Mechanisms of Tumor Necrosis Factor-a Alteration of PMN ... - NCBI

American Journal ofPathology, Vol. 136, No. 4, April 1990 Copyrigbt i American Association ofPathologists

Mechanisms of Tumor Necrosis Factor- a Alteration of PMN Adhesion and Migration Jonny L. Salyer, John F. Bohnsack, William A. Knape, Ann 0. Shigeoka, Edward R. Ashwood, and Harry R. Hill From the Division of Clinical Immunology, Microbiology, and Virology, Departments ofPathology and Pediatrics, University of Utah, Salt Lake City, Utah

We have investigated the effects ofrecombinant human tumor necrosis factor-a(rhTNFa) on polymorphonuclear leukocytes (PMNs), concentrating on the mecbanisms involved in the alterations of PMN-directed migration and adherence by this cytokine. RhTNFa profoundly suppressed PMN chemotaxis toward FMLP by 80%. At similar concentrations, it enhanced adhesion to gelatin-coated plastic dishes by more than tenfold and increased the expression of the CD) l b antigen to 182% of the control. The monoclonal antibody 60.1, which is directed against the alpha chain of the CDl1b/ CD18 complex, completely blocked rhTNFa, induced inhibition of the chemotactic response to FMLP, and rhTNFa induced hyperadherence, suggesting that these effects were related to rhTNFa's effects on CD) l b antigen expression. Thefluid state of the PMN membrane was also decreased by rhTNFa. N-butanol, a known membranefluidizer, partially inhibited the effect ofrhTNFa on membranefluidity and chemotaxis and completely reversed its effects on adherence and the expression ofthe CD) l b antigen. Pentoxifylline, an agent that has previously been studied for its ability to prevent some effects ofrhTNFa on PMNs, completely prevented the effect of rhTNFa on chemotaxis, the expression of the CDllb antigen, and membrane fluidity. Pentoxifylline partially prevented changes in adherence caused by this cytokine. Increased CD) lb antigen expression caused by rhTNFa may result in enhanced PMN adhesion and suppression of migration. These events may, in turn, lead to the accumulation ofPMNs on the vascular endothelium, resulting in the extensive vascular and tissue damage that is seen in gram-negative sepsis. (Am J Pathol 1990, 136:831-841)

Tumor necrosis factor-a (TNFa) is a cytokine produced by mononuclear cells that has a wide spectrum of activity. Initially it was reported to cause hemorrhagic necrosis of some tumors.1-3 In addition this cytokine was found to play a major role in the development of cachexia.4 5 Tumor necrosis factor-a has also been implicated as the principle mediator in endotoxemia, which involves extensive polymorphonuclear (PMN)-mediated vascular and tissue damage.r9 A number of investigators have examined the effects of TNFa on PMN function and found that TNFa is a potent activator of the human PMN.1O15 Sullivan and coworkers16 have reported that TNFa inhibits the chemotaxis of human PMNs and that this effect can be reversed by the methylxanthine derivative, pentoxifylline. In the present study, we have explored the mechanisms by which this monokine alters PMN migration by determining its effects on membrane fluidity, adhesion, and the expression of an adhesive glycoprotein found on the PMN membrane. In addition we studied the ability of butanol, a known membrane fluidizer and pentoxifylline (Trental, Hoeshst-Roussel, Pharmaceuticals, Somerville, NJ) to reverse the effects of rhTNFa on each of these PMN parameters to elucidate the mechanisms of action of TNFa.

Materials and Methods Leukocyte Preparation Whole blood was obtained from peripheral veins of healthy adults in acid citrate dextrose (Becton Dickinson, Rutherford, NJ). The blood was allowed to settle in 1% dextran (Pharmacia, Piscataway, NJ). After approximately 15 minutes, leukocyte-rich plasma was obtained from the upper two thirds of the suspension and centrifuged on Ficoll-Hypaque discontinuous density gradient (PharSupported by U.S. Public Health Service Grants Al13190 and Al19094. Accepted for publication November 27, 1989. Dr. Salyer received the ASCP/CAP Pathology Resident Research Award for this work at the 1988 Fall Meeting, Las Vegas, Nevada, October

21,1988. Address reprnt requests to Jonny L. Salyer, M.D., Division of Clinical Immunology, Microbiology and Virology, 58114 Department of Pathology, University of Utah School of Medicine, Salt Lake City, UT 84132.

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macia). The pellets were harvested and contaminating erythrocytes were hypotonically lysed. The leukocyte suspensions contained more than 98% PMNs as judged by morphologic examination. The PMNs were then suspended in a modified Hank's balanced salt solution (HBSS) containing 124 mmol/l (millimolar) NaCI, 4 mmol/l KCI, 0.64 mmol/I NaH2PO4, 0.66 mmol/I K2HPO4, 15.2 mmol/l NaHCO3, 10 mmol/l Hepes buffer, 5.56 mmol/l glucose, 1.6 mmol/l CaCI2, and 1% human albumin (Cutter Biological, Berkeley, CA).

Recombinant Human TNFa RhTNFa, lot number Db303B, was generously provided by Cetus Corporation, Emeryville, California. It was supplied in sterile vials containing lyophilized rhTNFa with a protein concentration of 0.29 mg per vial. The bioactivity was 6.5 X 106 units per vial as determined by cytotoxic activity against L929 cells. The endotoxin content was 0.058 ng per vial as determined by the Limulus amebocyte lysate assay. Lyophilized rhTNFa was reconstituted with phosphate-buffered saline (PBS) pH 7.4 and stored at -700C.

Reagents Pentoxifylline (Trental) was provided in powdered form by William J. Novick, Ph.D., of Hoechst-Roussel Pharmaceuticals, Inc., Somerville, NJ. Solutions of pentoxifylline were prepared with HBSS for the chemotaxis, adherence, and membrane fluidity assays and with PBS for the receptor studies. Butanol was obtained from Sigma, St. Louis.

Chemotaxis Assay Chemotaxis was assessed in a 48-well micro chemotaxis chamber (Neuroprobe Inc., Cabin John, MD) using 5-y pore-size micropore filters (Millipore Corp., Bedford, MA). Polymorphonuclear leukocyte suspensions (50 ML) were added to the top chamber, 30 1,L of 1 X 10-8 mol/l (molar) f-methionyl, I-leucyl phenylalanine (FMLP) (Sigma, St. Louis, MO) were added to the bottom chamber. The chambers were incubated for 2 hours at 37°C in a humidified chamber with 5% added C02. Filters were removed, fixed and stained with hematoxylin. Polymorphonuclear leukocytes, which were found at the leading front of the filter were counted using a 1 Ox ocular and 45X objective in 10 random fields. Experiments were performed in quadruplicate for each variable and the mean determined. In each assay untreated PMNs migrating toward FMLP were

used as a positive reference. To determine the dose response of rhTNFa on chemotaxis of PMNs, 2.5 x 106 PMNs/mL were exposed to concentrations of 10-17 to 1 -6 mol/l rhTNFa and incubated for 30 minutes at 37°C with constant agitation. The ability of n-butanol to reverse the effects of rhTNFa on PMNs was studied by incubating 2.5 X 106 PMNs/mL with 10-8 mol/l rhTNFa for 30 minutes at 37°C followed by exposure to various concentrations of n-butanol for 30 minutes at 370C. To determine the effect of pentoxifylline on chemotaxis of rhTNFatreated cells, 2.5 X 106 PMNs/mL were initially incubated with various concentrations of pentoxifylline for 30 minutes at 370C. The PMNs were subsequently exposed to 10-8 mol/l rhTNFa for 30 minutes at 370C.

Adherence Assay Polymorphonuclear leukocyte adherence to gelatincoated plastic dishes was evaluated as previously described.17 Tissue culture wells measuring 16 mm in diameter (Nunc, Roskilde, Denmark) were coated with 0.5 mL of gelatin at 50 gg/mL in HBSS for 2 hours at 370C and washed immediately before the assay. Purified PMNs were labeled with 111lndium oxine for 5 to 15 minutes at 370C and the unbound "'Indium was removed by washing. 1111n-PMNs were adjusted to 5.5 X 106 PMNs/mL in HBSS with 0.5% human serum albumin. The dose response of rhTNFa on adherence of PMNs was determined by adding 225 AL of "11n-PMNs to the gelatincoated wells with 25 AL of the various concentrations of rhTNFa or buffer. In experiments using pentoxifylline or nbutanol, 1111n-PMNs were first incubated with pentoxifylline or n-butanol for 30 minutes at room temperature before addition to the gelatin-coated wells and exposure to rhTNFa. In some experiments, PMNs were exposed to rhTNFa for 30 minutes and subsequently incubated with the monoclonal antibody 60.1 for 10 minutes at room temperature before addition to the wells. The antibody was present throughout the assay. PMNs were allowed to adhere for 30 minutes at 370C. Nonadherent cells were then removed by aspiration and a single wash with HBSS. Adherent cells were harvested by two successive cycles of 1 mol/l NH40H lysis and vigorous scraping with a cottontip applicator. The percentage of the cells adhering in each well was determined relative to the total number of counts added to that well.

Antibodies Mol (Coulter Immunology, Hialeah, FL), an antibody directed against the specific alpha chain of the CD1 1b/

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CD1 8 complex, CD1 lb, was used in the immunofluorescent flow cytometry studies. The antigen recognized by Mol is expressed by peripheral blood monocytes, certain macrophages, granulocytic cells (from myelocytic to mature PMNs), and a subset of null cells. A purified mouse IgM, which recognizes nonhuman tissue, MsIgM (Coulter Immunology), was used as a control reagent for nonspecific cell-surface staining with the primary monoclonal antibody Mol. Lyophilized GAM-FITC, (Coulter Immunology) a fluorescein-labeled whole-molecule IgG goat antibody against mouse immunoglobulin, was reconstituted in 500,uL sterile distilled water and diluted 1:80 in diluting media containing Medium RPMI 1640 with L-glutamine and sodium bicarbonate (Flow Laboratories, McLean, VA), 5% fetal calf serum (Row Laboratories), and 0.5 g/L sodium azide (Sigma St. Louis, MO). Aliquots were stored at -200C. The monoclonal antibody 60.1 was provided by John Harlan and Patrick Beatty of the University of Washington and the Fred Hutchingson Cancer Center, Seattle Washington. This is an IgG antibody directed against an epitope on the alpha chain of the CD1 1 b/CD1 8 heterodimer, which inhibits PMN adhesive functions in response to some agonists.18

Immunofluorescence Flow Cytometry Polymorphonuclear leukocytes were prepared as described then suspended in PBS at a concentration of 2 X 1 o6 in 200 uL. To determine the dose response of rhTNFa on the expression of the CD1 lb antigen, 2 X 1 06 PMNs were exposed to 10-15 to 10-6 mol/l rhTNFa in a total volume of 200 ,uL for 30 minutes at 370C. To determine the effects of varying times of exposure to rhTNFa, 2 x 1 06 PMNs were subjected to 10-8 mol/l rhTNFa for 5, 15, 30, 60, 90, and 120 minutes at room temperature in a total volume of 200 uL. To examine the effects of pentoxifylline and n-butanol on the expression of the CD1 1b antigen by rhTNFa-treated PMNs, 2 x 106 cells were incubated with pentoxifylline or n-butanol for 30 minutes, followed by a 30-minute exposure to rhTNFa at room temperature. In all experiments PMNs incubated with PBS alone served as the negative control. After appropriate incubations, 5 ML of either Mo1 or MsigM monoclonal antibody from a 100-test vial size was mixed with PMNs, incubated for 15 minutes at 4°C, and washed twice with PBS. Indirect immunofluorescence staining was carried out by adding 100 ,uL of GAM-FITC to PMNs and incubating for 15 additional minutes at 40C. Residual red blood cells were lysed with a 1:25 dilution of Immunolyse (Coulter Immunology) in PBS and cells were fixed with 250 ML Fixative (Coulter Immunology), washed twice, and resuspended in 100 MAL PBS. The antibody-labeled

cells were analyzed using an argon laser EPICS C Cytofluorograph (EPICS Division of Coulter Corp.) with excitation at 488 nm and emission at 520 nm. Polymorphonuclear leukocytes were selected and gated according to their forward angle and 90-degree light-scatter characteristics. A minimum of 2000 cells for each sample was analyzed and fluorescent data were collected on a three-decade log scale. The data were displayed as one-parameter histograms plotting log green fluorescence versus cell number. Positive cells were those in which green fluorescence intensity was greater than that observed with the MsIgM negative control. Fluorescence intensity in each experiment was evaluated by determining the mean fluorescence channel of positive cells and converting the log channels to a linear scale between 0.1 and 100, which was then reported as percentage of relative fluorescence in arbitrary units.

Membrane Fluidity Assay Membrane fluidity was evaluated using the fluorescent dye, C6-NBD-phosphatidylcholine (C6-NBD-PC), which was obtained as a solution in chloroform under nitrogen (category number 10130 Avanti Polar Lipids, Birmingham, AL). The dye was stored at -20°C until needed and aqueous solutions were made in TDx buffer (Abbott Laboratories, Irving, TX), as previously described.'9 Total fluorescence intensity and polarization were determined with an Abbott TDx fluorescence polarimeter (Abbott Laboratories). Triton X-1 00, a surfactant, was used as the polarization control (Sigma Chemical Co., St. Louis, MO). PMNs were prepared as described above and resuspended in HBSS to a concentration of 5 X 106 cells/mL. TDx buffer (1 mL) was mixed with 500 uL of PMNs. After background fluorescence polarization and intensity were established, the fluorescent dye (final concentration 0.5 mg/mL) was mixed with the cell suspension. After 6 to 7 minutes incubation, total fluorescence polarization and intensity was determined. Temperature was maintained at 34.0 ± 0.50C. Net polarization was calculated as previously described.19 Duplicate samples were run for each variable. The dose-response effect of rhTNFa on membrane fluidity was evaluated by exposing PMNs to concentrations of 10-12 to 10-6 mol/l rhTNFa or HBSS for 30 minutes at 370C. The effect of pentoxifylline and n-butanol on the membrane fluidity of rhTNFa-treated PMNs was determined by incubating PMNs with varying concentrations of pentoxifylline or n-butanol for 30 minutes at 370C, followed by incubation with 1 -8 mol/l rhTNFa for an additional 30 minutes at 370C. Using a computerized program, polarization was converted to net polarization.19

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treated with concentrations of 1 10 to 1 o-6 mol/l rhTNFa (7.0 ± 2.0, 6.2 ± 1.4, 5.0 ± 1.2, 5.8 ± 2.0, 3.2 ± 1.7 cells per 10 high-power fields, respectively) was not significantly different than the migration of untreated cells (5.5 ± 0.9).

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1a-17 10-15 ia-13 10-11 10-9 10-7 Molar Concentration of rhTNF-alpha Figure 1. The effects of rhTNFa on PMN chemotaxis. The results

represent data from four experiments using PMNs from four individuals withfour replicatesfor each variable. *P < . 001, **P < 0.05.

Statistics The results are reported as the mean ± the standard error of the mean. P values were determined using two-tailed paired and unpaired t-tests.

Next we investigated the effect of rhTNFa on PMN adhesion. 11lndium-labeled PMNs were incubated with rhTNFa and adherence to gelatin-coated wells was assessed. Polymorphonuclear leukocytes incubated with concentrations of 10-10 to 10-7 mol/l rhTNFa demonstrated marked enhancement of adhesion to the gelatincoated wells (Figure 2). Approximately 80% of PMNs exposed to these concentrations of rhTNFa were adherent as compared to only 7% adherence with untreated PMNs. A mild enhancement of PMN adhesion to 37% was observed at a concentration of 1 0-1 mol/l rhTNFa. Concentrations less than 10-" mol/l rhTNFa demonstrated no effect on PMN adherence. In comparison PMNs incubated with 10-8 mol/l FMLP, a potent chemoattractant, demonstrated an enhancement of adhesion to 63%.

Results The Effect of rhTNFa on Chemotaxis We first examined the effect of rhTNFa on the ability of PMNs to migrate in a chemotactic gradient. Normal adult PMNs were exposed to rhTNFa or HBSS, placed in the top compartment of the chemotaxis chamber, and directed migration toward FMLP (10-8 mol/l) was evaluated. Chemotaxis was profoundly suppressed at concentrations of 1 0-9 to 10-6 mol/l rhTNFa to approximately 20% of the untreated cells (Figure 1). Mild inhibition of migration was observed at concentrations of 10-12 to 1 0`' mol/l rhTNFa. There was no effect on the chemotaxis of PMNs, however, at concentrations of less than 10-12 mol/l rhTNFa. Exposure of the cells to rhTNFa followed by washing had essentially the same effect, indicating that there was no need for the cytokine to be present in the reaction mixture. In a series of four experiments, rhTNFa (10-10 to 10-6 mol/l) was added to the bottom of the modified Boyden chemotaxis chamber. In none of these experiments was rhTNFa found to stimulate directed migration. Studies of the effett of rhTNFa on nondirected migration were performed by incubating PMNs with rhTNFa, placing the cells in the top compartment of the chemotaxis chamber and evaluating migration toward HBSS. In quadruplicate studies, nondirected migration of PMNs

The Effect of rhTNFa on the Expression of the CD1 lb Antigen on PMNs To explore the mechanisms of this profound inhibition of migration and enhancement of adherence, we studied the effects of rhTNFa on expression of the major adhesive glycoprotein, the CD1 1 b/CD18 complex on the PMN surface by flow cytometry. RhTNFa stimulated a dramatic

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antibody 60.1 completely reversed the rhTNFa-induced hyperadherence. Only 26.7% (P < 0.001, compared to rhTNFa-treated cells) of the cells were adherent when PMNs were treated with rhTNFa followed by the 60.1 monoclonal antibody.

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Figure 3. The effects of rbTNFa on the expression of the CD11b antigen on PMNs. Results represent data from six experiments using PMNsfrom six individuals. *P < 0. 001, * *P < O.01, ***P