48, 288-295 (1979)
of Lymphocyte Reactivity in Vitro by Autologous Polymorphonuclear Cells (PMN)
CLEMENT C. S.Hsu,
MWYEH B. Wu, AND JUANITA RIVERA-ARCILLA
Section of Infectious Diseases -Hypersensitivjty, Department of Medicine, Columbus -Cuneo -Cabrini Medical Center, and Samual J. Sackett Research Laboratories, Northwestern University Medical School, Chicago, Illinois 60611 Received February 5, 1979 The influence of autologous polymorphonuclear cells (PMN) on lymphocyte reactivity was investigated by monitoring the uptake of tritiated thymidine by unstimulated, phytohemagglutinin (PHA)-stimulated, and fetuin-stimulated lymphocytes in vitro. Addition of PMN at PMN-to-lymphocyte ratios (P:L) of 0.5 to 2.0 progressively inhibited lymphocyte reactivity. Soluble extracts, obtained by sonication and ultracentrifugation (lOO,OOOgfor 90 min) of PMN, also inhibited lymphocytes. The PMN-derived inhibitor(s) is noncytotoxic, heat labile (56°C for 60 min), resistant to freeze-thawing (20 cycles), and appears to be nondialyzable. Inhibition was more marked when the factor was added at the initiation of lymphocyte cultures than when added with the tritiated thymidine 24 hr prior to cell harvest. Thus thymidine released by PMN which diluted the radiolabeled nucleotide and degradation of the tritiated thymidine did not explain these results. Lymphocytes incubated for 3 days in the medium containing the inhibitor reacted normally to PHA following washing, indicating that inhibition was reversible. These results suggest that a PMN-derived lymphocyte inhibitor(s) may modulate lymphocyte-mediated immune reactivity.
INTRODUCTION Several reports have documented the effects of PMN on lymphocyte proliferation in vitro. Some (1, 2) suggest that PMN may enhance lymphocyte responses to PHA or to allogeneic cell stimulation, while others (2-6) indicate that PMN inhibit lymphocyte reactivity. A more recent study (7) demonstrated that PMN contain both lymphocyte inhibitory and enhancing factors. Neutral proteinases in PMN have also been shown to stimulate lymphocytes (8). We investigated the effect of autologous PMN and sonicated extracts of PMN on lymphocytes cultured with and without mitogens. The results indicate that PMN contain a soluble, heat-labile factor(s) that reversibly inhibits lymphocyte reactivity. MATERIALS
Cell culture techniques have been described (9, 10). Heparinized venous blood from normal donors was allowed to stand at 37°C for approximately 2 hr to sediment erythrocytes (RBC). The leukocyte-rich plasma was separated and mononuclear leukocytes were isolated by Ficoll-Hypaque density gradient centrifugation (11). 288
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Mononuclear cells, consisting of approximately 80% lymphocytes, were isolated from the interphase of the Ficoll-Hypaque gradient and washed, and cell counts performed. For differential cell counts, 3% acetic acid containing crystal violet was used to stain and classify cells microscopically as lymphocytes, monocytes, or PMN. The absolute number of viable cells was counted using 0.4% trypan blue in 0.85% NaCl solution (12). A minimum of 200 cells was counted. The isolated cells were cultured at a concentration of 2 x lo6 lymphocytes/ml of Roswell Park Memorial Insititute (RPMI) medium 1640 containing antibiotics and 20% pooled normal human plasma. Cultures were performed in triplicate or quadruplicate in microculture plates with round-bottom wells. One-hundred eighty microliters of the cell suspension was distributed to each well. Mitogens were added in lo-$ volumes to produce specified final concentrations. Mitogens employed were PHA-P (Difco, Detroit, Mich.) and fetuin prepared by Spiro’s method (9) (Grand Island Biological Co., Grand Island, N.Y .), both of which have been shown to activate T lymphocytes. Autologous PMN preparations (see below) were added in 20-~1 volumes. Appropriate amounts of RPM1 medium 1640 were added to control cultures. The cultures were maintained in 5% C02-95% air atmosphere at 37°C for 3 days for PHA-stimulated cultures, and 6 days for fetuin-stimulated and unstimulated cultures. Twenty-four hours before harvest, 10 ~1 of [methyl-H3]thymidine (specific activity 1.9 Wmmol, SchwarzJMann, Division of Becton, Dickinson Co.) was added to each well to a final concentration of 1 &i/ml. Cells were harvested by mass automated sample harvester II (Flow Laboratories, Rockville, Md.) and processed with hydroxide of hyamine (10X) (Packard, Downers Grove, Ill.) and Bray’s solution as described (11). Tritiated thymidine uptake was determined in a Packard scintillation counter (model 3982) and the mean counts per minute of triplicate cultures was used to express the results. Isolation
PMN were obtained from the gravity-separated leukocyte-rich plasma after centrifugation on a Ficoll-Hypaque gradient. PMN sedimented to the bottom of the gradient, were collected, and were washed with RPM1 medium 1640. Contaminating RBC were lysed using hypotonic saline or NH&l solution (12). Less than 4% of the cells in the PMN suspension were lymphocytes; monocytes were rarely noted. After lysis of RBC, PMN were washed three times and suspended in RPM1 medium 1640 at 40 x 10Vml. To prepare PMN extracts, PMN were disrupted with a Sonifier (Branson Instruments, Stanford, Conn.) at approximately 20 k cycle/set for 15 sec. The suspension was then ultracentrifuged in a model L ultracentrifuge (Beckman Instruments, Lincolnwood, Ill.) at 100,OOOg for 90 min. The supernatant fluid was collected and passed through a Millipore filter (0.45 pm) before use in experiments requiring PMN extracts Deviation from the above methods will be indicated in the results. RESULTS The effect of addition of autologous PMN on lymphocyte response to fetuin is shown in Fig. 1. Addition of PMN at P:L ratios of 0.5 to 2.0 resulted in progressive inhibition of the response. Addition of PMN to unstimulated 6-day cultures also revealed an inhibitory effect on spontaneous lymphocyte proliferation (Fig. 2).
HSU, WU, AND RIVERA-ARCILLA 20 18 16 14 8 0 *
12 8 4 2
FIG. 1. Autologous PMN inhibition of fetuin-stimulated lymphocyte response as monitored by tritiated thymidine uptake. Each circle represents the mean cpm of triplicate cultures. PMN were added to Ficoll-Hypaque-separated lymphocyte cultures at PMN-to-lymphocyte ratios (P:L) of 0.5 to 2.0.
To examine the lymphocyte viability in cultures with excess autologous PMN, the viability of lymphocytes of 20 unstimulated 6-day cultures of Ficoll-Hypaque gradient-separated cells (mean + SD of P:L = 0.04 + 0.04), and that of 30 cultures of gravity-separated leukocytes (mean ? SD of P:L = 1.26 + 0.88) were compared. The percentages of viable lymphocytes were, respectively, 86% (SD = 28%) and 78% (SD = 24%) of the initial cell number. This difference was not statistically significant (P > 0.05). The inhibitory effect of PMN, heated PMN, and PMN sonicates on lymphocyte response to various doses of PHA is shown in Fig. 3. Inhibition was noted after heating at 56°C for 30 min (experiments B, C, D, and F) or sonication of cells (experiments B, C, D, and E). After both procedures, no viable PMN were detected by trypan blue stain. In three of four experiments, heating partially abolished inhibition (experiments C, D, and F); in contrast, sonication enhanced the inhibitory effects (experiments B, C, and E). The inhibitory factor liberated by PMN was soluble. After ultracentrifugation of sonicated PMN, the supernatant fluid (i.e., PMN extract) had the same inhibitory effect as the sonicated cell suspension (Table 1). The PMN-derived inhibitor
8 02 x E I
FIG. 2. Inhibition of spontaneous lymphocyte DNA synthetic activity by autblogous PMN after 6 days of incubation.
PHA Dose (&ml)
FIG. 3. Autologous PMN inhibition of PHA-stimulated lymphocyte response. Sonicated PMN (0 - - - 0) and heated (at 56°C for 30 mm) PMN (0 - - - Cl) were added at a P:L ratio of 2.0. TABLE
Comparison of Autologous Lymphocyte Inhibitory Effect of Sonicated PMN and That of Sonicated and Ultracentrifuged (100,000g for 90 min) PMN Extract” PHA dose (&ml) PMN (p:L = 2.0)
No PMN added Sonicated PMN PMN extract
1,551 141 82
9,528 341 97
22,370 409 164
33,355 229 100
a Lymphocytes were stimulated by PHA and their responses were monitored by tritiated thymidine uptake. PMN preparations were added at a PMN-to-lymphocyte ratio (P:L) of 2.0. Results are expressed as mean cpm of triplicate cultures.
HSU, WU, AND RIVERA-ARCILLA
Fetuin(mglml):O 7rratmmt PMN
2 Of ht.:
56-C 60 mln
66-Z 60 mln
60% 13 mm
FIG. 4. Influence of dialysis, 20 freeze-thaw (F-T) cycles. and heating of autologous PMN extract (PMN Ext.) on inhibition of fetuin-stimulated lymphocyte responses. Columns represent mean cpm of triplicate cultures and vertical bars represent maximum variation of cpm among the three.
appeared nondialyzable, resisted freeze-thawing, and was heat labile at 56°C for 60 min (Fig. 4). To investigate the mechanism of PMN inhibition of lymphocyte reactivity, the PMN extract was added to cultures at the beginning of incubation (Day 0) or with the radioactive thymidine (Day 2 for PHA-stimulated cultures and Day 5 for fetuin-stimulated cultures). The results (Tables 2 and 3) indicated that addition of the PMN extract at Day 0 resulted in a more marked decrease in counts per minute than addition at Day 2 or 5. The PMN inhibition of lymphocyte responsiveness was reversible. In four experiments lymphocytes were preincubated for 3 days with autologous PMN extract. The cells were washed three times and cultured for three more days with PHA. Preincubation of lymphocytes with PMN extract produced little inhibition of lymphocyte response to subsequent PHA stimulation (Fig. 5). Lymphocytes cultured with the PMN extract during the preincubation period and with PHA were TABLE
Effects of Addition of Autologous PMN Extract to PHA-Stimulated Lymphocyte Cultures at Day 0 or 2 of Incubation” PHA dose (&ml) PMN (PL = 2.0) None Added at Day 0 Added at Day 2 0 Results expressed as cpm
0 100 46 60
3,315 310 I,%2
8,585 3,130 5,146
23,427 6,361 15,992
Effects of Addition of Autologous PMN Extract to Fetuin-Stimulated Lymphocyte Cultures at Day 0 or 5 of Incubation” P:L ratio PMN
Added at Day 0 Added at Day 5
a Results expressed as cpm. Dose of fetuin: 4 mg/ml.
inhibited and lymphocytes handled in the same manner but cultured PMN extract responded normally to the mitogen.
DISCUSSION These results clearly demonstrate that autologous PMN inhibit lymphocyte reactivity in vitro as monitored by tritiated thymidine uptake. The inhibitory effect appears to be due to release of a soluble factor(s) present in PMN. Addition of the
FIG. 5. Reversibility of lymphocyte inhibitory effect of autologous PMN extract (at a dose equivalent to P:L of 2.0). Lymphocytes were preincubated in RPM1 medium 1640 containing 20% pooled normal human plasma with PMN extract for 3 days. After washing three times with RPM1 medium 1640, the lymphocytes were further cultured in the same medium for 3 days without PMN extract and stimulated with three different doses of PHA. The lymphocyte responses (C, - - - 6) are compared with: (1) those of lymphocytes incubated with PMN extract for 3 days, washed, and further cultured with PMN extract and PHA (0 l ), and (2) those of lymphocytes not incubated with PMN extract either before or after washing of the cells, but stimulated with PHA after the washing (0 0).
HSU, WU, AND RIVERA-ARCILLA
PMN extract 24 hr prior to harvest resulted in a smaller decrement in counts per minute than addition at the initiation of the culture. Therefore, the factor was not degrading the radiolabeled thymidine and was not PMN-derived thymidine which diluted the concentration of the tritiated thymidine added to the culture. Cytotoxicity was not the primary mechanism for the lymphocyte inhibition, since the inhibitory effect was reversible and addition of PMN did not significantly affect the viable lymphocyte count after 6 days of coincubation. Neutralization or destruction of the mitogenic activity of PHA or fetuin by the factor did not explain the inhibitory effect, as spontaneous lymphocyte proliferation in pooled normal human plasma was also depressed. Spontaneous lymphocyte transformation, noted in many disease states and in normal subjects (13-20), probably reflects the degree of lymphocyte activation in vivo at the time cells are obtained. Activated lymphocytes secrete a mitogenic factor that induces other lymphocytes to proliferate (21-27). The PMN-derived lymphocyte inhibitor may destroy the lymphocyte mitogenic factor or coat lymphocyte surfaces to prevent cell contact with mitogens. Alternately, the inhibitor may suppress the metabolic activity of the lymphocytes. The inhibitor is likely to be protein in nature, because of its heat lability. It is yet to be determined whether the PMN-derived lymphocyte inhibitor is active in vivo, but clinical studies suggest this possibility. Anergy has been found in patients with leukocyte counts above 15,000/mm3 (28). Furthermore, reactivation of latent pulmonary tuberculosis or aggravation of chronic active tuberculosis in patients with pyogenic pneumonia has been noted (29). It is also well recognized that herpes labialis frequently develops during pyogenic infections (30). It is tempting to speculate that the PMN-derived inhibitor suppresses T-lymphocyte reactivity which is responsible for delayed skin hypersensitivity reactions and resistance to tuberculosis and viral infections. ACKNOWLEDGMENTS The authors are indebted to Dr. M. Carruthers and Dr. J. P. Phair for corrections of the manuscript. The work was supported by a grant (AI 12085) from the National Institute of Allergy and Infectious Disease, National Institutes of Health, and a grant from the Leukemia Research Foundation, Inc.
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