Inhibition of human lymphocyte reactivity by plasma fibronectin in vitro J.C. SCHULTZ AND N.T. SHAHIDI The effect of purified human plasma fibronectin (FN) on the reactivity of human lymphocyte-rich mononuclear cells to mitogens and allogeneic cell interactions was studied. Concentrations of FN from 25 to 100 pg per 250 FL of culture consistently depressed phytohemagglutinin (PHA) responses. To exert an inhibitory effect, FN must be present within 20 hours after the addition of PHA to cells, and, therefore, it appears to interfere with early events in the transformation process. Increasing the concentration of PHA failed to reduce the inhibitory effect of FN, which suggests that the depressed response was not the result of FN-PHA complex formation, which would reduce the amount of mitogen available for stimulation. This possibility was supported by the finding that FN also inhibited the mixed lymphocyte response (MLR), in a reaction that was not dependent on the activity of soluble antigen or mitogen. In contrast, the stimulation of lymphocytes to undergo transformation that is induced by the nonlectin mitogen, sodium periodate, was unaffected by FN. Periodate-treated cells are, however, already stimulated to undergo transformation, prior to their exposure to FN. FN did not interfere with the activity of interleukin-2, nor did it indirectly regulate lymphocyte responses by modifying the production and/or effect of humoral regulato factors released from the adherent accessory cells (macrophages). These studies x o w that FN is a potent immunosuppressive agent in vitro. TRANSFUSION 1990:30:791-798.

FIBRONECTIN (FN) IS A large plasma glycoprotein that exhibits a wide variety of biologic activities and molecular interactions.’.* These properties include cell-to-cell and cell-to-substratum adhesion, morphology, migration, malignant cell transformation, phagocytosis, and hemostasis. Recent studies have also shown that FN interacts with the various components involved in the immune response. FN is synthesized by fibroblasts and macrophages3 and is present on the surfaces of macrophagesY4monocytes,’ and neutrophikG More recently, Klingemann et aL7 reported the presence of FN on normal human peripheral blood B cells but not on OKT3positive T lymphocytes or Leu 11-positive natural killer cells. Cseh et al.* found that approximately 20 percent of normal B cells and 7 percent of the null cells contain surface-bound FN. FN fragments function as chemoattractants for monocytesg and as mitogenic agents for fibroblasts.1° Martin et al.” reported stimulation by plasma FN of human monocyte- or macrophage-derived growth factor production. Purified rat plasma FN has been reported to induce nonspecific transformation of rat lymphoid cells from spleen and lymph nodes in the absence of mitogen or antigenI2 and to depress the blastogenic response of the

lymph node cells to mitogen (phytohemagglutinin [PHA], concanavalin A, and bacterial lipopoly~accharide)’~~’~ and alloantigens in the mixed lymphocyte reaction (MLR).” In contrast, purified human FN has been reported to restore the defective MLR of patients’ peripheral blood lymphocytes after marrow grafting and to augment the MLR of lymphocytes from healthy donors, but it was without effect on PHA- and OKT-3-induced lymphocyte proliferation.16 These studies were, however, performed with a single concentration of FN (25 kg/mL), which is well below physiologic concentrations. In another article in this issue of TRANSFUSION,17 we showed that FNcontaining blood products, plasma, and cryoprecipitate obtained from healthy donors depress PHA-induced lymphocyte transformation and that selective removal of FN results in a marked reduction in the inhibitory capacity of the product. The aim of this article is to present the results of our studies on the effect of various concentrations of purified human plasma FN on the reactivity of normal peripheral blood lymphocytes to lectin (PHA) and nonlectin (periodate) mitogens and alloantigens in the mixed lymphocyte reaction. Materials and Methods

Sodium metaperiodate (NaIO,) was obtained from Fisher Scientific Co. (Fairlawn, NJ) and cell culture medium, anlibiotics, and trypan blue from Grand Island Biological Co. (Grand Island, NY). [Methyl-3H]-thymidine([3H]TdR)(6.7 Ci/mmol) was purchased from New England Nuclear (Boston, MA). Re-

From the Dcpartrncnt of Pcdiatrics, University of Wisconsin, Madison, Wisconsin. Supportcd by Grant CA-05436 from thc National lnstitutcs of Health. Rcccivcd for publication January 2, 1990; revision received April 6, 1990, and acccptcd May 9, 1990.

791

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combinant interleukin-2 (IL-2) was obtained from Cetus (Emelyville, CA). Fetal calf serum (FCS) was obtained from HyClone (Logan, UT) and human plasma FN (Product No. F2006) and PHA (Product No. L-9132) from Sigma Chemical Co. (St. Louis, MO). The FN was dissolved in 0.1 M NaCl containing 0.05 M Tris-HCI, pH 7.5, at a concentration of 1 mg per mL. Using

I%) of 13.5, we determined FN 1cm concentrations by their absorbance at 280 nm. Protein homogeneity was ensured by crossed immunoelectrophoresis and polyacrylamide gel electrophoresis.’8 an absorption coefficient (E

Monoiiuclear cell isolation We obtained peripheral blood from healthy donors and collected it in preservative-free heparin (20 U/mL, final concentration). Mononuclear cells (MNCs) were obtained essentially as described by Boyum.” We carefully harvested the lymphocyte-rich MNCs from the interface and washed them by resuspension in phosphate-buffered saline lacking Ca2+ and Mg2+ (PBS); then we recentrifuged and counted them in a hemocytometer.

Periodate stimulatioii We performed periodate oxidation by incubating the washed lymphocyte-rich MNCs at a concentration of 20 x loh cells per mL in freshly prepared 0.4 mM NaIO, for 30 minutes at 4°C. The reaction was terminated by centrifugation at 400 x g for 10 minutes at 4°C and resuspension first in PBS and then in RPMI-1640. We suspended the cells for culture in RPMI1640 supplemented with penicillin (100 U/mL), streptomycin (100 pg/mL), amphotericin B (0.25 pg/mL), and plasma. We prepared cultures in triplicate in 96-well flat-bottom microtiter plates at a concentration of 5 x lo5 cells per well and adjusted final volumes to 250 pL. After incubation for 48 hours at 37°C in humidified air containing 5 percent CO,, [3H]TdR (2 pCi/ 25pL) was added to each well. After incubating the cells for an additional 6 hours, we collected them from the wells onto glass-fiber filter paper (Reeve Angel, 934AH, Whatman Inc., Clifton, NJ) using a semiautomatic microharvesting device. The filters were washed with 0.15 M NaCI, air-dried, and transferred into vials containing 2 mL of scintillation fluid consisting of 5 g of 2,5-diphenyloxazole and 0.1 g of phenylosazolylphenyloxazolylphenyl per liter of toluene. The cellassociated radioactivity was measured in a liquid scintillation spectrometer with a counting efficiency of 43 percent.

PHA stimulation We suspended washed lymphocyte-rich MNCs for culture in RPMI-1640 supplemented with penicillin (100 U/mL), streptomycin (100pg/mL), and amphotericin B (0.25 pdrnL) and incubated the cultures in 96-well flat-bottom microtiter plates. Each well contained 0.10 mL of cell suspension (2.5 x lo5 cells). Plasma, PHA, and FN were added simultaneously. We adjusted final volumes to 250 p L with RPMI1640 and incubated the cultures for 72 hours. [3H]TdR (2 pCi/ 25pL) was added 10 cell cultures during the final 6 hours of the incubation. Variations on the above three procedures are stated. The cells were collected from the wells onto glass-fiber filters, washed, and assayed for radioactivity as described above.

MLRs MLRs were prepared in 96-well flat-bottomed microtiter plates. The MLR consisted of 50-yL aliquots, each of 0.75 x

TK,\NSFUSION

\‘d30. Nn. 9-1990

lo5 responder peripheral blood MNCs (PBMNCs) and 1 x lo5 of irradiated (1000 rad) stimulator PBMNCs. We added various concentrations of purified FN (19-94 pdculture) to the MLR, supplementing cultures with 10 percent FCS rather than human plasma to minimize the amount of additional FN added. The FN content of FCS is substantially lower (4-6 pg/mL) than that of human plasma (260-380 pg/mL). We made adjustments for the amount of NaCl and Tris-HCI added to cultures with increasing volumes of FN solution by adding 0. l M NaCl in 0.05 M Tris-HCI, pH 7.5, to control cultures (minus FN) for each concentration of FN tested.Final volumes were adjusted to 0.25 mL with RPMI-1640. We assayed replicate cultures for tritiated thymidine incorporation after 6 days of incubation and determined the regulatory effects of FN by comparing the MLR response in the absence of FN. We assessed lymphocyte proliferative activity by measuring the incorporation of [3H]TdR (2 pCi/25 pL) added to cultures 7 hours prior to collection of cells. Cells were harvested and [3H]TdR levels measured as described above.

Blast cell assay for IL-2 activity To prepare the blast cells, we suspended PBMNCs (1 x loR)in 1 mL of RPMI-1640 supplemented with 5 percent FCS and PHA (8 pg) and cultured them for 72 hours at 37°C in humidified air containing 5 percent CO,. The stimulated cells (T lymphoblasts) were harvested by centrifugation (400 x g for 10 minutes) and washed with RPMI-1640. Stimulation of DNA synthesis was determined on the 3-dayold IL-2-dependent T lymphoblasts described above by nieasurement of [3H]TdR uptake. We suspended washed cells (4 x lo4) in RPMI-1640 supplemented with 5 U of recombinant IL-2 or in RPMI-1640 for controls when IL-2 was omitted and various concentrations of FN (25, 50, and 75 pg) in a final volume of 150 pL. The cultures were incubated at 37°C in humidified air containing 5 percent CO,. [3H]TdR (0.5 pCi; 6.7 mCi/mmol), added in a 25 pL volume of RPMI-1640, was present during the final 8 hours of the 48-hour incubation. We harvested the cells and assayed them for radioactivity as described above.

Preparation of FN-coated microtiter plates A 96-well flat-bottomed microtiter plate (Falcon 3040) was treated with 4 pg of FN per cm2 as follows. We added a 0.1mL aliquot of a solution of 20 pg of FN per mL of RPMI1640 to each well. For the control wells, RPMI-1640 was added when FN was omitted. The plate remained at room temperature for 45 minutes to permit binding of the FN to the growth surface of the wells. The FN solution was removed with a Pasteur pipet. We performed PHA stimulation of lymphocytes in the presence of 5 percent pl;ism;i, as described above.

Assay of purified FN for mitogenic activity Experiment 1. Fifty pg of FN was delivered to cultures in a 50-pL volume of 0.05 M Tris-HCI, pH 7.5, containing 0.1 M NaCI. Cultures were supplemented with 5 percent FCS and volumes adjusted to 250 pL with RPMI-1640. Adjustments to control cultures (minus FN) were made by the addition of 50 p L of 0.05 M Tris-HCI, pH 7.5, containing 0.1 M NaCI. [3H]TdR (1.25 pCi added in a volume of 25 pL of PBS) was present during the final 9 hours of the 100-hour incubation period. Experiment 2. PBMNCs were suspended for culture at a concentration of 2.5 x IF per mL of RPMI-1640. One hundred-

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FN INHlBlTlON OF LYMPHOCYTE TRANSFORMATION

pL aliquots were transferred to titer wells. Purified human FN (200 pg) was dissolved in 1 mL of 0.05 M Tris-HCI, pH 7.5, containing 0.1 M NaCI, and aliquots were added to the wells. Cultures were supplemented with 10 percent FCS and volumes adjusted to 250 p,L with RPMI-1640 after correcting for NaCl and Tris-HCI buffer as described in Experiment 1. During the final 8 hours of the 72-hour incubation, 0.5 pCi of ['HITdR was present.

Results Effect of FN on PHA-induced lymphocyte transformation The effect of purified FN on PHA-induced transformation of human peripheral blood lymphocytes was measured. AS seen in Table 1, increasing concentrations of FN depressed PHA-induced mitogenesis in a concentration-dependent manner. It should be noted that the inhibitory concentrations of FN fall below and within the average concentration range of FN found in plasma. Normal plasma FN levels range from 260 to 380 pg per mL.2n Effective inhibitory concentrations of FN shown in this report range from 25 to 100 pg per culture (100400 pg/mL). Concentrations of FN greater than 100 pg per culture were not tested.

Effect of time of addition of FN on inhibition of PHA stimiilatioii To determine whether FN was inhibiting an early or late event during the transformation of lymphocytes, we added 50

Table 1 . Effect of fibronectin (FN) on phytohemagglutinin (PHA)-induced lymphocyte transformation Concentration of FN (Lglculture)

[3H]TdRincorporation (mean countshin +. SD Percentage of of 3 determinations) inhibition

Experiment 1 0 5 10 20 25 30 40 50 60

Without PHA or FN

107,081 +. 13,244 91,139 f 2246 94,572 f 7382 88,107 f 8363 71,061 2 8546 69,232f 1976 65,211 f 3649 61,178 f 3949 55,885 f 1954 2249 f 443

0 15 12 18 34 35 39 43 49

Experiment 2 0 2 5 10 20 40 60 80 100

34,422 f 31,447 f 33,884 f 32,066 f 33,550 f 24,284 f 18,952 -c 14,457f 11,704 f

2221 1449 215 1075 5138 1712 744 139 785

0 9 2 7 3 30 45 58 66

'Experiments 1 and 2 were performed with different preparations of FN and donor cells. Data shown are representative of six separate experiments. A one-factor analysis of variance suggests that the presence of FN does have a significant (p0.50). Optimal concentrations of PHA, as determined in preliminary experiments, range between 1.5 and 2.5 pg per culture (6-10 pg/mL).

Effect of FN on periodate-induced lymphocyte transformation PBMNCs induced to undergo transformation by periodate treatment were cultured in the presence of various concentrations of purified FN (12-62 pg/culture). As seen in Table 2, periodate-induced stimulation was unaffected by the presence of concentrations of FN that inhibited the lectin (PHA) mitogen-induced response and MLR. Sodium periodate was selected as a stimulus for transformation, because this nonlectin mitogen, unlike PHA, need be present for only a short period of time (minutes) to commit the responding cells to increased DNA synthesis. Commitment is the point at which a cell becomes stimulated and no longer depends upon the presence of the mitogenic agent. The mitogen can then be easily and completely removed from the stimulated cells before they are exposed to FN,23 and therefore the effects of FN on already stimulated cells can be measured in the absence of any contact between the mitogen and the FN.

Effect of FN on the MLR T h e potential immunoregulatory effect of purified FN on cell-to-cell interactions in the MLR was studied. We added various concentrations of FN (19-94 pglculture) at the start of the incubation period, and they remained throughout incubation. Table 3 illustrates the potent inhibitory activity of FN on the MLR at the end of 6 days of incubation. Concentrations of FN between 19 and 94 pg per culture evoked significant inhibition of the MLR. A two-factor analysis of variance suggests that the incorporation of ['HITdR in the M L R does depend on FN, with an overall significance for the effect of FN at p 0.20); however, there does appear to be sig-

794

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SCHULTZ AND SHAHIDI Table 2.

Concentration of FN (pglculture)

0 12 22 27 32 42 54 62

Vnl. 30, No. 9-1990

Effect of fibronectin (FN) on sodium periodate-induced lymphocyfe transformation*

NalO, stimulation [3H]TdR incorporationt (countslmin f SD) 9492 9228 9558 7296 8024 9860 8717 8751

f 1453

0 3

f 1735 f 2739 f 650 ?

0

785

1092

Percentage of inhibition

25,516 NT* NT NT NT 18,745 15,110 10,044

14 16 0 8 8

f 3965 f 998 ?

PHA stimulation [3H]TdRincorporationt (countshin f SD)

Percentage of inhibition

0

25 41 61

'Phytohemagglutinin (PHA)- and sodium metaperiodate (Nal0,)-treated peripheralblood mononuclear cells were cultured simultaneously and under identical Conditions. Adjustments were made for the increased amounts of NaCl and Tris-HCI added to cultures with increasing volumes of the FN solution by the addition of 0.1 M NaCl in 0.05 M Tris-HCI, pH 7.5. All cultures contained peripheral blood plasma at a final concentration of 5 percent. The amount of FN present in the plasma would represent an increase of only 3.5 pg per culture from the values shown. A one-factor analysis of variance supports the hypothesis that the presence of FN does not have a significant effect (p > 0.75) on perlodate-Induced lymphocyte transformation. The periodate stimulation data shown are representative of two separate experiments. The PHA stimulation data shown are representativeof six separate experiments. tMean f SD of three determinations. *Not tested.

Table 3. Effect of purified fibronectin (FN) on the mixed lymphocyte reaction

Culture conditions

[3H]TdR Percentage incorporation' of (countslmin f SD) inhibition

Buffer alone 50,718 f 10,712 Buffer plus FN (19 pglculture) 34,527 f 17,314

32

Buffer alone 45,469 f 15,754 Buffer plus FN (38 pglculture) 18,611 f 1492

59

Buffer alone 29,162 rt Buffer plus FN (75 pg/culture) 12,024 f

7282 3296

59

Buffer alone 23,185 f Buffer plus FN (94 pglculture) 6307 ?

6962 1682

73

Table 4. Effect of fibronectin (FN) on the activity of recombinant interleukin-2 (IL-2)

Culture conditions Blast cells alone Blast cells plus IL-2t 11-2 FN (25 pg/culture; 166 pglml) 11-2 FN (50 pLglculture; 333 pglmL) 11-2 + FN (75 pglculture; 500 d m L )

+ +

[3M]TdR incorporation' (countshin f SD) 234

?

44

41,636 f 5661 44,057 f 7858

1 OO%* 106%

36,792 f 491 3

88%

27,712 f 8035

66%

'Mean f SD of three determinations. Because the variance In the error term from repeat runs was observed to increase with FN concentration, the (3H]TdRresponse was expressed in logarithmic units. Accordingly, significance values were also based on the log of the response. In any case, the change in significance due to response transformation is minimal.

'Mean ? SD of three determinations. Data shown are representative of three separate experiments. tlL-2 was present at a concentration of 5 U per culture (33 U/ mL) in all cultures. *The percentage of PHJTdRincorporation,taking as 100 percent that obtained in the presence of IL-2 alone.

nificant (p p > 0.10). Paired comparisons of the control versus each concentration level of FN support the hypothesis that, in concentrations found in human plasma (166 and 333 p/mL), FN does not affect the activity of IL-2 (p >0.40). However, the IL-2 activity at 500 p g of FN was found to be significantly different (p ~ 0 . 0 5 )from that of the control. A 34-percent reduction in the IL-2-dependent [3H]TdR uptake occurred in cultures containing 500 p.g of FN per m L of culture, a concentration well above that found in plasma. These results suggest that FN i s not exerting its inhibitory effect on lymphocyte reactivity by interfering with lymphokine (IL-2) activity. We have not, however, measured the effect of FN on IL-2 production in mitogen-stimulated or MLR cultures.

Effect of FN on IL-2 activity The following experiment was performed to determine if the inhibitory effect of FN on lymphocyte reactivity was the result of interference with the lymphocyte stimulatory factor, IL-2, which i s produced and utilized during lymphocyte transformation. We cultured 3-day-old PHA-stimulated lymphoblasts in the presence of human recombinant IL-2 and increasing concentrations of FN. Cells cultured in the absence of IL-2 served as a control. The T lymphoblasts require the presence of IL-2 for growth. As seen in Table 4, tritiated thymidine incorporation i s stimulated by IL-2. Concentrations of FN (166 and 333 p g h L of culture) that were depressive to the mitogen response and MLR had little or no inhibitory effect on the activity of IL-2. A one-factor analysis of variance suggests that FN concentration may have some effect on the activity

Preparation of FN rnonocyte-conditioned riiedin We investigated the possibility that FN stimulated the plastic-adherent accessory cells (macrophages) present in the lymphocyte-rich MNC preparation to synthesize and release soluble inhibitors of PHA-induced lymphocyte transformation. We plated

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FN INHIBITION O F LYMPHOCYTE TRANSFORMATION

2 mL of PBMNC suspension (4 x loh cells in RPMI-1640) into each of three 5 x 14-mm flat-bottomed plastic culture wells (total growth surface of the wells, 9.6 cm2) of a 6-well plate (Falcon 3046). The remaining wells received 2 mL of RPMI-1640 each (no cells added). After 1 hour at 37°C in humidified air containing 5 percent CO,, the growth medium was carefully removed with a Pasteur pipette. We removed nonadherent cells from the wells by vigorous washing of the wells three times with RPMI-1640. RPMI-1640 was immediately added to all wells. We added FN (400 p,g) and PHA (16 Fg), either alone or in combination, to both control (minus cells) wells and plastic-adherent cell-containing wells and adjusted final volumes to 2 mL with RPMI-1640. After 24 hours of culture at 37°C in humidified air containing 5 percent CO,, supernatants were collected by centrifugation at 500 x g for 10 minutes. Supernatants from control wells were treated identically. We added various concentrations of the adherent cellconditioned media and the media from control wells (minus cells) to fresh PBMNCs suspended in RPMI-1640 supplemented with 5 percent FCS and PHA (8 pg/mL) (Table 5). The incorporation of [3H]TdR by cells cultured in the media conditioned by adherent cells exposed to FN, alone or in combination with PHA, and also by cells stimulated with PHA in the absence of FN did not differ from that obtained with control media (p>0.20), as determined by a two-factor ANOVA for each of the three different conditioned media. These results were consistent for all media concentrations (10-40%) tested, which suggests that the inhibitory effect evoked by FN may involve interaction with the accessory cell (macrophage/monocyte) but not the enhanced production of soluble inhibitors released as a result of the FN-adherent cell interaction.

Assay of purified FN for mitogenic activity The incubation of purified FN (10-50 pg/culture) with increasing numbers of PBMNCs (1 x 105 to 9 x lO'/culture) in the absence of exogenous stimuli, such as mitogen, did not result in increased levels of [3H]TdR incorporation as compared to control (minus FN) cultures, which indicates a lack of mitogenic activity (Table 6). Doses of FN from 0 to 15 p,g per 250 pL of culture also did not affect the incorporation of [3H]TdR. With cell concentration as a blocking factor, a IWO-

factor ANOVA supported the hypothesis that the effect of FN as a mitogenic agent is not significant (p>O.15).

Effect of plastic-adherent FN on PHA-induced lymphocyte transformation Plasma FN has a strong affinity for cell culture plastics. It is not known whether soluble or plastic-adherent FN, or both forms, can inhibit mitogen responses. To test the effect of plastic-adherent FN on PHA-induced lymphocyte transformation, we coated the 96-well microtiter plate with 4 pg of FN per cm2 of growth surface of the well; we immediately added cell suspensions and supplemented them with PHA (10 & n L culture) and 5 percent plasma. At the end of the 72-hour incubation period, ['HITdR incorporation by cells cultured in FN-coated wells (170,870 & 18,879) did not differ significantly from that by cells cultured in uncoated (minus FN) wells (165,843 ? 18,879), which indicates that the plastic-adherent FN was without effect on the PHA response.

Discussion In this article, it is shown that human plasma FN is capable of depressing PHA-induced lymphocyte transformation, as determined by a reduction in the incorporation of [jH]TdR into DNA. This immunoregulatory effect results from the activity of purified preparations of FN. Protein homogeneity was assured by disc gel electrophoresis, gradient polyacrylamide gel electrophoresis, and crossed immunoelectrophoresis. The inhibitoIy activity of FN was dose-dependent (Table 1) and was not the result of a cytotoxic effect on responding cells (not shown). For optimal expression of inhibition, it is necessary that F N be present during the early stages of lymphocyte activation b y PHA. Maximum inhibition (67%) occurred when FN w a s added to cultures within 1 hour after the addition of PHA; the addition of FN within 20 hours after that of PHA resulted in a significant inhibition (>50%) (Fig. 1).

Table 5. Effect of fN*-adherent cell-conditioned media on PHAt-induced lymphocyte transformation

Culture conditions PHA alone PHA plus FN (50 pg/culture) PHA plus FN (100 pglculture) PHA plus FN-Ad+cell-CM§ PHA plus PHA-Ad+cell-CM PHA plus PHA and FN-Ad'cell-CM

rH]TdR incorporation* lcountslrnin 2 SDI

Control media ~

288,616 t 31,234 107,366 t 12,950 70,133 f 3679 (10%)11 241,009 f 29,521 (20%) 233,046 f 21,193 (40%) 168,731 f 17,429 (10%) 310,666 t 15,880 (20%) 316,691 2 12,048 (40%) 331,269 t 8150 (10%) 275,358 2 10,260 (209'0) 248,713 -t 3795 (40%) 206,395 f 9741

~~~

(10%)11 204,585 (20%) 242,975 (40%) 190,210 (10%) 313,616 (20%) 308,466 (40%) 313,108 (10%) 277,000 (20%) 249,445 (40%) 198,451

f 27,060 2 10,092 f 4786

t 12,865 2 18,987 2 7294 f 5555 f 8190 f 25,677

'Fibronectin. tPhytohemagglutinln. *Mean f S D of three determinations. Data shown are representative of four separate similar experiments. §Ad+cell-CM represents RPMI-1640 culture medium conditioned by plastic-adherent peripheral blood mononuclear cells cultured in the presence of FN and PHA, alone and in combination as indicated. IlValues in parentheses represent conditioned media concentrations tested.

796

SCHULTZ A N D SHAHIDI

Table 6. Assay of purified fibronectin (FN) for mitogenic activity

Experiment number

Peripheral blood mononuclear cells (per 250 pL culture)

1

FN concentration (pg/250pL of culture)

[3H]TdR incorporation* (countshin f SD)

x 105

0 308 ? 137 0 1 1 04 ? 349 0 3639 -+ 708 0 5957 f 1234 0 8585 f 2537 1 x 106 50 444 f 246 3 x 105 50 1384 ? 778 5 x 105 50 3529 -t 426 7 x 105 50 6591 f 513 9 x 105 50 6853 f 578 0 6686 2 802 2 2.5 x lo6 2.5 x 10' 10 8027 2 1927 2.5 x 10' 15 7672 2 1438 'Mean 2 SD of three determinations. Data represent two separate experiments. 1 3 5 7 9

70c

* I

I

0

I

I

10

x 105 x 105 x 105 x 105

I

1

I

I

I

I

I

I

I

1

50 60 30 40 20 Time of Addition of FN (hrs)

I

I

70

FIG. 1. Effect of dclaying the addition of purified FN to PHAstimulated peripheral blood mononuclear cells. Aliquots (0.1 mL) of cell suspension (2.5 x los cells in RPMI-1640) were addcd to microtifer wells. Cultures werc supplemented with 10 pcrccnt fctal calf serum. FN (50 pL; 50 pg) was addcd at thc start of the culturing of the PHA-stimulated cells (timc zero) and at the times indicated. All volumcs wcre adjusted to a final volume of 200 pL with RPMI-1G40 before thc addition of FN. Cells not exposed to FN incorporated 331,669 counts per minute of YHlTdR.

The finding that FN added late to the culture system fails to depress significantly the response of cells may be based on culture conditions that differ from those in effect when FN, added at the initiation of incubation, evokes a potent inhibitory effect. FN has a strong affinity for cell culture plastics, therefore the amount of FN in

TKhNSFUSION Vol. 30. No. 9-1980

soluble or plastic-adherent form may vary with the length of time the FN is present in culture. In a preliminary experiment, lymphocyte transformation induced by PHA, carried out in titer wells already coated with FN, was found not to differ from that obtained in untreated wells, which suggests that the soluble form of FN is inhibitory. The amount of FN bound to the titer well reaction surface after the addition of soluble FN to the PHA-stimulated cell cultures was not determined. Therefore, it is possible that the amount of FN coated on the wells may be noninhibitory and may not be comparable to the amount present when FN is added directly to the cell cultures in a soluble form. That the presence of FN prior to cell activation is required to evoke an inhibitory effect is not unique to FN but is, rather, similar to the case with other normal immunosuppressive plasma protein^.^^-^^ It has been suggested that exogenously added FN is bound and subjected to endocytosis by cultured macrophages.3 Preliminary studies by Lause et aI.l5 showed, at the electron microscopic level, the uptake of FN by peritoneal exudate cells and the macrophage cell line P388D1. It has been suggested that the inhibition of PHA- and concanavalin A-induced stimulation of human peripheral blood lymphocytes by certain other purified normal plasma glycoproteins simply reflects an interaction between mitogen and soluble inhibitor that reduces the amount of mitogen available to the cells for ~ t i m u l a t i o n .This ~~ mechanism of action was initially supported by our findings, because concentrations of FN that depressed the PHA response were without effect on lymphocyte transformation induced by the nonlectin mitogen sodium periodate (Table 2). However, increases in the concentration of PHA failed to reduce the relative percentage of inhibition, which indicated that the inhibition did not appear to be the result of FN's complexing with PHA. This was further supported by our finding that purified plasma FN is a potent inhibitor of the cell-to-cell interactions in the MLR (Table 3), an assay not dependent on the activity of soluble antigen or mitogen. Although a single reportl6 indicated that exogepous human FN (25 p&mL) enhances the MLR of human PBMNCs, we have been unable to demonstrate any stimulatory effect at any of the concentrations tested (19-94 p,g FN/culture). Studies by others2' showed that the mechanism of periodate stimulation is an indirect one that is analogous to an MLR. An indirect mechanism can be described as the reaction of a mitogenic agent with one cell that causes a second cell to undergo transformation. The inability of purified FN to depress periodate-induced lymphocyte transformation may be due to the oxidation and subsequent alteration of FN-binding sites on participating cells (lymphocytes and/or macrophages) or to the changing of the relationship or arrangement of other cell-surface glycoproteins so as to prevent positive interactions. Period-

TRANSFUSION 1990-Val. 30, No. 9

FN INHIBITION OF LYMPHOCYTE TRANSFORMATION

ate is an oxidative mitogen that under the proper conditions is relatively specific for vicinal hydroxyl groups of carb o h y d r a t e ~ .The ~ ~ oxidation of sialic acid residues of lymphocyte cell surface glycoproteins triggers the transformation. Interaction between the periodate-treated lymphocytes and plastic-adherent cells (macrophages) is required for m i t o g e n e s i ~ .Syngeneic ~~ lymphocytes recognize these changes as foreign and react against them, as in an MLR. Another possibility is that FN is without effect on cells already stimulated to undergo transformation, as is the situation with the periodate-treated lymphocytes. The lymphocyte-rich MNCs were treated with periodate under conditions (30 minutes at 4°C) that produce a *maximal response. The periodate was completely removed from the oxidized cells prior to their exposure to FN, which allowed the effects of FN on already stimulated cells to be measured directly in the absence of any contact between the mitogen and the FN. This concept that FN may be involved (directly or indirectly) in the series of events required for the initiation of lymphocyte transformation is further supported by our findings that FN must be present during the first hours of lymphocyte activation by PHA or alloantigens in the MLR. The addition of FN 20 to 24 hours after the stimulation of cells is without effect on the stimulated cells. FN was shown to be without effect on the activity of IL-2 (Table 4). This was anticipated, as FN was without effect on periodate-induced lymphocyte transformation. The production of IL-2 by T cells and its utilization are requirements for lymphocyte transformation in both lectin (PHA) and nonlectin mitogen (including periodate) stimulation and the MLR. FN appears to be exerting its inhibitory effort on lymphocyte transformation prior to cell activation; IL-2 production and use occur later in the series of events leading to cell transformation and proliferation. We have demonstrated that purified plasma FN interferes with early events in the reactivity of lymphocytes to mitogens and alloantigens. PHA and periodate are Tcell mitogens, and, because FN does not seem to bind to purified human T cell^,^.^ it may be exerting its action on the accessory cell. As mentioned earlier, macrophages and monocytes have membrane receptors for FN. Purified plasma FN has been shown to activate monocytes and macrophages to produce a growth factor for fibroblast and aortic smooth muscle cells1' and to enhance the secretory activity of inflammatory rat peritoneal exudate macrophages and the release of interleukinl.30The nondialyzed conditioned media obtained from cocultures of macrophages and FN in the rat system and, in our studies, from human plastic-adherent MNCs failed to influence PHA responses of cells. Thus, the inhibitory effect exerted by FN does not appear to occur with the enhanced production of soluble inhibitors released as a result of adherent cell-FN interaction. The mechanism

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of inhibition of lymphocyte proliferation by FN may result from the interference of recognition sites on the macrophage membrane. The effect of purified rat plasma FN on rat lymphocyte reactivity, described by Lause and c o - w ~ r k e r s , l ~has -'~ certain features in common with the results obtained in our studies. FN depresses both mitogen-induced lymphocyte transformation and MLR at physiologic concentrations. The inhibition of PHA responses by FN cannot be overcome by increasing the PHA concentrations. The time of addition of FN to cultures is critical: both systems require early addition of FN. The inhibitory effect evoked by FN appears to be mediated at the accessory cell but not by the enhanced production of soluble inhibitors released as a result of the FN-macrophage interaction. We were, however, unable to demonstrate the mitogenic effect of FN in the absence of exogenous stimuli and in the presence of high concentrations of lymphoid cells, as described by Lause et a1.lZ Evidence has been obtained that demonstrates that FN fragments can be produced by proteolytic cleavage that display functional activities not shown by intact FN. For example, fragments have been isolated from FN preparations following either cathepsin D and G or plasmin digestion that demonstrate an ability to influence cellular growth; these include heparin-binding fragments of FN, which are mitogenic for hamster fibroblasts,1° and fragments that are inhibitors of endothelial cell growth.3' Spontaneous fragmentation of FN preparations may account for the mitogenic activity, especially because electrophoretic analysis of several rat plasma FN preparations revealed the presence of FN f r a g m e n t ~ . ' ~ JKlingemann ~ et a1.16 described an in vitro stimulatory effect of FN on the proliferation of lymphocytes from patients with graftversus-host disease after marrow grafting and from healthy donors in mixed lymphocyte cultures (MLC) at a concentration of 25 pg of FN per mL of culture, the only concentration described. The FN failed, however, to enhance PHA-induced mitogenesis and did not show mitogenic activity when tested in the absence of exogenous stimuli (mitogen), results that are in agreement with our findings. It is not known why, in the studies of Klingemann et a1.,16 exogenous FN enhanced the MLR already at a below-physiologic concentration of 25 k g per mL or why exogenous FN does not influence PHA- and OKT-3-induced lymphocyte proliferation. The addition of FN or FN fragments to titer wells coated with CD3 antibody and containing highly purified CD4 cells induced the activation of CD4 cells, whereas neither FN (or fragments) or CD3 antibody alone can do it." FN was effective at concentrations of 0.4 to 50 k g per mL of culture, with optimal response obtained with concentrations as low as 10 k g per mL. The effect of physiologic concentrations of FN on cell activation was not described.

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SCHULTZ AND SHAHIDI

In summary, we have no evidence to support any beneficial effect of FN on lymphocyte reactivity. We have, however, shown here that purified FN is a potent immunosuppressive agent in vitro and in our other article17 that the removal of endogenous FN from blood products, such as plasma, significantly reduces its immunosuppressive effects on PHA responses.

References 1. Hynes RO, Yamada KM. Fibronectins: multifunctional modular glycoproteins. J Cell Biol 1982;95:369-77. 2. Mosher DF. Physiology of fibronectin. Annu Rev Mcd 1984;35:56175. 3. Alitalo K, Hovi T, Vaheri A. Fibroncctin is produccd by human macrophages. J Exp Med 1980;151:602-13. 4. Hormann H, Jelinic V. Fibronectin, VII. Binding of cold-insoluble globulin and of dcnatured collagen by macrophagcs. Hoppe Seylers 2 Physiol Chem 1980;361:379-87. 5 . Bcvilacqua MP, Amrani D, Mosesson MW, Bianco C. Receptors for cold-insoluble globulin (plasma fibronectin) on human monocytes. J Exp Med 1981;153:42-60. 6. Hoffstein ST, Weissmann G, Pearlstein E. Fibroncctin is a functional component of the surfacc coat of human ncutrophils. J Cell Sci 1981;50:315-27. 7. Klingemann HG, Ebert J, Deeg HJ. Fibronectin is prescnt on Bcells but not on OKT 3-positive T-lymphocytes or LEU ll-positive natural killer cells. J Lcukoc Biol 1986;40:491-5. 8. Cseh K, Jakab L, Torok J, et al. Fibronectin on thc surface of human lymphocytes. lmmunol Lett 1985;9:301-5. 9. Norris DA, Clark RAF, Swigart LM, et al. Fibronectin fragment(s) are chemotactic for human peripheral blood monocytcs. J Immunol 1982;129:1612-8. 10. Savill CM, Ayad SR. The mitogenic activity of a heparin-binding fibronectin fragment (Mr 35,000) produced by cathepsin D digestion. Anticanccr Res 1986;6:321-6. 11. Martin BM, Gimbrone MA Jr, Majeau GR, Unanue ER, Cotran RS. Stimulation of human monocytdmacrophage-derived growth factor (MDGF) production by plasma fibronectin. Am J Pathol 1983;111:367-73. 12. Lause DB, Bcczhold DH, Doran JE. Induction of lymphocyte blast transformation by purified fibronectin in vitro. J Immunol 1984;132: 1294-9. 13. Lause DB, Doran JE, Houston JA, Beezhold DH. Modulation of rat lymphocyte transformation by plasma fibronectin. J Reticuloendothel SOC1983;34:437-48. 14. Lause DB, Doran JE, Houston JA. Interaction of plasma fibronectin i n the in vitro allograft response. Transplantation 1982;34:147-50. 15. Lause D, Beezhold D. Regulation of lymphocyte reactivity by plasma fibronectin: cellular requirements. Immunol Invest 1985;14:299-314.

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16. Klingemann HG, Tsoi MS, Storb R. Fibroncctin rcsforcs defcctivc in vitro proliferation of patients’ lymphocytes after marrow grafting. Transplantation 1986;42:412-7. 17. Schultz JC, Shahidi NT. Influence of fibronectin-containing blood products on lymphocyte reactivity. Transfusion 1990;30:799-807. 18. Weiss RE, Reddi AH. Isolation and characterization of rat plasma fibronectin. Biochem J 1981;197:529-34. 19. Boyum A. Isolation of mononuclear cells and granulocytes from human blood. S a n d J Clin Lab Invest 1968;21(Suppl 97):77-98. 20. Mosesson MW, Umfleet RA. The cold-insoluble globulin of human plasma. 1. Purification, primary characterization, and rclationship to fibrinogen and other cold-insoluble fraction componcnts. J Biol Chem 1970;245:5728-36. 21. Sasaki MS, Norman A. Prolifcration of human lymphocytes in culture. Nature 1966;210:913-4. 22. Soren L. Variability of the time at which PHA-stimulafcd lymphocytes initiate DNA synthesis. Exp Cell Rcs 1973;78:201-8. 23. Prcsant CA, Parker S . Surface alterations in calf lyrnphocyfcs oxidized by sodium periodate. J Biol Chem 1976;251: 1864-70. 24. Cooperband SR, Badger AM, Davis RC, Schmid K, Mannick JA. The effect of immunorcgulatory alpha-globulin (IRA) upon lymphocytes in vitro. J Immunol 1972;109:154-63. 25. Curtiss LK, Edgington TS. Regulatory serum lipoprotcins: regulation of lymphocyte stimulation by a spccics of low dcnsity lipoprotein. J Immunol 1976;116:1452-8. 26. Hubbard WJ, Hess AD, Hsia S , Amos DB. The cffccts of clcctrophoretically “slow” and “fast” a-2 macroglobulin on mixcd lymphocytc cultures. J Immunol 1981;126:292-9. 27. Chase PS. The effects of human serum fractions on phytohcmagglutinin- and concanavalin A-stimulatcd human lymphocytc cultures. Cell Immunol 1972;5:544-54. 28. Bcycr CF, Bowers WE. Periodate and concanavalin A induce blast transformation of rat lymphocytes by an indirect mcchanisni. Proc Natl Acad Sci USA 1975;72:3590-3. 29. Biniaminov M, Ramot B, Novogrodsky A. Effcct of macrophages on periodate-induced transformation of normal and chronic lymphatic leukaemia lymphocytes. Clin Exp Immunol 1974;16:23542.

30. Beczhold DH, Lause DB. Stimulation of rat macrophagc interleukin 1 secretion by plasma fibronectin. Immunol Invest 1987;16:437-49. 31. Homandberg GA, Williams JE, Grant D, Schumachcr B, Eisenstein R. Heparin-binding fragments of fibronectin are potcnt inhibitors of endothelial cell growth. Am J Pathol 1985;120:32732. 32. Matsuyama T, Yamada A, Kay J, et al. Activation of CD4 cells by fibronectin and antLCD3 antibody. A synergistic cffccf mcdiated by the VLA-5 fibronectin recepfor complex. J Exp Mcd 1989;170:1133-48. John C. Schultz, PhD, Associate Researcher, Departmcnt of Pcdiatria, Division of Pediatric Hematology/Oncology, University of Wisconsin-Madison School of Medicine, 600 Highland Avcnuc, Room H4/475 CSC, Madison, WI 53792. [Reprint requests] Nasrollah T. Shahidi, MD, Professor, Departmcnt of Pcdiafrics, Division of Pediatric Hematology/Oncology.