ENHANCEMENT OF LYMPHOKINE-ACTIVATED T KILLER CELL TUMOR NECROSIS FACTOR RECEPTOR mRNA TRANSCRIPTION, TUMOR NECROSIS FACTOR RECEPTOR MEMBRANE EXPRESSION, AND TUMOR NECROSIS FACTOR/LYMPHOTOXIN RELEASE BY IL-lP, IL-4, AND IL-6 IN VITRO'

Co-culture with IL-2 can induce human T lymphocytes to proliferate and become nongenetically restricted, lymphokine-activated killer (LAK) cells in vitro. Our studies were conducted with long term cultured, human T-LAK celk from peripheral blood, which are 95 to 99% CD3+. We found that proliferating 7- to 10-day human T-LAK cells express TNFR, by using a lZ5I-TNF binding assay. Additional treat-ment of these cells with the cytokines IL-la, IL-4, or IL-6 rapidly up-regulated 55-kDa TNFR mRNA transcription and doubled TNFR membrane expression. Further studies revealed that these cytokines also increased the release of TNF and lymphotoxin (LT). Antibody neutralization studies indicated that IL-1 induces release of both TNF and LT; however, JL-4 and IL-6 induce primarily LT release. These results further support the concept that these cytokines are involved in the

T cells: LT. lymphotoxin. lymphoid cells (4,5) that cause a wide range of biologic activities in vivo and in vitro. In vivo, TNF is known to be involved in inflammation (6), wasting, and shock (7). In vitro, TNF has direct cytotoxic effects on certain transformed cell lines (8) and is also known to stimulate fibroblast growth (9, 10) and regulation of cell surface Ag expression (1 1, 12). This cytokine also possesses multifunctional immunoregulatory activities (1 3), which include activation of NK cells (14) and macrophages (15) and induction of growth and function of B and T lymphocytes in vitro (1 3, 16).
The biologic effects of TNF/LT, both in vivo and in vitro, are initiated by their first binding to specific cell membrane receptors 117). It has recently been shown that mammalian cells can express one or both of two different TNF/LT receptors, one of 55 kDa (1 8) and one of 68 to 75 kDa (1 9). These membrane proteins show some homology in their extracellular domains, but their intracellular domains are totally different in amino acid sequence. The fact that they are two different receptors was not detected by almost all previous investigators. It has been demonstrated that human T-LAK cells express the TNFR but unstimulated T cells do not (1 3, 20). However, it is not yet known whether these cells express one or both receptors. It has also been shown that TNF and other cytokines are released by human T cells after activation in co-culture with IL-2 in vitro (21). Furthermore, it is known that TNF and IL-2 synergize to augment proliferation of T cells (1 3) and cytotoxic activity of human T-LAK cells (22,23). It is becoming apparent that released cytokines and their membrane receptors are involved in T cell proliferation, differentiation, and expression of cytolysis in vitro. We were interested in further examining the role of cytokines and their membrane receptors in the differentiation of T-LAK cells in vitro.
We established the baseline expression of the TNFR on T-LAK cells and then determined the effects of selected cytokines on mRNA transcription of the 55-kDa TNFR, membrane TNFR expression, and TNF/LT release. We found that cytokines IL-lD, IL-4, and IL-6 up-regulate 55-kDa TNFR transcription, TNFR expression, and TNF/LT release in human T-LAK cells in vitro. TNF and LT may be important immunoregulatory molecules in the function and growth of T-LAK cells. Collection of human PBMC. Human PBMC were collected from peripheral venous blood of normal donors, which was defibrinated, diluted with a n equal volume of PBS, and separated by density gradient centrifugation on 1.077 Histopaque (Sigma). The PBMC were collected from the interface of the plasma and Histopaque and further washed, to remove the remaining Histopaque, by alternate centrifugation (500 x g for 10 min). After the final centrifugation, the cell pellet was resuspended in AIM-V with 2% FCS and the cells were counted using a SPotlite hemacytometer. These cells were 99 to 100% viable, as determined by trypan blue exclusion test.

Cells
Generation of T-LAK. Human PBMC were initially cultured in AIM-V with 2% FCS. supplemented with 400 U/ml human rIL-2 and 0.4 pg/ml PHA. The T-LAK cells were cultured by a method similar to that described by Ingram et al. (3), at a cell density of 2.0 x lo6 cells/ml. in a T-75 tissue culture flask. After 48 to 72 h at 37°C a cell sample in trypan blue was counted on a SPotlite hemacytometer and resuspended at 0.5 X lo6 viable cells/ml in complete medium, which contained AIM-V with 2% FCS plus 400 U/ml rIL-2. These T-LAK cell cultures were recultured every 48 h in fresh complete medium, at a density of 0.5 x lo6 cells/ml.

Flow cytometric analysis.
Cell suspensions were analyzed by FACScan (Becton-Dickinson, Mountain View, CA). The FACscan was calibrated with the Autocomp program and optimized for each sample tested. Forward scatter thresholds were set in order to eliminate debris and dead cells. Particles with unusually low forward and side scatter were considered to be nonviable and were not counted. Surface phenotypes on lymphocytes were analyzed by staining the cells for surface Ag with the following mAb: (a) CD3 (pan-TI, (b) CD4 (helper-inducer). and (c) CD8 (cytotoxic-suppressor). The mAb were fluorescein conjugated and were obtained from Becton Dickinson Immunocytometry Systems. Cells were incubated with mAb for 30 min on ice and were washed twice with PBS with 0.2% sodium azide, by alternate centrifugation (5 min at 400 x g ) and resuspension. Cells were fixed in 1.0% paraformaldehyde solution and stored at 4°C until further analysis. Data were expressed as percentage of positive cells for corresponding surface Ag.
L929 cytotoxic assay. T cell culture supernatants were assayed the method of Gatanaga et al. (24). Briefly, L929 cells (8 x lo4 cells/ for TNF or LT activity against the L929 mouse fibroblast cell line by well), culture supernatant, and actinomycin D (0.2 pg/well) were incubated at 37°C for 18 h in 96-well flat-bottomed microcytotoxicity plates (Corning), in 200 pl/well RPMI 1640 with 10% FCS. After incubation, the supernatants were aspirated and the plate was stained with 1 % crystal violet for 5 min at room temperature. Excess dye was rinsed from the plate with water, and the plate was allowed to dry and was solubilized with 100 p1 of acidified methanol (100 amount of TNF and LT bioactivity was determined by comparing mM HC1 in methanol). Absorbance was measured at 600 nm. The activity with a standard curve made with rTNF-a and rLT (Genentech). Antibody neutralization studies were carried out in the same manner: however, antiserum was added in conjunction with the culture supernatants. Immune polyclonal antisera were generated against human rLT and rTNF. in NZW rabbits, by the method of Angeles, CA). After the dialysis. rTNF was concentrated using a m.w. cut-off Spectra-por dialysis tubing (Spectrum Medical Inc., Los Speed Vac concentrator (Savant, NY). The final concentration of rTNF was 2 mg/ml. The rTNF iodination procedure was performed as described by Bolton and Hunter (26). rTNF ( 100 pg) in 20 pl of 0.1 M sodium borate buffer, pH 8.5. was added to the dried iodinated ester '251-Bolton-Hunter reagent and the reaction mixture containing Bolton-Hunter reagent and Iz5I (DuPont). Free iodine was removed by gel filtration over a NAP-5 column (Pharmacia LKB Biotechnology, Inc.. Piscatway. N J ) with PBS containing 5 mg of gelatin. Fractions of 150 p1 were collected, and 1-pl aliquots were assayed by Clinigamma counter (Pharmacia). Peak fractions were pooled, and the specific activity was determined to be 5400 cpm/ng. The labeled material was stored at 4°C and used within 1 month.
Binding studies and Scatchard analyses. One million cells were incubated with binding buffer (RPMI 1640 with 10% FCS) in 24-well plates and then incubated with 0.5 to 30 ng of lz5I-TNF, in the presence or absence of a 100-fold excess of unlabeled TNF. After a 3-to 4-h incubation at 4°C binding buffer was removed and the cells were washed two to four times with ice-cold PBS (27). Cells were solubilized in 1 ml of 0.3 N NaOH and radioactivity was determined by Clinigamma counter (Pharmacia). Cells were incubated at 37°C for 4 h before the binding assay when the effects of IL-1p (50 ng/ml), IL-4 (20 ng/ml), and IL-6 (50 ng/ml) on rTNF binding were examined. Specijic oligonucleotide probefor the 55-kDa TNFR. The oligonucleotide 60-mer for the human TNFR probe 5"GCCACACACGGT-CCT-3' (amino acids 67 to 86) (18) was synthesized by using a Biosearch 8600 DNA synthesizer. The oligonucleotide probe was end-labeled by [T-~'P]ATP (New England Nuclear, Boston, MA), using T4 polynucleotide kinase (28).

RNA isolation and Northern blot analysis.
Total cellular RNA was isolated from T-LAK cells by the guanidinium isothiocyanate/ cesium chloride method and was quantitated spectrophotometrically as previously described (29). Integrity of the RNA was confirmed with ethidium bromide (0.5 mg/ml). RNA (5 to 10 pg) was electrophoresed in 2.2 M formaldehyde-l.2% agarose gels, transferred to nitrocellulose filters (Schleicher & Schuell. Keene, NH), and baked at 80°C for 2 h, as previously described (30). The filters were hybridized with 32P-labeled TNFR-specific synthetic probe. After hybridization, blots were washed at 25°C with 2X SSC, 0.1 % SDS, for 30 min, and then with 0.1 x SSC. 0.1 % SDS, for 30 min. The blots were then exposed to X-Omat XAR-5 (Kodak Co., Rochester, NY) X-ray film at -70°C.

Expression of T lymphocyte surface phenotypic markers by 7-10-day T-LAK cells. T-LAK cells were generated as described in Materials and Methods
and were subjected to phenotypic analysis on day 7 and day 10 of culture. Shown in Figure 1 are the data obtained from phenotyping of one individual donor: however, these results are representative of data from all normal individuals (1 0 subjects) examined. These data reveal that CD3+ T cells dominate (90-95%) the culture by days 7 to 10 and, although not shown, remain the predominant cell type throughout the culture period (days 15 to 21). The ratio of CD4+ to CD8+ cells in these cultures from a single donor remained relatively constant: however, they vary from individual donor to donor.

Capacity of IL-1 /3, IL-4, and IL-6 to induce the release of TNFILT by T-LAK cells in vitro.
T-LAK cells were incubated with the cytokines IL-10. IL-4, or IL-6 (in addition to IL-2) for 24 h, and then cell-free supernatant cytotoxic activity was measured in the L 9 2 9 assay, as described in Materials and Methods. Supernatants from cells incubated with cytokines for a period of 4 h did not  show any cytolytic activity. Figure 2 shows that supernatants from T-LAK cells incubated with these cytokines for 24 h produce cytotoxic activity against L929 cells. Supernatants from T-LAK cells incubated with IL-4 produced the highest amount of cytotoxic activity. followed by IL-6 and IL-1p. However, control T-LAK cells incubated with IL-2 alone produced much less. Additional studies revealed that the cytotoxic activity of all T-LAK supernatants was blocked 75 to 95% by the anti-LT antiserum, and 45% of the cytolytic activity produced by IL-2-and IL-1 @-stimulated cells was neutralized by anti-TNF antiserum. Thus, the majority of the cytolytic activity of these supernatants was due to LT and not TNF.

Effects of IL-lp. IL-4. and IL-6 on the expression of the TNFR on T-LAK cells. The expression of TNFR on
human T-LAK cells (days 7 to 17) was examined in a ' "1-TNF-binding assay, as described in Materials and Methods. The expression of TNFR was examined at 4 and 24 h after incubation with the cytokines IL-lp, IL-4, and IL-6 (in addition to IL-2). Results from one experiment are shown in Figure 3

Effects of IL-l@, IL-4. and IL-6 on 55-kDa TNFR mRNA expression by T-LAK cells.
Human T-LAK cells were exposed to the different cytokines and then tested for 55-kDa TNFR mRNA expression by Northern blot analysis, as described in Materials and Methods. Results in Figure  4A demonstrate that T-LAK cells cultured with IL-2 expressed mRNA for the 55-kDa TNF/LTR. However. after   a 4-h incubation with IL-l@, IL-4. or IL-6 (in addition to IL-2). a n up-regulation in 55-kDa mRNA was evident. Duplicate samples were run on the same gels to permit quantitation of the amount of RNA loaded/lane. The gel was cut and stained with ethidium bromide. Results are shown in Figure 4B. The samples all produced 2 8 s ribosomal RNA bands in similar regions and of similar intensity.

DISCUSSION
The generation of T-lymphokine-activated killer cells, expressing T cell phenotypes, from human peripheral blood has been described (31). These non-MHC-restricted T-LAK cells exhibit a broad range of cytotoxic activity against panels of target cells in vitro (2). Whereas IL-2 stimulates proliferation of T-LAK cells, other cytokines. such a s TNF, have been shown to be involved in the expression of cytolytic activity in vitro (22,23). Little is known, however, with regard to the effects of various cytokines on the control of TNF/LT release and membrane receptor regulation.
Much effort is now being aimed at defining the function and regulatory activity of cytokine receptors on T lymphocytes. Others have suggested that up-regulation of TNF membrane receptors is involved in the autocrine/ paracrine pathway operative after IL-2 stimulation and that interaction of TNF/LT with these receptors is important in T cell proliferation, differentiation, and expression of functional ability (13, 20). Fresh unstimulated PBL do not express TNFR of any type (13, 20). Although it is not yet apparent which class of receptors are expressed, Owen-Shaub et al. (20) demonstrated that lymphocyte TNFR expression is regulated by IL-2 concentration in vitro. However, although the presence of surface TNFR on activated T-lymphocytes has been established, the mechanisms of receptor regulation are unknown.
It has been shown that various cytokines can influence both proliferation and expression of T cell function. 1Llp, IL-4, and IL-6 have been shown to induce human and murine thymocyte proliferation (32)(33)(34). Suda et al. (34) reported that IL-4 induced murine T cell proliferation and IL-lp together with IL-6 enhanced proliferation induced by IL-2. It is also known that activated T cells have the capacity to produce IL-2, IL-4, IL-6. and TNF/LT and that these cytokines are involved in an autocrine loop of T cell expression of cytotoxicity and proliferation in vitro (22,23,35).
We report here that TNFR number, 55-kDa mRNA expression, and TNF/LT release can be up-regulated, in response to IL-lP, IL-4, and IL-6 incubation, on human T-LAK cells co-cultured with IL-2. Although these studies do not distinguish whether up-regulation of receptors involves 55-or 75-kDa receptors, or both, we noted dramatic up-regulation within 4 h. In fact, by Scatchard analysis the number of sites/cell for IL-I@-, IL-4-. and IL-6-treated cells increased almost 2-fold. In addition, we found that 4-h incubation of day 10 T-LAK with IL-l@, IL-4, and IL-6 resulted in an up-regulation of 55-kDa TNFR mRNA expression. This finding agrees with the Scatchard analysis data, in that TNFR mRNA enhancement correlates with an increase in TNFR membrane expression. It is interesting that opposite results are observed with stimulated human monocyte-like cell lines; namely, LPS, PMA, and IL-lP rapidly down-regulate TNFR membrane expression. It is also interesting to note that T-LAK cell TNF/LT release was not apparent at 4 h but was up-regulated by each cytokine after 24 h. IL-4 was the most effective in up-regulating TNF/LT release. Both IL-2 and IL-I P induced about equal amounts of TNF and LT; however, IL-4 and IL-6 induced predominantly LT. It is evident and perhaps slightly significant that there is some selective induction of LT/TNF by these cytokines.
The evidence presented here further supports the concept that IL-2 is sufficient to induce T cell proliferation but is not the sole cytokine involved in the regulation of TNF/LT release or TNFR synthesis and membrane expression. IL-16, IL-4, and IL-6 can also be involved in the up-regulation of the TNFR at the level of both mRNA transcription for the 55-kDa receptors and membrane expression. In the latter case, it is not yet clear whether this is selective expression of 55-kDa or 75-kDa receptors or up-regulation of both. It is not yet clear whether the T-LAK cell can express the 75-kDa receptor. The exact mechanism involved in the immunoregulation and differentiation of T-LAK effector cells and the specific role of the TNFR is unclear; however it is apparent that cytokines are important in regulating the magnitude of the immune response.