Mechanisms of action of “lymphocyte-activating factor” (LAF)

CELLULAR

IMMUNOLOGY

Mechanisms

56, 68-79 (1980)

of Action of “Lymphocyte-Activating

Factor”

(LAF)

IV. Differential Stimulation of T Lymphocytes by Induced Macrophage Enzymes (Catheptic Carboxypeptidase B and Serine Proteases)’ S. P. KATZ,~ T. SHIMAMURA,~ J.-P. DESSAINT,~ D. BRAVERMAN,~ AND B.H. WAKSMAN Departments of Pathology and Biology (Immunology Sectionj, Yale School of Medicine, New Haven, Connecticut 06510 Received September 12, 1979; Accepted February 9, 1980 PHA and concanavalin A are unable to induce DNA synthesis in a substantial proportion of highly purified rat lymph node T cells in serum-free culture. Addition of dialyzed macrophage supernatant (LAF or TAF) to these mitogen-stimulated cells, even as a 12-hr pulse starting at 14 hr, gives a stronly potentiated response, while LAF (TAF) alone is nonmitogenic. Thus mitogen provides a first signal and LAF (TAF) a second signal in time. General protease inhibitors (trasylol, soybean trypsin inhibitor, and especially e-aminocaproic acid) markedly inhibit both the low response to mitogen alone and the LAF effect, while certain more narrowly specific inhibitors (phenylmethylsulfonyl fluoride, tosyl-lysl-chloromethyl ketone, iodoacetate) do not. This finding suggests that the LAF effect involves protease action. The LAF effect has been shown to parailel the carboxypeptidase B content of various LAF preparations and can be mimicked by commercial pancreatic carboxypeptidase B. A lesser LAF effect is also induced by such serine proteases as trypsin, chymotrypsin, and plasmin, none being mitogenic alone. Thus LAF (TAF) may represent the combined action of several enzymes. These findings are contrasted with the demonstrated ability of many serine proteases to stimulate DNA synthesis, i.e., to serve as first signal, in B cells. LAF (TAF) acts synergistically with commercial carboxypeptidase B. This implies that they may act on different target sites in the cell membrane and that triggering of an adequate number of sites is required for an effective second signal. LAF (TAF) interaction with T cells is not cell cycle specific since activity is absorbed by unstimulated cells and by cells at various times after mitogen exposure.

INTRODUCTION Recent studies of the macrophage supernatant activity designated LAF (lymphocyte-activating factor)6 or TAF (T-cell-activating factor), defined by its I Supported by USPHS Grants AI-06455 and CA-23887. * Recipient of USPHS Research Fellowship AI-05470. Present address: Department of Tropical Medicine, School of Public Health and Tropical Medicine, Tulane University, New Orleans, La. 70112. 3 On leave from Department of Microbiology, Tokai University School of Medicine, Isehara 259- 11. Japan. 4 Recipient of Research Grant GA HS 7811 from the Rockefeller Foundation. On leave from the Center d’Immunologie et de Biologic Parasitaire, Institut Pasteur, 20 Boulevard Louis XIV, 59012 Lille, France. 5 Present address: Harvard College, Cambridge, Mass. 02138. 6 Abbreviations used in this paper: BAF, B-cell-activating factor; B cells or B lymphocytes, thymus-independent lymphocytes; CPB, carboxypeptidase B; Con A, concanavalin A; Acpm, cpm 68 0008-8749/80/150068-12$02.00/O Copyright 0 1980 by Academic Ress, Inc. All rights of reproduction in any form reserved.

LAF AND MACROPHAGE

ENZYMES

69

ability to potentiate T-lymphocyte responses to antigen or mitogen without being mitogenic alone (l-4), have established the following significant points. LAF acts on stimulated T cells in late Gr, causing a calcium-dependent rise in cyclic GMP which appears closely linked to the later initiation of DNA synthesis and mitosis. LAF action is inhibited by substrate analogs of catheptic CPB (carboxypeptidase B) and partially mimicked by the action of commercial pancreatic CPB. Since, however, catheptic CPB did not appear to account for LAF action entirely (3), we looked into the possible role of other macrophage proteases in triggering T cells. In addition, the exact time of interaction of LAF with the plasma membrane of mitogen-stimulated T cells was examined, both by absorption experiments with cells in different phases of the cell cycle and by examining the LAF effect on lymphokine production, which is initiated in early or mid-G, (see 5). Data concerning these two aspects of LAF action are presented here. MATERIALS

AND METHODS

Reagents. RPM1 with penicillin and streptomycin (Associated Biomedic Systems, Buffalo, N.Y.); Eagle’s minimal essential medium (MEM), and heat-inactivated fetal calf serum (FCS) Microbiological Associates, Bethesda, Md.) dialyzed 24 hr and Nalgene-filtered; pancreatin (Grand Island Biological Co., Grand Island, N.Y.); Phytohemagglutinin-P (PHA) and concanavalin A (Con A) (Difco Laboratories, Detroit, Mich); [3H]thymidine (3HTdR) (2 Ci/mmol) and [3H]leucine (3HL) (5.0 Ci/mmol) (New England Nuclear, Boston, Mass.); pancreatic (porcine) carboxypeptidase B (CPB) (specific activity 140-150 U/mg, residual serine protease blocked by DFP), plasmin (fibrinolysin) (0.3 U/mg), trypsin (14100 IJ/mg), cY-chymotrypsin (44 U/mg), Pronase (0.7 U/mg), trasylol, soybean trypsin inhibitor (SBTI), tosyl-lysyl-chloromethyl ketone (TLCK), phenylmethylsulfonyl fluoride (PMSF) (kept as a stock solution (x 250) in acetone and always controlled with acetone 1:250 alone), sodium iodoacetate, 1, lo-phenanthroline, l -aminocaproic acid (EACA), dithioerythritol (all from Sigma Chemical Co., St. Louis, MO.); and CdCl, (Fisher Scientific Co., Fairlawn, N.J.). Purified thrombin (3312 U/mg) was a gift of Dr. John W. Fenton II, New York State Department of Health, Division of Laboratories and Research. All proteases and protease inhibitors were used as freshly prepared solutions in RPMI. LAF production. LAF was prepared as in previous studies (1, 2) by 24 hr incubation of washed peritoneal exudate macrophages (3-day exudates, induced by injection of beef heart infusion in 10% protease peptone (Difco), incubated at 3 x 107/ml in 6 ml warm RPM1 in plastic petri dishes, 60 x 15 mm (Falcon, Oxnard, Calif.) for 2 hr, then washed to remove nonadherent cells) in 6 ml serum-free RPMI. The supernatant was cleared by centrifugation, dialyzed 24 hr at 4°C against 50 vol of RPMI, sterilized by filtration (0.45 pm, Millipore Corp., Bedford, Mass.), and stored at -20°C. minus background; EACA, e-aminocaproic acid; FCS, fetal calf serum; 3HTdR, [3H]thymidine; 3HL, [3H]leucine; A or hcpm, cpm observed with all reagents added simultaneously minus cpm observed with mitogen alone and other reagents alone; LAF, lymphocyte-activating factor; LNC, lymph node cells; LT, lymphotoxin; M4, macrophage(s); M@, macrophage depleted; PHA, phytohemagglutinin P; PMSF, phenylmethylsulfonyl fluoride; SBTI, soybean trypsin inhibitor; TAF, T-cell-activating factor; TLCK, tosyl-lysyl-chloromethyl ketone; T cells or T lymphocytes, thymus-derived lymphocytes.

70

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ET AL.

Cells. All tests were carried out on pooled cervical, axillary, and inguinal lymph node cells (LNC) of 2- to 3-month-old DA rats of both sexes, bred and maintained in the Division of Animal Care, Yale University, Cell viability (trypan blue) was always >95%. Spleen cells were used in one experiment. Macrophage-depleted (M+-) LNC were usually prepared (2) by incubating 2 ml of LNC suspension (1 x 108/ml) in RPM1 containing 10% FCS on 5 ml prewarmed, acid-washed glass beads (type 100-5005, Minnesota Mining and Manufacturing Co., St. Paul, Minn.) in a lo-ml glass syringe for 30 min at 37”C, eluting with 30 ml of the same medium, and washing and resuspending in serum-free RPMI. The cell yield was 50-60% of which < 1% were macrophages (by acridine orange staining) or B lymphocytes (by direct fluorescence staining with fluorescein-labeled rabbit anti-rat IgM) (Miles Laboratories, Elkart, Ind.). In some experiments LNC (or spleen cells) depleted on glass beads were purified further by passage through a nylon wool column using the technique of Julius et al. (6). The yield was about 30% of the glass bead-passed cells, with fewer than 0.5% macrophages or B cells with LNC. Alternatively macrophages were removed by three successive 60-min incubations of 6 ml of the original LNC suspension (at 1 x lO’/ml in RPM1 and FCS) on 60 x 15-mm plastic petri dishes. The yield was 60-70%, with l-2% M+ and B cells. Cell culture. Cultures were set up (2) in triplicate in flat-bottomed microculture plates (Costar, Cambridge, Mass.). Each well contained 2.5 or 5 x lo5 LNC or M@ LNC in a total volume of 0.08 ml serum-free RPMI, with or without suboptimal concentrations of PHA (0.25 or 0.5 pi/ml) or Con A (0.5 pg/ml) and with or without LAF (25% v/v), CPB, or other enzymes or enzyme inhibitors added at 0 or 14.hr. Cultures were incubated at 37°C in humidified 5% CO,-air, pulsed with 1 $Zi 3HTdR at 38 hr, and harvested at 48 hr on glass fiber filters (Whatman, Clifton, N.J.) with a MASH II (Microbiological Associates, Bethesda, Md.). The filters were counted for 3H in a Beckman liquid scintillation system (Beckman Instruments, Palo Alto, Calif.). When LAF, enzymes, or enzyme inhibitors were used as a 14- to 26-hr pulse, the cultures were washed once at 26 hr and fresh medium containing mitogen alone was added. In one experiment cultures were pulsed with [3H]leucine rather than 3HTdR and harvested in the usual manner. Results are expressed as Acpm after subtraction of baseline 3HTdR incorporation. Acpm was calculated (1) from Acpm for cultures with mitogen plus LAF or reagents by subtracting Acpm values obtained with mitogen alone and with the LAF or other reagents alone. A hcpm ratio was calculated in one experiment as the ratio of the Acpm values for mitogen plus LAF and ACPM for mitogen alone. The SE of individual determinations was always <20%. Measurement of LAF “binding.” M+ LN cells, stimulated with suboptimal PHA for various intervals up to 24 hr, were incubated with LAF at 4 or 37°C for 90 or 120 min. In some experiments, PHA at the same concentration was present during the incubation; in other experiments, EACA or other proteolysis inhibitors. The supernatants were added directly or, in the experiments where inhibitors were used, after 72 hr dialysis, to assay cultures of M+ LNC, cultured with suboptimal PHA, pulsed with 3HTdR at 44-48 hr, and processed in the usual way. Where PHA was present during absorption, its presence was taken into account in determining the final concentration of mitogen in the assay cultures. Absorbed LAF was always added to the assay at 25% (v/v), calculated on the basis of original LAF. In some

LAF AND MACROPHAGE TABLE

71

ENZYMES

1

LAF Effect on Purified T Cells” aHTdR incorporation Mitogen

Expt II

(Acpm x 10m3)

LAF

Expt I

Expt III

0

+

0.9

1.6

0.2

PHA

0 +

8.0 34.0

15.1 34.4

5.2 7.9

Con A

0 +

7.2 15.8

3.3 9.7

3.1 6.9

” LNC, purified on glass beads (Expts I and II) or glass beads and nylon wool (III). M4 and sIg+ cells cl.0 and <0.5%, respectively. LAF was added as 12-hr pulse starting at 14 hr (I and II) or was added between 14 hr and end of culture (III).

experiments a dummy LAF preparation (RPM1 only) was carried through the complete adsorption procedure and finally added to assay cultures to provide a realistic negative control. Assay of carboxypeptidase B. The enzyme was measured photometrically by hydrolysis of hippuryl-L-arginine (Sigma) using the technique of Folk et al. (7). RESULTS LAF action on purified T lymphocytes. Two techniques were used in the present experiments to purify LN T-cell suspensions: passage through a glass bead column or passage successively through columns of glass beads and nylon wool. Both reduced the M+ and B-lymphocyte levels to less than 2% and usually less than 1%. However, the cell yields in repeated experiments were approximately 60 and 18%, respectively, of the original. Thus the nylon wool removed two-thirds of the T cells. The PHA and Con A responses were low with both types of purified T cells, but the LAF effect was very much reduced only with nylon-purified cells (Table 1). By implication, the T-cell population adherent to nylon includes the subpopulation most responsive to the combination of mitogen and LAF or an auxiliary cell type other than the macrophage. (Adherence to plastic, used in one experiment, removes somewhat fewer cells (both B and T) than glass beads.) With spleen cells, the B-cell depletion was less complete; about 22 and 3% sIg+ cells, respectively, remained in purified suspensions prepared with the two techniques. The LAF response nevertheless was completely eliminated in both populations (see below). As established in earlier experiments (1,2), LAF was effective if added at 14 hr (late G,), even as an abbreviated (12 hr) pulse. Proteases and LAF action. A number of general protease inhibitors were tested for their ability to inhibit the mitogenic effect of PHA and LAF on M$- LN cells. Trasylol, SBTI, and EACA were found to inhibit the responses, both to PHA alone and to PHA + LAF, significantly while PMSF, TLCK, and iodoacetate produced minor degrees of inhibition. These inhibitors were active even though added 14 hr after mitogen.

72

KATZ

ET AL.

Since EACA appeared to be the most efficient inhibitor of LAF action, its properties were examined in more detail. This agent inhibited completely the response to Con A and Con A + LAF (Table 2); it was strongly inhibitory at concentrations as low as 5 x 10e3 M. Since on the other hand the responses to PHA and PHA + LAF were only partially suppressed by 10-l M EACA, it appeared that PHA and Con A might act by different pathways or on different cells. The inhibition of responses to the two mitogens alone, in systems containing neither macrophages nor LAF, implied that each mitogen may itself act via a proteolytic event of some sort (lysosomal discharge ?) at the surface of the cell, occurring or acting after 14 hr. The high degree of inhibition obtained with EACA focused our attention on catheptic carboxypeptidase B and on serine proteases as possible mediators of LAF activity. The presence of CPB in standard LAF preparations was established by their ability to split hippuryl-L-arginine (Fig. 1). In agreement with published descriptions of this enzyme (7-9), activity shows an acid pH optimum, is increased in the presence of dithiothreitol and dithioerythritol, and is inhibited by the zinc chelator phenanthroline as well as by EACA and CdCl,. In a separate note, (3), we have presented data showing that LAF activity of successive LAF preparations tested on M4- LN cells parallels roughly their CPB content. LAF activity could be absorbed on EACA (a CPB substrate analog) columns. Furthermore commercial porcine pancreatic CPB added to PHA-stimulated LN cells mimicked the action of LAF. Several serine proteases, obtained commercially, were tesed and found to exert significant “LAF” activity on M4 and B-cell-depleted LN or spleen cells stimulated with PHA (Table 3). In general they showed little mitogenic activity for T cells when used alone, in agreement with earlier reports (IO- 12). None of these, however, enhanced DNA synthesis to the same extent as LAF (added at 14 hr). At high concentrations several of the serine proteases, trypsin in particular, inhibited both spontaneous and PHA-induced DNA synthesis (data not shown). It should be noted that, while none of this group of enzymes was, properly speaking, a M4, product, catheptic serine proteases have been identified (9, 13) and TABLE Inhibition

of Responses

2

to Mitogens Percentage

EACA concentration CM) 5 x 10-S 10-Z 5 x 10-Z 10-i

PHA I

41 35

II -14 -3 -17 47

PHA III

I

31

55 39

and LAF inhibition

by EACAa

of response

+ LAF II 6 19 8 40

Con A + LAF

Con A III

61

II 89 58 86 100

III

-32

II 35 36 62 91

III

64

(2 Results of three experiments with LN cells, depleted by use of glass beads and nylon wool (I) or glass beads alone (II and III) and stimulated with suboptimal PHA (0.25 or 0.5 @ml) and Con A (0.5 &ml). PHA responses in absence of EACA were, respectively, 2.9, 16.6, and 5.1; PHA + LAF 8.8, 38.7, and 31.3; Con A (in II and III only) 5.6 and 0.9; Con A + LAF 37.7 and 26.8. In all cases LAF was added, with or without EACA, at 14 hr.

LAF AND MACROPHAGE

73

ENZYMES

160

I

2 MINUTES

3

4

5

FIG. 1. Assay of CPB activity in LAF. 0.1 ml undiluted LAF (activity index 2.85 f 0.43) incubated with 2.9 ml hippuryk-arginine in 0.025 A4 Tris-HCl, containing 0.1 M NaCl. Absorbance read at 30-set intervals at 254 nm and 25°C. Symbols: -, hydrolysis at pH 7.65; - - -, at pH 5.5; -.-, at pH 5.5 in presence of 0.06 M dithioerythritol. ---, . , and -’ .-, inhibition by CdCI, lo-” M, EACA 10e2 M, and the zinc chelator phenanthroline 10m4M.

plasminogen activator, which produces plasmin in the presence of plasma, is a well-known product of activated M+ (14). Synergistic action of macrophage supernatants and CPB. Table 4 presents data obtained in two experiments to test similarities and differences between supernatants designated LAF and commercial CPB as secondary signals. The two reagents clearly differed qualitatively; in both experiments LAF appeared most effective when added early while the optimum time for CPB varied but tended to be at 14 hr. When the two were used simultaneously or in succession on mitogen-stimulated cells, they produced a response substantially greater than the sum of responses to either alone, i.e., apparent synergy. [3H]Leucine incorporation was substantially increased by 40-48 hr in the groups showing increased DNA synthesis (data not shown). This result could not be obtained if LAF and CPB simply acted on separate target populations. They must have acted, at least in part, on the same cells, producing an increase comparable to what is obtained by merely raising the concentration of LAF (Fig. 2). By implication, they acted on different target sites. Since they did not induce any DNA synthesis alone or in combination in the absence of mitogen (data not shown), it is clear that all three agents affected the same cells, mitogen providing a first signal and LAF and/or CPB the second. This was particularly striking with Con A which by itself gave no DNA synthesis in M&depleted cells (Table 4). “Binding” of LAF by mitogen-stimulated T cells. It was possible to remove LAF activity from M$I supernatants by incubation with PHA-stimulated LN cells. Such cells, in the continued presence of PHA, reduced the stimulated DNA synthesis attributable to LAF by 40-50%. By interpolation on a titration curve obtained with the same LAF preparation, this reduction was shown in one experiment to

74

KATZ ET AL.

represent removal of more than 70% of the actual LAF activity (Fig. 2). LAF “binding” was found to take place not only with cells in late G1, i.e., at 14-22 hr, but also with cells harvested much earlier after exposure to mitogen or indeed with unstimulated cells. LAF was bound at 4 and 37°C and binding was not affected by the presence of EACA or by PMSF (data not shown). Effect of LAF and CPB on early lymphokine production. LAF and CPB, at concentrations known to serve as-second signals in triggering DNA synthesis, failed to enhance 24-hr lymphotoxin production by whole LNC or purified T cells, stimulated with optimal concentrations of mitogens. Attempts to demonstrate lymphotoxin production by cells stimulated with LAF alone gave equivocal results. DISCUSSION The present study extends our evidence that the so-called LAF (or better TAF (4)) activity of crude macrophage supematants may be mediated by catheptic TABLE “LAF”

3

Activity of Various Proteases Added at Start of Culture Stimulation of 3HTdR incorporation

Enzyme

Concentration” in two experiments b-&ml)

M+- LN cells’

(cpm

x

10e3)*

M+- spleen cellsC

A

A

A

50 l-50

-0.5 6.2

1.6 4.7

-

-

0.1

3.9(4.4)

1.0-5.0 0.1-2.5

-0.1 1.9

3.1 4.2

1.0 0

2.8(3.0)

Chymotrypsin

1.0-5.0 0.5-5.0

-0.3 -0.1

3.8 2.7

0.6 -0.5

6.5 5.8(4.3)

Thrombin

0.1-50 5.0-50

-0.1

Plasmin Trypsin

Pronase LAF”

0.1-50 0.1-2.5 25% (v/v)

-

1.2

-

-

1.2

0.6

-0.14 0.2

21.4 6.9

-0.4

0.4

A

-

1.2(O) - (0) O.O(-0.08)

a Concentrations range in which positive responses were observed. All enzymes were tested at 0.1, 0.5, 1.0, 2.5, 5.0, and 50 pg/ml in two separate experiments. b A, cpm value with enzyme alone minus background. A, cpm value with PHA + enzyme minus values with PHA and enzyme tested separately. Only maximum A and X are given (- means no positive response). Background values varied between 0.5 and 1.2 x lo3 cpm (2.3 for nylon-purified spleen cells). PHA responses (Acpm) in the two experiments were 8.7 and 1.9 x IO3 for M4-LN cells and 17.7 and 8.9 (7.1) x lo3 for M+- spleen cells. c Depletion on glass bead column. B lymphocytes (sIg+) were 1.6% in depleted LN cells and 22.4 and 8.0% in depleted spleen cells. Values in ( ) obtained with nylon wool-purified spleen cells, containing 2.9% B lymphocytes. ri LAF was added at 14 hr.

LAF AND MACROPHAGE

75

ENZYMES

proteases. Our previous paper presented evidence favoring carboxypeptidase B as the principal active constituent of LAF (3). The use of more or less specific inhibitors appeared to rule out significant contributions of acid or thiol proteases such as cathepsin D or C or of cathepsin A to LAF activity. Serine proteases (plasmin, trypsin, chymotrypsin) were shown in the present study to exert a low to moderate degree of potentiating activity on mitogen-stimulated T cells containing negligible percentages (~0.5%) of macrophages or of sIg+ B cells. General protease inhibitors were able to inhibit the LAF activity of macrophage supernatants to a substantial degree. The most active inhibitor tested was EACA, which acts on both CPB and serine esterases like plasmin (8, 9). Nevertheless LAF tended to parallel CPB activity in a series of macrophage supernatants tested, and the use of pancreatic CPB mimicked LAF action almost quantitatively (3). Thrombin and pronase, on the other hand, were essentially inactive, and strong inhibitors of thrombin activity (PMSF, TLCK; these also inhibit macrophage elastase and cathepsin A) failed to inhibit LAF. We cannot rule out conclusively at present the possibility that one or more nonenzymatic constituent(s) of test supernatants possess LAF activity. The results with purified T cells stand in marked contrast to the finding in many laboratories that serine proteases are direct (first signal) stimulants of B cells. Plasmin, trypsin, chymotrypsin, thrombin, pronase, and elastase are all active (lo-12), and stimulation is reduced by more or less specific inhibitors such as EACA, TLCK, a-antitrypsin, and leupeptin and by a variety of substrate analogs (15- 17). Stimulation has been studied with highly purified B cells and spleen cells of athymic (nude) mice and assayed by DNA synthesis and/or clonal expansion of IgM plaque-forming cells. Serine proteases also stimulate fibroblast proliferation TABLE Nonequivalency

4

of LAF and CPB in Potentiating Responses to Mitogens” Reagents added

0 hr: 14 hr:

LAF -

LAF

CPB -

CPB

Both -

Both

LAF CPB

CPB LAF

Cultures with PHA 1” II III

4.5 7.2

2.0 4.0 1.6

0.8 4.4

2.1 3.2 1.4

9.5 8.7

9.4 14.5 4.1

12.8 9.7

2.9 4.7

Cultures with Con A I II III

6.1 8.4

2.4 7.0

-0.3 3.0

-0.6 1.6 1.2

20.2 33.7

3.6 19.5 9.5

12.0 14.6

2.0 10.2

n Values are given for A: cpm x 10m3with mitogen + LAF and/or CPB minus cpm values obtained with each stimulus used alone. Values given in experiment III are for [3H]leucine incorporation at 40-48 hr. * (I) Experiment with glass bead-purified LN cells. Background, 0.33; PHA, 4.7; Con A, 0.48 x lo3 cpm. (II) Experiment with LN cells purified by adherence to plastic. Background 2.4; PHA, 5.9; Con A, 0.59 x lo3 cpm. (III) Glass bead-purified LN cells. Background, 2.4; PHA, 3.5; Con A, 1.6 x IO3 cpm.

76

KATZ ET AL. oo--q

x

-

Acpm

LConA + LAF) (LAF only) X cpm IPHA + LAF) (PHA + absorbed

I d---L-

c----D------

5

LAFI

7.5

IO 12.5

25 28

50

100

FIG. 2. Plot of Acpm obtained with Con A + LAF (0 0) or with LAF alone (0 - - - 0) against the amount of LAF in culture, plotted on a logarithmic scale, is nearly linear. A good linear relation is shown between Acpm, obtained with PHA + LAF, and the log concentration of LAF (0 Cl) in another experiment. Interpolation of hvalue obtained with absorbed LAF sample in this same experiments (*) leads to estimate of residual LAF in sample, in this case 28%.

(18-21); such stimulation has usually been interpreted as release from contact inhibition, a concept difficult to apply to B-lymphocyte triggering. Since cell stimulation in vivo takes place in the presence of serum proteins, the production of plasmin consequent to release of plasminogen activator from participating macrophages or indeed by B cells themselves (14, 22) may dominate the lymphocyte stimulation picture. The possible relation of BAF (B-cell-activating factor) produced by macrophages (23,24) to enzyme systems like those discussed here remains conjectural. Wood and his colleagues (23) report that BAF is distinct from plasminogen activator and its action is not affected by inhibitors such as SBTI and PMSF. The substrate(s) for LAF action in the T or B-cell membranes is (are) unknown, and it is not clear whether there may be a LAF-binding site distinct from the substrate molecule(s). Absorption of LAF activity by activated T cells was demonstrated in the present study and was shown not to be inhibited by EACA, a substrate analog of CPB, or by PMSF7. It was also possible to absorb LAF activity on EACA Sepharose columns and elute it with free EACA (3). (Similar experiments with specific serine protease substrates or substrate analogues were not attempted.) This result suggests that the binding and enzymatic sites may be the same. On the other hand, a number of immunologically significant mediators with enzymatic activity, such as activated complement components and certain lymphokines, have been found to interact with separate binding and enzymatic sites in the cell membrane or to bind to a site in the cell membrane and act enzymatically on a substrate in the fluid phase. As an example, human LIF (leukocyte migration inhibitory factor) binds to a membrane site which appears to involve a-L-fucose and/or N-acetyl-D-galactosamine (25, 26) and is inhibited by serine esterase ’ The possibility that LAF was destroyed rather than bound must be considered, though proteolytic breakdown appeared to be ruled out by the failure of EACA and PMSF influence “binding”.

LAF

AND

MACROPHAGE

ENZYMES

77

inhibitors such as DFP and PMSF (27), yet shows a strong substrate specificity for arginine esters and amides (27,28). The enzymatic target of catheptic CPB in LAF must be C-terminal lysine or arginine peptides (8,9). Serine protease target(s), on the other hand, might be almost any cell membrane protein. Studies of fibroblast stimulation by serine proteases (18-20) have tended to stress the possible role of a special membrane protein variously known as CSP (cell surface protein), fibronectin, LETS (large external transformation sensitive) protein, or simply as “Z,” though recent experiments cast doubt on this simple suggestion (reviews in (21)). In spite of the evidence that a substantial part of LAF action is attributable to catheptic CPB, LAF and pancreatic CPB acted synergistically when added to mitogen-triggered T cells, producing a striking increase in both DNA and late protein synthesis above what was seen with either alone. Thus LAF and pancreatic CPB differ, either in the fact that other proteases (presumably se&e proteases, on the evidence given here) are present in the LAF preparation in addition to CPB or more probably by virtue of differences in the binding properties of catheptic and pancreatic CPB or in their access to substrate molecules in the target cell membrane. (Another possibility is that CPB prepares or improves receptor sites for enzymatically inactive molecules with LAF activity.) Thus the crucial element in cell stimulation (i.e., provision of a second signal to mitogen-stimulated cells) is probably the number of sites affected. These results are reminiscent of those obtained independently by Moller and Greaves (29,30) several years ago using two or more mitogenic agents simultaneously on a single cell population. The marked synergy observed led to the inference that different binding sites were involved and that “the cell can count.” The present results also emphasize the well-known heterogeneity of the LN T-cell population in that some cells appear capable of responding to PHA alone, others to PHA + LAF, still others to PHA + LAF + CPB, etc. It is noteworthy that the low degree of stimulation produced in T cells by mitogen alone in the absence of macrophages may itself be completely inhibited by such agents as EACA. This result lends credence to the suggestion that mitogen may cause a proteolytic event at or near the cell membrane at the appropriate time, possibly consequent to a discharge of lysosomes (31, 32). Similarly blast transformation of B lymphocytes stimulated with pokeweed mitogen is effectively inhibited by protease inhibitors (16). LAF, in our hands, has never shown itself to be mitogenic for T cells alone, nor has pancreatic CPB (40), the combination of LAF with CPB, nor any of the serine esterases which were tested. These agents provide an effective second signal to cells initially triggered with mitogen or, as suggested in work from other laboratories (33), antigen. This result is in contrast with the ability of agents which split membrane carbohydrates, notably periodate (34, 35) and the combination of neuraminidase and galactose oxidase (35, 36) to trigger T lymphocytes (the latter may act, however, by an indirect pathway) and in contrast, as well, with the ability of the serine proteases as a group to stimulate B lymphocytes. Here another comparison with earlier results obtained with mitogens suggests itself: the well-known ability of PHA and Con A to trigger T cells in soluble form contrasted with their inability to stimulate B cells (to which they bind equally well) unless displayed on a solid carrier such as plastic or Sepharose beads (29, 37). Since we now know that most or all T-cell triggering by PHA and Con A requires macrophage

78

KATZ

ET AL.

participation, specifically as a source of proteases, it is ajustified conjecture that the B cells themselves, when exposed to these agents on Sepharose or plastic, may discharge lysosomal hydrolases and thus trigger themselves. The fact that LAF and CPB raise cGMP in late G, in the prepared T cell and that this rise may determine later DNA synthesis (2)8 must also be juxtaposed with observations suggesting that an early rise in cGMP is produced in B cells by effective mitogens and may determine later DNA synthesis (38). A second group of experiments, in the present study, was directed to the question of timing of the action of LAF (or pancreatic .CPB) in relation to the cell cycle of stimulated T lymphocytes. It seemed clear that LAF is bound by cells in any phase of the cycle after exposure to mitogen or indeed by unstimulated cells. This “binding” is followed by prompt entry of Ca2+ from the ambient medium.s Stimulation of DNA synthesis, on the other hand, takes place only in cells first exposed to mitogen and is associated with a Caz+-dependent rise in cGMP in late Gi, i.e., starting 12- 14 hr after mitogen (1, 2). Indeed LAF is effective if added to stimulated cells only after 14 hr, as in many of the present experiments. This finding is in accord with results obtained in other laboratories showing that triggering of DNA synthesis by mitogen (39, 40) or antigen (41) requires two successive stimulatory signals and that the second of these, starting at 15- 18 hr, may require or be mimicked by the action of macrophage products (41, 42). ACKNOWLEDGMENTS We thank Dr. John W. Fenton II for the purified thrombin. It is a particular pleasure to acknowledge the enthusiastic and knowledgeable help of Carol Lanza in all technical aspects of this study.

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