed into 5-ml syringes and washed with 0.05 M Tris-HCl, pH 7.2. EF (8.2 ml) was filtered over each IgG-Sepharose column, washed with the same buffer, and eluted with 4 M guanidine HCl, pH 7.3. Aliquots from the void volume and from the guanidine HCl wash were tested for EF activity. Reaction of PEC extracts with antibodies against several guinea pig proteins. Anti-guinea PF IgG, anti-AT III IgG, and anti-fibrinogen IgG were obtained by
178
REMOLD,
SHAW,
AND
DAVID
affinity chromatography of 2.0 ml crude antiserum on a protein A-Sepharose column (1 X 5 cm) (22). IgG was eluted from the column with 0.1 M glycine HCl buffer, pH 3.0, and immediately adjusted to pH 7.0 with NaOH. The eluted IgG was concentrated to 2 ml. PEC extract (2.5 ml) was incubated with 0.5 ml IgG for 60 min at 4°C. As an additional control, IgG was incubated in buffer alone. Ten percent heat-killed suspension (0.5 ml) of Stuphylococcus aureus Cowan I (a gift from Dr. E. Remold-O’Donnell) was then added to the solution, and they were mixed at 4°C for 30 min. The suspensions were then centrifuged at 9OOg for 10 min. No IgG could be detected in the supernatants after treatment with Staphylococcus aureus by gel diffusion. The supernatants were dialyzed against MEMPS and assayed for MIF-enhancing activity. Surface fluorescence staining of peritoneal macrophages with anti-PF serum. Normal rabbit serum ( 100 ~1) or rabbit anti-guinea pig PF serum were incubated with 100 ~1 of a fluorescein-labeled protein A solution (1.1 mg/ml) at 4°C for 30 min, as previously described (23). One hundred microliters from serial dilutions (1:l to 1:64) of the incubation mixture was incubated with 5 X lo6 washed guinea pig peritoneal exudate cells at 4°C for 30 min. The cells were then washed with PBS four times and examined for surface fluorescent staining. Glycolipid extraction. PEC supernatants obtained by hypotonic treatment were extracted using a modification of the method of Esselman et al. (24). In brief, 4 ml of chloroform/methanol (2 v/v) was added to a lyophilized preparation of the PEC supernatants obtained from 6 X 10’ cells. The suspension was sonicated at 4°C for 1 min using a Model 250 ultrasonic cleaning bath (E/MC, Division of Rai Research, Fisher Scientific, Medford, Mass.) and centrifuged. The pellet was reextracted with chloroform:methanol. The combined supernatants were filtered on a medium-porosity sintered glass filter and evaporated to dryness. The glycolipids were dissolved in sterile MEM-PS by sonication for 15-30 set and incorporated into liposomes using the method of Kinsky (25). The liposomes were assayed for enhancement of MIF response (7). Flotation of PEC extracts in salt solutions with increasing densities using a procedure developed for the separation of lipoprotein classes. PEC extracts were centrifuged in NaCl-NaBr solutions of increasing density (26). Sequential fractionations were carried out in solutions with densities of 1.0063 (0.196 A4 NaCl), 1.067 (0.0786 M NaBr), and 1.210 (2.962 M NaBr) g/ml in a SW 50.1 rotor (Beckman Instruments, Palo Alto, Calif.) at 45,000 rpm for 23 hr at 9°C. The resulting pellets and supernatants were dialyzed against MEM-PS and assayed for enhancement of the MIF response. Determination of protein. Protein was measured by the method of Lowry et al. (27). Measurement of pinocytosis. Pinocytosis of PEC was measured according to the method of Steinman and Cohn (28) as applied to guinea pig macrophages (29). RESULTS Enhancement
of the MIF Response of Macrophages
by PEC Extracts
Supernatants obtained after incubation of peritoneal exudate cells in MEM-PS for 1 hr at 37°C were found to markedly enhance the response of fresh PEC to a limiting amount of the lymphocyte mediator MIF. The results of these experi-
A FIBRONECTIN-LIKE
CELL
SURFACE
COMPONENT
179
EXPEUIMEN T NUMBER
FIG. 1. Effect of PEC supernatants on a low dose of MIF. MIF activity is shown on the ordinate; the abscissa shows six experiments. The stippled bars represent the response to a low dose of MIF on normal macrophages. The cross-hatched bars represent the response of macrophages in the presence of 0.5 ml PEC supernatants diluted with 0.5 ml MEM-PS. Note that the presence of PEC supernatants increases the response of macrophages to MIF.
ments are shown in Fig. 1, where it can be seen that optimal increase in the MIF response was found when 0.5 ml of PEC supernatant/ml of assay medium was used. An amount of 1.0 ml was also effective (results not shown), but amounts smaller than 0.5 ml did not result in enhancement of MIF response. More consistent enhancing activity was obtained when casein- or oil-induced PEC were extracted with cold hypotonic buffer (see Materials and Methods). Figure 2 shows six such experiments where 0.35 ml PEC extract caused a marked enhancement of migration inhibition. Similar results were obtained when PEC were extracted with 6 it4 urea in the cold. Urea extract (0.7 ml/ml) containing 350 pg protein enhanced the response of macrophages to a limiting concentration of MIF (2 and 5% I) to 43 and 289’0 I respectively. It should be noted that PEC extracts alone in several concentrations had no MIF-like activity (data not shown).
:-~- -L 0 10 zo M/f
30
-IL 40 50
ACr/vlTY
60 70 /%I)
FIG. 2. Enhancement of the MIF response of macrophages by hypotonic PEC extracts. The abscissa shows MIF activity; the ordinate six experiments. The amounts of PEC extract added per milliliter medium is indicated on the left. Note that in all experiments as little as 0.35 ml PEC extracts/ml medium induced an enhanced response of the macrophage to a low dose of MIF.
180
REMOLD,
SHAW, TABLE
Restoration
of the Response
of Trypsinized
AND
DAVID
1 Macrophages
to MIF
Percentage Experiment 1 2 3 4 5 6 7 R Migration b Amounts
inhibition of trypsinized of PEC extract included
Ob 14 17 0 11 15 3 16
by PEC
Extracts”
inhibition
0.3sb
o.7b
24 22 42
34 23 45 44 52 24 40
macrophages exposed to 150 ~1 MIF per ml medium. in the assay medium in milliliters per milliliter of medium.
Hypotonic treatment of lymph node cells and PMN also released a substance which enhanced the migration inhibition of macrophages. Inhibition in the presence of a limiting concentration of MIF (0 and 13% I) was increased by lymph node cell extracts to 45 and 67% I and by PMN extracts to 59 and 49% I. Restoration of the Response of Trypsin-Treated by PEC Extracts
Macrophages
to MIF
It was shown in earlier studies that macrophages treated with trypsin fail to respond to MIF (30, 31). Since the EF described above augments the response of normal macrophages to MIF, we asked whether it would also restore the response of trypsinized macrophages to MIF. When trypsinized PEC, which were unresponsive to MIF, were incubated with PEC extracts, the response to MIF was consistently restored (Table 1). Specific Absorption of EF Activity Guinea Pig Plasma Fibronectin
from
PEC Extracts
by Antibodies
against
Fibronectin plays an important role in a variety of cellular functions, such as aggregation and motility. Therefore, it was of interest to investigate whether EF activity is associated with a fibronectin-like macrophage surface component. To test this, we incubated PEC extracts containing EF activity with antibodies to PF. PEC extracts were incubated with rabbit anti-guinea pig PF IgG, and, as a control, with normal rabbit IgG and rabbit anti-guinea pig AT III IgG. After incubation, the IgG was removed by addition of heat-killed Staphylococcus aureus Cowan I and subsequent centrifugation. In a second set of controls, anti-guinea pig PF IgG and anti-guinea pig AT III IgG were incubated with PBS alone and then precipitated with Staphylococcus aureus. In each case, the supernatants were dialyzed and tested for migration inhibition enhancing activity. The results of 10 such experiments are summarized in Fig. 3. It can be seen that enhancing activity is abrogated when PEC extracts are absorbed with anti-PF IgG, but is unaltered in all controls.
A FIBRONECTIN-LIKE EC
PRETREATMENT
-
NOW
CELL
SURFACE
COMPONENT
181
~-~-
onf~-ATil
FIG. 3. Effect of anti-PF IgG on the MIF-response enhancing activity of EF. PEC extracts (EF, 0.7 ml/ml medium) or PBS as a control (-) were incubated with medium alone (none), normal rabbit IgG (normal IgG), anti-AT-III IgG (anti-AT III), and anti-PF IgG (anti-PF) for 60 min at 4°C. The solutions were then incubated with heat-killed Staphylococcus aureus and centrifuged. The supernatants were tested for enhancement of MIF response using a limiting concentration of MIF (0.2 ml/ml medium) on normal macrophages. The columns summarize the MIF activity of 10 experiments (abscissa). The standard error is indicated. Note that treatment of PEC extracts with anti-PF IgG abolished the enhancing effect.
Fibrin is also known to be a macrophage surface component (32). It is of note that absorption of PEC extracts with anti-guinea pig fibrinogen-IgG did not remove EF activity: in three experiments the macrophage response to a low amount of MIF (13,0, and 5% I) was augmented by addition of PEC extracts pretreated with anti-fibrinogen IgG to 62, 56, and 53% I, respectively, whereas PEC extracts pretreated with anti-PF IgG lacked the MIF-response enhancing effect (16, 18, and 19% I). We conclude from these experiments that EF serologically cross-reacts with plasma fibronectin, but not with fibrinogen. In a similar set of experiments we tested the effect of anti-PF IgG on the capacity of PEC extracts to restore the response of trypsinized macrophages to MIF. The results, shown in Table 2, indicate that restoration of MIF response to trypsinized macrophages is abolished by incubation of these extracts with anti-PF IgG. In contrast, after treatment with normal rabbit IgG, the MIF response is restored. In other controls normal rabbit IgG or anti PF IgG was incubated in MEM-PS alone and was then precipitated with heat-killed Staphylococcus aureus. The supernatants did not restore the response of macrophages to MIF indicating that abolishment of restoration of the MIF response to trypsinized macrophages is not due to material present in the supernatant after precipitation with Staphylococcus aureus. These data indicate that both activities, MIF enhancing and restoring activity, are associated with the same substance. Since EF can be absorbed out by anti-PF antibodies, we questioned whether purified plasma fibronectin from guinea pig plasma is able to substitute for EF, i.e., is PF able to enhance the response of normal macrophages to MIF. When guinea pig plasma fibronectin was added to PEC at a concentration of 100 or 50 pg/ml medium, no enhancement of the MIF response was seen (experiments not shown). In three additional experiments, plasma fibronectin did not restore the response of trypsinized PEC to MIF (17, 13, and 19% I in the controls versus 2, 17, and 26% I using 100 yg/ml plasma fibronectin and 15, 22, and 10% I using 50 pg/ml plasma fibronectin). As a positive control, 0.7 ml PEC extracts/ml me-
182
REMOLD,
Effect
of Antibody
Treatment
SHAW,
AND
TABLE
2
on the Capacity of PEC Extracts Trypsinized Macrophages to MIF” Percentage
Experiment
PBS + normal rabbit-IgG
I 2 3 4
DAVID
7 4 19 10
EF + normal rabbit-IgG 41 20 32 35
to Restore
the Response
of
inhibition PBS + antiPF-IgG 9 2 4 7
EF + antiPF-IgG 18 11 21 6
a Migration inhibition of trypsinized macrophages in the presence of 150-300 WI/ml MIF and various pretreated PEC extracts. Aliquots (2.5 ml) of PEC extract (EF) were incubated with S-10 @I normal rabbit IgG or with 5-10 rg rabbit anti-guinea pig PF-IgG for 60 min at 4°C. Subsequently heat-killed S~uphylococcus nrrrelcs (0.5 ml of a 10% solution) was added and after 30 min at 4°C the suspensions were centrifuged. The supernatants were tested for their capacity to restore the response of trypsinized macrophages to MIF. In parallel control samples PBS replaced the PEC extracts. Note that the lacking MIF response of trypsinized macrophages (PBS + normal rabbit-IgG) is restored when PEC extracts are added (EF + normal rabbit-IgG). This effect is abrogated when the PEC extracts were absorbed with anti-PF IgG (EF + anti-PF IgG).
dium enhanced the migration inhibition significantly to 41, 42, and 33% I. These results show that although antiplasma fibronectin antibodies cross-react with EF, plasma fibronectin is not capable of augmenting or restoring the migration inhibition of macrophages. It is, therefore, not identical with EF. To further study the relationship between fibronectin and EF, experiments were carried out to see if plasma fibronectin competes with EF for a common binding site. Preincubation of macrophages with plasma fibronectin consistently prevented enhancement of the MIF response due to EF (Table 3). When macrophages were preincubated with 200 pg/ml myoglobin (used as a control) they still responded to EF. This experiment suggests strongly the EF and plasma fibronectin compete for a common binding site on the macrophage. Furthermore, when anti-PF IgG was absorbed out with purified plasma fibronectin, it failed to remove EF activity from PEC extracts, indicating that anti-PF IgG and not a contaminating antibody is responsible for the abolition of EF (three experiments, data not shown). In addition, anti-PF-IgG-PF complexes did not absorb out EF activity making it unlikely that EF binds nonspecifically to immune complexes. Cell Surface Location
of EF
The conditions used to extract EF from the cells were such that cell breakage was minimal. To demonstrate this, the activity of the cytoplasmic enzyme lactate dehydrogenase was measured in PEC extracts. Activity of lactate dehydrogenase in hypotonic PEC extracts was 7.0 f 0.01% (SE) of the activity in the corresponding cell homogenates (eight determinations). These values are similar to those obtained from isotonic macrophage supernatants (data not shown) and suggest that EF activity is derived from the cell surface. In order to establish this point more fully, we investigated whether material which crossreacts serologically with plasma
A FIBRONECTIN-LIKE
CELL TABLE
Abrogation
of EF Activity
by Preincubation
SURFACE 3 of Macrophages Percentage
Ob Experiment I 2 3 4 5 6
Medium 1 0 9 0 0 0
183
COMPONENT
with
Plasma
Fibronectin”
inhibition 3oob
2oob EF
Medium
EF
Medium
EF
35 36 39 39 30 34
1 0 4 0 0 18
17 21 24 7 0 15
18 0
4 9
-
a Macrophages (6 X 10’ cells/ml) were preincubated with 200 pg/ml HBSS and 300 Kg/ml plasma fibronectin for 1 hr at 37°C. They were then assayed for their response to a limiting concentration of MIF in the presence (EF) and absence (medium) of 0.7 ml PEC extract. Note that macrophages preincubated with plasma fibronectin show decreased or no EF-dependent enhancement of MIF response. b Amounts of plasma fibronectin (micrograms per milliliter) used for preincubation.
fibronectin is present on the macrophage cell surface. Oil-induced PEC, consisting of 90% macrophages, were incubated for 30 min at 4°C with rabbit anti-guinea pig-PF IgG complexed with fluorescein-conjugated protein A and then extensively washed (see Materials and Methods). Cells treated in this manner consistently showed fluorescent staining on their surface (Fig. 4.1). The staining pattern, as seen in Fig. 4.1, is patchy, and the patches have a diameter of 0.4-0.8 pm. 34 f 1% of the PEC are stained with anti-PF antiserum (three experiments). On the other hand, cells incubated with normal rabbit serum complexed with fluoresceinconjugated protein A did not show staining of the cell surface (Fig. 4.3). These results are similar to the recent findings of Colvin et al. (33) and demonstrate that fibronectin-containing material is a component of the macrophage cell surface. It should be noted that trypsinized macrophages do not show fluorescent staining by anti-PF IgG, indicating that the structures specific for anti-PF IgG are trypsin sensitive. EF Activity Glycolipids,
is Not Caused by the Action of Esterase Inhibitors, or Lipoproteins
In previous studies, we showed that pretreatment of macrophages with proteinase inhibitors enhances the response of macrophages to a limiting concentration of MIF (5). It was therefore of importance to investigate (a) whether hypotonic PEC extracts contain esterase inhibitors and (b) whether EF activity can be attributed to the action of an esterase inhibitor. A small amount of esterase inhibitors in hypotonic extracts from PEC was found; undiluted PEC extracts when added to ‘251-fibrin plates (34) inhibit the activity of low concentrations of trypsin (0.1 pg/ml), whereas higher trypsin concentrations (30-60 pg/ml) were not inhibited. We were, however, unable to remove the esterase inhibitory activity from the PEC extracts by absorption with anti-PF antibodies (H. G. Remold and J. E. Shaw, manuscript in preparation). We also found that
FIG. 4. Fibronectin-like material on the surface of guinea pig peritoneal macrophages. 1. Cells incubated with rabbit-anti-guinea pig PF serum complexed with fluorescein-conjugated protein A (see Materials and Methods). Note that the cell surface is stained. 2. A cell photographed in the plane of the cell surface. Note the patchy distribution of the fluorescent stain on the cell surface. 3. Cells incubated with normal rabbit serum complexed with fluorescent protein A. No fluorescent surface stain can be seen. X900.
A FIBRONECTIN-LIKE
CELL
SURFACE
COMPONENT
185
esterase inhibitors did not restore the response of trypsinized macrophages to MIF. For instance, trypsin-treated macrophages that did not respond to a large amount of MIF (16% I) did respond when 0.7 ml PEC extract was added per ml medium (65% I). AT III (100 pg/ml), soybean trypsin inhibitor (100 pg/ml), and DFP ( lop4 M), which are potent serine esterase inhibitors, were unable to restore this response (21, 8, and 0% I, respectively). These experiments demonstrate that the action of EF is not that of an esterase inhibitor. Higgins et al. (7) showed that macrophage glycolipids enhance the response of macrophges to MIF. To investigate whether EF activity is due to the action of a glycolipid, PEC extracts were fractionated using an extraction method for glycolipids (see Materials and Methods). No biological activity was recovered in the glycolipid fraction, demonstrating that EF is not a glycolipid (six experiments, not shown). Since lipoproteins have immunoregulatory effects (35), we also investigated whether EF is a lipoprotein. PEC extracts were fractionated using a method developed for the separation of lipoprotein classes (26). In three experiments the response to a limiting amount of MIF (10, 0, and 5% I) was not enhanced when the fractions with a density of 1.0063 (21, 0, and 0% I), 1.067 (15, 0, and 0% I) m 1 were tested. EF activity accumulated in the pellet and 1.210(12,0,andO%I)g/ of the densest fraction (1.210 g/ml), which contains components denser than lipoproteins. The densest fraction enhanced the response to a low concentration of MIF from 10, 0, and 5% I to 70, 23, and 37% I, respectively. These experiments show that EF does not have the characteristics of a lipoprotein. DISCUSSION The experiments described here demonstrate that hypotonic extracts from guinea pig peritoneal exudate cells contain a material which enhances the response of normal macrophages to MIF and which restores the MIF response of trypsinized macrophages. The data also show that antibodies against purified plasma fibronectin absorb out both activities from PEC extracts, suggesting that they are related to each other and are associated with the same substance. This substance is referred to here as MIF-response enhancing factor, or EF. In earlier studies, we found that the response of macrophages to MIF is enhanced by three groups of substances, proteinase inhibitors (5), reagents which alter cell surface components, such as diazotized sulfanilic acid (6), and fucogangliosides from the macrophage cell membrane (36). The EF described here is not an esterase inhibitor, nor a glycolipid, indicating that the action of EF on the macrophage differs from the action of esterase inhibitors or glycolipids. There are several lines of evidence to indicate that EF activity is associated with a substance related to fibronectin. First, EF activity can be absorbed out by antiPF IgG. Second, plasma fibronectin competes with EF for a common binding site on the macrophage, and third, trypsin treatment abolishes the response of macrophages to MIF. Concomitantly, the capacity of the cells to stain with fluorescent anti-PF IgG is also abolished by trypsin. On the other hand, plasma fibronectin cannot substitute for EF; therefore it is not identical with it. Plasma fibronectin does not enhance the response of normal macrophages to MIF, nor does it restore the MIF response of trypsinized mac-
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rophages. Furthermore, plasma fibronectin is relatively easy to purify, but we were unable to purify EF by conventional methods such as gel filtration, sucrose density gradient centrifugation, or electrophoresis, probably because of its strong tendency to aggregate. For example, affinity chromatography of EF on anti-PF-IgG-sepharose columns was inconclusive, because EF, due to its high affinity to particulate matter, absorbed to normal-IgG-sepharose columns as well as to anti-PF-IgG-sepharose columns. It could be eluted from both resins with 4 M guanidine HCl. It was reported that anti-PF IgG was also found to react with human neutrophil surfaces (37). These cells displayed immunofluorescent staining for fibronectin which, surprisingly, persisted after trypsin treatment. The reported resistance of the fibronectin-like structure to trypsin on the neutrophil strongly indicates that this material is modified, possibly by cross-linking. In addition, a cold insoluble protein with an apparent MW of 125,000 was found on mouse lymphocytes which plays a role in the regulation of the immune response (38). The findings presented in this study extend the importance of fibronectin-like substances to the physiology of the macrophage. Further studies will be necessary to characterize the material associated with the peritoneal macrophage and its role in the mechanism of the MIF-macrophage interaction. It will also be necessary to investigate whether other macrophage functions, such as tumoricidal capacity, are dependent on this cell surface component. At first sight the function of the fibronectin-like substance on macrophages seems to be different from the known role of fibronectins in other cells. One function of fibronectin can be interpreted as enabling cells to respond to messages from other cells through cell to cell contact. For example, addition of cell surface fibronectin to transformed fibroblasts restores their morphology, adhesiveness, and contact inhibition of movement (10). In our experiments, fibronectin enables macrophages to respond to a soluble mediator released by lymphocytes. These substances are messengers allowing the transfer of a signal through the surrounding medium. We see no basic difference between messages delivered by cell to cell contact and messages conveyed by means of lymphokines. An important role for fibronectin in the macrophage response to lymphocyte mediators should not then be too surprising. ACKNOWLEDGMENTS This work a grant-in-aid
was supported by United States Public from the American Heart Association.
Health
Service
Grants
AI121
10 and AI07685
and
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