Ability of dialysates containing transfer factor to induce lymphocyte activating factor by human mononuclear cells

CELLULAR

IMMUNOLOGY

45,133-141

(1979)

Ability of Dialysates Containing Transfer Lymphocyte Activating Factor by Human ATSUSHI

TOGAWA,

JOOST J. OPPENHEIM,’

Factor to Induce Mononuclear Cells

AND CHARLES

H. KIRKPATRICK

Laboratory of Microbiology and Immunology, National Institute of Dental Research, Laboratory of Clinical Investigation, National Institute of Allergy and Infectious Diseases, National Institute of Health, Bethesda, Maryland 20014 Received

September

and

25, 1978

Dialysates of human leukocyte lysates containing transfer factor (TF,) stimulated human mononuclear cells (MNL) to produce lymphocyte activating factor (LAF). Both unfractionated and adherent MNL cultures were stimulated by TF, to produce a factor which was mitogenic for murine thymocytes and had the biochemical characteristics of LAF as determined by Bio-Gel P-100, DEAE cellulose, and hydroxylapatite chromatography. Fractionation of TF, on Sephadex G-25 showed that the specific transfer factor activity of converting in viva skin tests was present in the major uv-absorbing peak, whereas the substance(s) that induced LAF activity was present in a number of the other fractions. Therefore, the capacity of TF, to induce monocytes to produce LAF is not a measure of classical transfer factor activity. However, this effect of TF, may instead participate in the nonspecific immunoenhancing effects of TF,.

INTRODUCTION Although dialysates of leukocyte lysates containing transfer factor activity (TF,)’ are immunologically active in converting the skin test reactions of unsensitized donors (1, 2), their biochemical composition and mechanism of action are still unknown. TF, has been reported to increase the levels of intracellular guanosine cyclic monophosphoric acid (cGMP) (3) which may modulate a number of immune responses (4). It has also been reported that cGMP itself or agents that increase cGMP in cells can stimulate monocytes to produce lymphocyte activating factor (LAF) (5), a substance which is mitogenic for mouse thymocytes (6, 7). In an effort to elucidate the mechanism of action of TF,, we have therefore tested the in vitro effect of TFd on LAF production by human mononuclear cells. TF, was found to be a potent stimulant of LAF production, although it did not have direct mitogenic activity. The resultant activity was virtually identical in its biochemical characteristics (molecular weight and behavior on DEAE cellulose and hydroxylapatite) to LAF produced in response to other stimulants (8). ’ Send reprint requests to Dr. Joost J. Oppenheim, Bldg, 30, Room 322, National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland 20014. p Abbreviations used: TFd, dialysates of human leukocyte lysates containing transfer factor; LAF. lymphocyte activating factor; MNL, human mononuclear cells; uv, ultraviolet light; cGMP, guanosine cyclic monosphosphoric acid; PMA, phorbol myristic acetate. 133 0008-8749/79/070133-09$02.00/O Copyright All rights

0 1979 by Academic Press, Inc. of reproduction in any form reserved.

134

TOGAWA,

OPPENHEIM,

AND

KIRKPATRICK

We have also studied the possible relationship between the induction of LAF and the specific skin converting effects of transfer factor in an effort to establish whether in vitro LAF production might provide a simple assay for in vivo transfer factor activity. MATERIALS

AND METHODS

Preparation of human leukocyte subpopulations. Peripheral blood mononuclear leukocytes (MNL) were obtained by centrifugation of heparinized whole blood through Ficoll-Hypaque gradients according to the method of Boyum (9). These unfractionated MNL usually consisted of 65-85% lymphocytes and 15-35% monocytes as determined from their morphology and phagocytic capacity. To prepare adherent cells, a suspension of 1 x 107/ml MNL was incubated at 37°C for 2 to 4 hr in petri dishes and the supernatants containing nonadherent cells were decanted. The resultant adherent MNL were washed three times and usually consisted of 75-85% phagocytic monocytes and 15-25% lymphocytes. In some experiments, the MNL were passed through nylon wool columns according to the method of Julius et al. (10) to obtain the nonadherent T cell-enriched subpopulation of MNL. These nonadherent cells were usually contaminated with 2-5% phagocytic monocytes. TFB preparation. TF, was prepared as previously described (3). Unfractionated MNL were disrupted by multiple cycles of freezing and thawing and then dialyzed against distilled water. The dialysate was lyophilized and reconstituted with either distilled water or phosphate-buffered saline so that the extract from 3 x IO8 cells was contained in each milliliter. An identical preparation of nonlymphoid cells was made by lysing, dialyzing, lyophilizing, and reconstituting WI-38 cells (HEM Research, Inc., Rockville, Md.) to a concentration of 3 X lo* cell equivalents/ml. Preparation of LAF-containing supernatants. For the production of LAF, unfractionated MNL (4 x 106/ml), adherent MNL (obtained from 1 x 10’ MNLl ml), or nonadherent T cell-enriched subpopulations (3.5 x 10Vml) were cultured with a 1: 10 dilution of TFd in RPMI-1640 medium containing 2% heat-inactivated human serum. After 48 hr of culture, the culture fluids were harvested by centrifugation (4OOg, 10 min). Preliminary studies indicated that 1: 10 dilutions of TF, were optimal in inducing LAF production and that 48 hr of incubation resulted in greater activity than 24 or 72 hr. Phorbol myristic acetate (PMA), a potent inducer of LAF production (17), was used as a positive control. Biochemical characterization of LAF activity and TFd. Supernatants containing LAF activity were concentrated lo- to 20-fold by Amicon ultrafiltration and applied to a 2.5 x 95-cm Bio-Gel P-100 (Bio-Rad Laboratories, Richmond, Calif.) column equilibrated in 0.05 M Tris, pH 7.5,0.1 M NaCl, and antibiotics. Chromatography was performed with reverse flow at a rate of approximately 17/ml/hr. The fractions containing lower molecular weight LAF (11) by bioassay were pooled, concentrated, and applied to a 0.7 x 19-cm DEAE cellulose column equilibrated in 1 mM sodium phosphate, pH 7.5. The column was washed with two column volumes of starting buffer (flow rate = 16 ml/hr) prior to initiation of a linear 1- 150 mM NaCl gradient (12). Peaks 2 and 3 from the DEAE cellulose column were pooled, concentrated, and layered on a l-ml hydroxylapatite column (HTP,

DIALYZABLE

TF INDUCES

135

LAF

Bio-Rad, Richmond, Calif.) equilibrated in 1 m&f sodium phosphate, pH 6.8. The column was washed with 3 ml starting buffer prior to initiation of a step gradient of 2 ml each of 10, 20, 40, 80, 160, and 320 mM sodium phosphate, pH 6.8 (12). TF, was also separated on a Sephadex G-25 fine (Pharmacia Fine Chemicals) column equilibrated with 0.02 M ammonium bicarbonate, pH 7.4, as previously described (3). LAF assay. Prior to LAF assay, all samples were supplemented with 5% human serum and dialyzed against RPMI-1640 overnight. LAF activity was assayed as previously described (8). Transferfactor activity in fractions from the Sephadex G-25 gelfiltration column. As previously described (3), fractions of TF,, from the Sephadex G-25 gel filtration column were pooled in two parts. One of them contained a pool of the 300- to 450-ml effluent volume. The other contained all the other fractions. Both pools were lyophilized and then reconstituted to 1 ml/3 x lo8 cells. Two milliliters of a pool was injected subcutaneously into immunologically deficient patients for assessment of transfer factor activity. Intradermal skin tests were applied 5-7 days later and the delayed cutaneous responses measured as previously described (3, 13). RESULTS Demonstration of LAF activity in supernatants cultured with TFd. In preliminary studies the optimal doses of stimulant, kinetics, and cell concentration for producing potent LAF were established (Table 1; data of cell concentration and kinetics not shown). MNL were usually cultured at 4 x 10Vml for 48 hr. Overall, 11 of the 12 batches of TF, tested over the past 2 years consistently stimulated TABLE

1

Comparison of LAF Production by MNL Induced by Dialyzable Extracts of Leukocytes (TF,) and Fibroblasts

Stimulant”

Dose

None PMA TF,

1 b&ml 1:5 dil 1:lO dil* 1:20 dil

Dialysate of extracted WI-38 fibroblasts

1:5 dil 1: 10 dil 1:20 dil

Medium controlr

[3H]TdR incorporation 2 SE by thymocytes incubated with 1:8 dilutions of supernatants of MNL 2,445 79,019 16,517 28,059 1,921

-+ 161 t 1674 2 3271 k 2459 + 342

2,776 t 223 7,979 5 692 1,733 ? 499 927 2 217

a Unfractionated MNL at 4 x lOVm1 were incubated with stimulant for 48 hr, and supematants were tested for mitogenic effect on thymocytes. * A 1:lO dilution in l-ml volume of MNL represents 10% of the TF, extracted from 3 x lOa leukocytes. c Background L3H]TdR incorporation by unstimulated C3H/HeJ thymocytes. The PMA and dialysates by themselves stimulated 5 1000 cpm of [3H]TdR uptake by the thymocytes.

136

TOGAWA,

OPPENHEIM, TABLE

LAF Production by Unfractionated Stimulant

Dose

None TFd Medium control

1: 10 dilb

AND KIRKPATRICK 2

MNL, Adherent MNL, and Nonadherent T Cell-Enriched Unfractionated MNL 350 2 3” 11,868 * 268 642 4c

Adherent MNL 1,611 ? 3.5 8,100 2 934

MNL

Nonadherent T cellenriched subpopulation 91 2 2 186 * 5

a Mean + SE cpm of [3H]TdR incorporated by C3H/HeJ mouse thymocytes incubated for 3 days and pulsed for the final 4 hr with 0.5 &i [3H]TdR. * Added 10% of TF, obtained from 3 x lo* leukocytes/ml. c Thymocytes incubated with stimulants only incorporated < 100 cpm [3H]TdR.

significant LAF production by MNL. The representative experiment in Table 1 shows that a 1:lO dilution (containing 0.3 x lo8 cell equivalents/ml) maximally stimulated LAF production. The TFd stimulated about one-third of the LAF activity produced in response to the optimal dose of the most potent inducer of LAF production, phorbol myristic acetate. To examine the immune cell specificity of this effect, we tested the effect of dialysates of extracts of WI-38 cells, a human fibroblast cell line, on MNL LAF production. These extracts were significantly less effective in inducing LAF activity than TF,. On a cell-equivalent basis, they had only 28% of the activity of TFd. LAF activity in response to the TF, appeared in supernatants of both unfractionated and adherent MNL. The nonadherent T cell-enriched subpopulation of MNL did not demonstrate any LAF activity (Table 2). When mouse thymocytes were incubated with TF, directly, fewer than 100 cpm of [3H]TdR were incorporated, indicating that the TF, itself was not responsible for the mitogenic activity. Biochemical characterization of TF&ducedLAF. The biochemical characteristics of TF,-induced LAF were investigated using Bio-Gel P-100, DEAE cellulose, and hydroxylapatite chromatography. Unfractionated and adherent MNL stimulated with TF, yielded varying amounts of two molecular weight species of LAF activity that eluted off Bio-Gel P-100 in the range of 16,000-22,000 and 60,000-70,000 daltons (Figs. 1 and 2). Both the unfractionated and the adherent MNL cultures showed similar elution profiles of mitogenic activity. The pattern of activity was detected both as a direct thymocyte mitogenic effect (-PHA) and as an enhancing effect of the supernatant on the PHA-induced thymidine incorporation by mouse thymocytes (+PHA). Only the low molecular weight LAF moiety obtained by the Bio-Gel P-100 column was subjected to further biochemical characterizations because we have observed that the high molecular weight LAF consists of low molecular weight LAF aggregated to serum component(s) (8). When the low molecular weight LAF derived from unfractionated MNL was chromatographed on DEAE cellulose, activity appeared in the column void volume and in two other major peaks in a broad salt gradient from 10 to 90 m&f NaCl (Fig. 3). A pool of the second and third peaks obtained from the DEAE cellulose column was combined and in turn eluted at concentrations of 80 and 160 mM sodium phosphate off the hydroxylapatite column (Fig. 4). These Bio-Gel P-100, DEAE, and

DIALYZABLE

I

TF INDUCES

LAF

137

+PHA

-PHA

100

150 EFFLUENT

FIG. 1. Bio-Gel MNL. Fractions PHA. Note that dalton) molecular in augmentation

200

250 VOLUME

300 (ml)

P-100 chromatography of supernatant from cultures of TF,-stimulated unfractionated were assayed for LAF activity at a 1:2 dilution in the presence and absence of 1 &ml TF,, induced production of both high (60,000-70,000 dalton) and low (16,000-22,000 weight LAF activity. Both fractions had activity in the direct assay (lower panel) and of PHA-induced mitogenic activity (upper panel).

hydroxylapatite chromatography elution profiles are characteristic of LAF produced by human MNL in response to a wide variety of stimulants (8). Fractionation of TFd by Sephadex G-25 column. The relationship of skin coverting activity and accumulation of cGMP to components of TF, that induce LAF production was studied by partially purifying the crude TF, using Sephadex G-25. Three different preparations of TFd were used for these experiments, and their efficacy in converting delayed cutaneous reactions in immunodeficient recipients with chronic mucocutaneous candidiasis is shown in Table 3. These TFd preparations were fractionated once (E) or twice and tested several times for their capacity to induce MNL from several donors to produce LAF. The reactions to the chromatography fractions of each preparation of TF, were quite variable. However, the capacity of a given fraction to stimulate LAF production by different donors was reproducible, and representative results are shown in Fig. 5. The

138

TOGAWA,

OPPENHEIM,

AND KIRKPATRICK

+PHA

80 c-eb

60

~

I

PHA

100

I

150

EFFLUENT

200

250

300

VOLUME

(ml)

FIG. 2. Bio-Gel P-100 chromatography of supematant of adherent MNL cultured with TF,. Fractions were assayed for LAF activity at a 1:2 dilution in the presence and absence of 1 wg/rnl PHA.

locations of the fractions that cause accumulation of cGMP in mononuclear cells are indicated, as is the fraction that contains the skin test converting activity (Fig. 5) (3). Unfractionated TF, consistently stimulated both unfractionated and adherent MNL to produce increased LAF activity. In the initial two experiments the Sephadex G-25 fractions that induced LAF activity eluted in the middle and latter

w +PHA

I

PHA

I

0

K 0 Lx ---80 ; j-605 5

10

FRACTION

15

20 NUMBER

25

30

t--40? I -20 j-4

5 z

FIG. 3. DEAE cellulose chromatography of the MNL-derived lower molecular weight LAF eluted from the Bio-Gel P-100. Fractions were assayed for LAF activity at a 1:2 dilution in the presence and absence of 1 &ml PHA.

DIALYZABLE

q

Sample Apphed

SODIUM

TF INDUCES

139

LAF

-PHA

1

10

20

PHOSPHATE

40

80

160

320

CONCENTRATION

(mM)

FIG. 4. Hydroxylapatite chromatography of LAF-containing peaks from DEAE. Fractions were assayed for LAF activity at I:2 dilution in the presence and absence of I &ml PHA.

parts of the column. However, in several later experiments, the induction of LAF activity appeared in different Sephadex G-25 pooled fractions in each experiment (Fig. 5). Therefore, multiple components in the TF,, which are distinct from the skin converting activity, appear to be responsible for inducing MNL LAF production. DISCUSSION TF, activated unfractionated and adherent human MNL to produce a factor which was mitogenic for mouse thymocytes. There is considerable evidence that TABLE

3

Conversion of Delayed Cutaneous Reactions in Immunodeficient

Recipients of TF,

Induration

(diam in cm) Recipient reactivity

Antigens” Candida albicans’

Streptokinase streptodomased

Dose 1: 100 dil

40 units

TF, preparation0

Donor reactivity

Pretreatment

Posttreatment

A-B C-D E

0.9 0.9

0 0

0.8

0

0

0

A-B C-D E

1.0 1.0

0

0.9 1.2 0.9

1.3

1.2 0

1.1

’ The donors of preparations A-B and C-D had positive reactions to tetanus toxoid (1 LFU) which failed to transfer to the recipients. The three donors were all PPD negative, and the recipients did not become PPD positive. b These three TF, preparations were fractionated either once (E) or on two different occasions, and the capacity of the fractions to convert skin tests and induce LAF production is shown in Fig. 5. c Obtained from Hollister-Stier, Spokane, Wash. n Obtained from Lederle Laboratories, Pearl River, N.Y.

140

TOGAWA,

OPPENHEIM,

AND KIRKPATRICK Histamine

“0 M.W.

Ascorbate

I

Markers

I cGMP

Biological

Serotonin I

I

Accumulation

Activities Skin Test %Y&%

12, a’-A 642p! P

*

--

a-B

Control

Whole TFd

0

100 EFFLUENT

2003004005006oa VOLUME

lmll

FIG. 5. LAF-inducing;activity of Sephadex G-25-fractionated TF,. The locations of the molecular weight markers and fractions containing various biological activities are also shown. TF,, from three different donors was fractionated on five separate occasions, and the LAF production to Sephadex G-25 fractions of the three TF, preparations is shown in panels A and B, C and D, and E, respectively.

the supernatant activity produced by TF, stimulated MNL is indeed LAF. (i) The factor is directly mitogenic for mouse thymocytes. (ii) Adherent, but not nonadherent, MNL, when stimulated by TFd, produce the factor. (iii) The biochemical characteristics of the factor, when analyzed by Bio-Gel P-100, DEAE cellulose, and hydroxylapatite chromatography, closely resemble those of LAF obtained by other workers (12, 14- 17). The relationship between three important biological effects of crude TF, was explored. Unfractionated TFd converts delayed skin test reactions, stimulates MNL LAF production, and elevates cGMP levels in monocytes. Investigation of these three activities in TF, reveals that they are dissociable by Sephadex G-25 chromatography. (i) The region from 300- to 450-ml effluent volumes which are equivalent to the major uv absorbing peak contains the component of TF, responsible for conversion of delayed skin reactions (Fig. 5). (ii) The induction of LAF activity was obtained with heterogeneous fractions and appeared in different pools of the Sephadex G-25 fractions in a number of experiments (Fig. 5). (iii) The region of the Sephadex G-25 column fractions which increases the cGMP level ranges from the void volume to the region of maximal uv absorption (Fig. 5). Crude TF, contains serotonin as well as ascorbate (3). High concentrations of both of these agents (e.g., 10V3M serotonin and 10e2 M ascorbate) are known to stimulate

DIALYZABLE

TF INDUCES

LAF

141

leukocyte cGMP production (18, 19) and MNL LAF production (20). However, the fractions of TF, eluting in the region containing serotonin did not increase cGMP and those fractions containing ascorbate and serotonin did not induce LAF activity, probably because the concentration of these agents present in the Sephadex G-25 effluent was too low. These data do not indicate any consistent relationship between the capacity of Sephadex G-25-fractionated TF, to produce LAF, to elevate cGMP, and to convert skin tests. We suspect that the LAF production by MNL in response to Sephadex G,, fractions of TF, is due to heterogeneous substances present in the leukocyte extracts. In fact in one experiment (Fig. 5E), the fractions were more stimulating than the original TFd, suggesting that inhibitory components were present. Furthermore such inhibitors could also be contributing to the varied effects of some of the Sephadex Gz, fractions. There have been several reports that TF, preparations augmented in vitro lymphoproliferative reactions to antigens (21, 22). This activity also did not cochromatograph with skin converting activity (23). It is possible that the components in crude TFd that induce LAF production may contribute to this nonspecific immunoenhancing effect of TF,. ACKNOWLEDGMENTS We appreciate the helpful comments of Drs. Steven B. Mizel and John J. Farrar, the morphological evaluation performed by Dr. Hinnak Northoff, the excellent technical assistance of Mrs. Suanne Dougherty and Ms. Lynn Greenberg, and are grateful to Mrs. Carrie McGahey, Mrs. Dorothy Earman, and Ms. Sybil Ceja for typing the manuscript.

REFERENCES 1. Lawrence, H. S.,ln “The Harvey Lectures,” Series 68, pp. 239-350. Academic Press, New York. 1974. 2. Ascher, M. S., Gottlieb, A. A., and Kirkpatrick, C. H. (Eds.), “Transfer Factor: Basic Properties and Clinical Applications.” Academic Press, New York, 1976. 3. Sandier, J. A., Smith, T. K., Manganiello, V. C., and Kirkpatrick, C. H., 1. Clin. Invest. 56, 1271, 1975. 4. Goldberg, N. P., and Haddox, M. K., Annu. Rev. Biochem. 46, 865, 1977. 5. Diamantstein, T., and Ulmer, A., Immunology 30, 741, 1976. 6. Gery, I., Gershon, R. K., and Waksman, B. H., J. Exp. Med. 136, 218, 1972. 7. Gery, I., and Waksman, B. H., J. Exp. Med. 136, 143, 1972. 8. Togawa, A., Oppenheim, J. J., and Mizel, S. B., J. Immunol., in press. 9. Boyum, A., J. Clin. Lab. Invest. 21, 31, 1968. 10. Julius, M. H., Simpson, E., and Herzenberg, L. A., Eur. .I. fmmunol. 3, 645, 1978. 11. Farrar, J. J., and Koopman, W. J.,ln “Biology of the Lymphokines” (S. Cohen, E. Pick, and J. J. Oppenheim, Eds.). Academic Press, New York, in press. 12. Mizel, S. B., Oppenheim, J. J., and Rosenstreich, D. L., J. Immunol. 120, 1504, 1978. 13. Kirkpatrick, C. H., Rich, R. R., and Smith, T. K., J. Clin. Invest. 51, 2948, 1972. 14. Gery, I., and Handschumacher, R. E., Cell. fmmunol. 11, 162, 1974. 15. Calderon, J., Kiely, J. M., Lefko, J. L., and Unanue, E. R., J. Exp. Med. 142, 151, 1975. 16. Koopman, W. J., Farrar, J. J., and Fuller-Bonar, J., Cell. fmmunol. 35, 92, 1978. 17. Mizel, S. B., Rosenstreich, D. L., and Oppenheim, J. J., Cell. Immunol., 40, 230, 1978. 18. Sandler, J. A., Clyman, R. I., Manganiello, V. C., and Vaughan, M., J. Clin. Invest. 55,43 1, 1975. 19. Sandler, J. A., Gallin, J. I., and Vaughan, M., unpublished observations. 20. Oppenheim, J. J., Mizel, S. B., and Meltzer, M. S.,ln “Biologyofthe Lymphokines” (S. Cohen, E. Pick, and J. J. Oppenheim, Eds.). Academic Press, New York, in press. 21. Ascher, M. S., Schneider, W. J., Valentine, F. T., and Lawrence, H. S., Proc. N&l. Acad. Sri. USA 71, 1178, 1974. 22. Burger, D. R., Vandenbark, A. A., Finke, P., Nolte, J. E., and Vetto, R. M., J. Immunol. 117,782, 1976. 23. Littman, B. H., Hirschman, E. M., and David, J. R., Cell. fmmunol. 28, 158, 1977.