The concept that T lymphocytes regulate neutro- phil function has an important implication in the understanding of the role of these cells in immunity.
0022-1767/92/1481-0177$02.00/0 THEJOURNAL OF lMMUNOLOCY Copyright Q 1992 by The Amerlcan
January 1 . 1992 Printed I n U.S.A.
Voi. 148. 177-181. No. I .
Assodationof immunologists
NEUTROPHIL STIMULATION AND PRIMING BY DIRECT CONTACT WITH ACTIVATED HUMAN T LYMPHOCYTES' JIAN-HUAZHANG,ANTONIOFERRANTE,'ANDRE-PATRICKARRIGO,
The concept that T lymphocytes regulate neutrophil function has an important implication in the understanding of the role ofthese cells in immunity against infection and in inflammatory diseases, but evidence forthis concept is primarily derived from the effects of lymphokines on neutrophils. We now present evidence to show that living or paraformaldehyde-fixed mitogen-activated T lymphocytes, as well as anactivated T cell line (HUT-78).induce by cell-cell contact, an oxygen-dependent respiratory burst measured by both the lucigenin-dependent chemiluminescence assay and superoxide production. Neutrophils reacted with purified human T lymphocytes which had been activated by culture in the presence of PHA and PMA for 72 h showed a marked and significant respiratory burst compared with neutrophils treated with T lymphocytes cultured in the absence of these mitogens. Similar results wereobservedwith the paraformaldehydefixed T cell line (HUT-78). Theability to stimulate neutrophils required intact paraformaldehyde-fixed T cells, and neutrophil stimulation failed to occur if the T cells and neutrophils were separated by membrane filters. mAb to TNF-a, and TNF-B blocked the ability of rTNF-a and TNF-flto stimulate neutrophils but did not block the neutrophil response induced by activated T cells. Pretreatment of neutrophils with the activated T lymphocytes enhanced the response to thetripeptide, FMLP. It is therefore conceivable that activated T lymphocytes attracted a t sites of inflammation influence neutrophil activity by direct plasmamembrane interaction which clearly represents an efficientmicrobialdefence mechanism, minimizing tissue damage during inflammation.
AND
JEAN-MICHEL DAYER
response can regulate the accumulation and functionof neutrophils (3-7). Further evidence for a role of T lymphocytes in neutrophil activationis derived from studies on the effects of T cell lymphokines on neutrophil function (8-1 1 ) . Both the release of oxygen-derived reactive species and lysosomal enzyme secretion from neutrophils can be augmented by IFN-7 (12, 13). IL-2 (9, 14). and lymphotoxin (13. 15. 16). These cytokines have also been shown to activate neutrophils for increased killing of bacteria (1 0, 17, 181, fungi (10, 19), amoebae (20). and Plasmodiurnfalcipurum (21, 22).In addition the T cell lymphokines can activate neutrophils for increased antibody-dependent cellular cytotoxicity (23) and increased degradation of proteoglycan in human and bovine articular cartilage[ 11, 24, 25). However, it is also appropriate to consider thatT cells adhere to neutrophils during inflammation and in this manner alter the neutrophil response. We now demonstrate that activated T lymphocytes are capable of activating neutrophils. MATERIALS AND
METHODS
Preparation of mononuclear leukocytes. The buffy coat of blood from healthy donors was diluted 1/4 in HBSS (GIBCO. Paisley. Scotland) and centrifuged on Ficoll-Paque media (Pharmacia, Uppsala. Sweden). The mononuclear leukocytes were harvested from the ban at the interface. washed three times in PBS (Dulbecco's, GIBCO).and resuspended in CM.3 The CM consisted of RPMI 1640 medium supplemented with 10% heat-inactivated FCS (GIBCO), 2 mM L-glutamine (GIBCO), 1 0 0 U/ml penicilIin, and 100 pglml of streptomycin (GIBCO). Purification oj'T lymphocytes. Mononuclear leukocytes were incubated in plastic dishes. the nonadherent cells were collected and passed through two cycles of nylon wool columns (26). The cells were also treated with L-leucin methyl ester to eliminate remaining monocytes (27). The cells were 94 to 98% CD2+, 83 to 94% CD3+, 2% CD14+ as measured on an EPICS V flow-cytometer and less than (Coulter, Hialeah FL) by indirect immunofluorescence method. T lymphocyte actiuation andfixation. The T lymphocytes were The importanceof T lymphocytes in regulating neutro- cultured in CM alone or in the presence of PHA (1 pg/ml) plus PMA phils responses has recently been emphasized (1). T cells (5 ng/ml) for 7 2 h a t 37°C unless specified otherwise. For fixation, T cells were washed in PBS and fixed with freshly prepared1% and neutrophils canbe found in the same cellular infil- the paraformaldehyde (extra pure. Merck) PBS in for 4 h. The fixed cells trates (2) and T lymphocytes via a cell-mediated immune were washed three timesin PBS. resuspended in CM, and incubated a t 37°C overnight. Then the cells were washed once more before use. Received for publication January 22. 1991. Preparation of neutrophils. Neutrophils were prepared by the Accepted for publication October 14. 1991. rapid single-step method (28). Freshly drawn blood from healthy The costs of publication of this article were defrayed in part by the volunteers was layered onto Ficoll-Hypaque of density 1.1 14 (Mopayment of page charges. This article must therefore be hereby marked nopoly resolving medium, Flow Laboratories, Scotland). After cenadoertisement in accordance with 18 U.S.C. Section 1734 solely to inditrifugation at 400 g/30 min the neutrophils resolved in the second cate this fact. ' This investlgation received financial support from the Swiss National leukocyte band. The cells were harvested. washed three times, and were of 297%purity. Science Foundation, the Foundation Elsie Carlos de Reuter Medical CenChemiluminescence.Thechemiluminescenceassaywasconter, the Australian National Health and Medical Research Council and 100 pl of 1 x IO7 neutrophils/ the UNDP/World B a n k p H 0 Special Programme for Research and Train- ducted as described previously (29). To ing in Tropical Disease [TDR). ml were added 100p1 of 1 X 10' T cells/ml and the volume made up 'Address all correspondence and reprint requests to Dr. A. Ferrante, to 500 w l . To this were then added500 PI of 250 Mg/mlof lucigenin Department of Immunology. Adelaide Children'sHospital, North Adelaide, South Australia. 5006, Australia. Abbreviation used in this paper: CM. complete medium
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NEUTROPHIL ACTIVATION BY T LYMPHOCYTES
(bis-N-methylacridinium nitrate, Sigma), thesample mixed, and during the interaction, we examined the effect of paraplaced immediately in a light-proof chamber (37°C water-jacketed formaldehyde-fixed T lymphocytes. The data presented incubator] of a luminometer (LKB, Walac, Finland) without further in Figures 2 and 3 show that activated T cells, when mixing. The chemiluminescence was measured in mV and results expressed a s initialmaximal rate, unless specified otherwise. In compared with nonactivated T lymphocytes, induced a experiments which tested for priming for a n FMLP response, the marked chemiluminescence response in human neutroneutrophils were pretreatedwith Tcellsfor 10 min before the phils. addition of 5 pM FMLP. Superoxide assay. Superoxide was measured by the cytochrome Neutrophils pretreated for 10 min with activated T cells c reductiop assay (30). To 100 p1 of 2 x lo' neutrophils/ml were showed an augmented response toFMLP which was sigadded 100 pl of 2 X lo7 T cells/ml, 200 p1 of cytochrome c (to give nificantly differentto the effects induced by nonactivated final concentration of 100 mM) with or without the addition of 20 pl of superoxide dismutase (80 pg) The volume made up to 500 pl and T cells. These findings were essentially confirmed by cells mixed. After 30 min of incubation a t 37OC the cells werepelleted using a fixed T cell line, HUT-78 (Fig. 4). Although the by centrifugation at 4°C and theA550of the cellfree supernatant read unstimulated HUT-78 caused a response in neutrophils, in a multiscan spectrophotometer. In experiments examiningpriming for a n FMLP response the neutrophils were pretreated for 10 the activated HUT-78 had a greatereffect and enhanced min with theT cells and then stimulated with 5 pM of FMLP. Cell culture In two-chamber plates. In some experiments the 6 I effects of physical separation of T lymphocytesand neutrophils was studied by using 24-well cluster disheswith 6.5-mm Transwell units (Costar Transwell Cell Culture Chambers). The wells of the plate were separated into upper andlower chambers by a layer of 3.0-pm pore Nucleopore membrane. To the bottom chamber of all wells were added 5 x lo6 neutrophils. Then the wells were divided into two sets. To each of one set was added either "nonactivatedor activated T lymphocytes in the upper chamber.T lymphocytes were similarly added to the other set of wells but in this casewere placed together with neutrophils in the lower chamber. Each well content of 2 ml received the appropriate concentrations of reagents for conducting a cytochrome c reduction assay (see above) and then incubated for 10 min at 37°C. After incubation the supernatants were transferred 3 15 30 to tubes andcentrifuged to remove cells. These were then read in a TIME (rnin) spectrophotometer to determinethequantity of superoxide proFigure 2. The effect of human peripheral blood T cells (paraformalduced. HUT-78 cell line. The human cutaneous T cell lymphoma cell dehyde-fixed) onneutrophil chemiluminescence recorded as the rate of chemiluminescenceoutput per unit time. The effect of varying the conline, HUT-78, was obtainedfrom the American Type Culture Collec- centration of activated T cells is shown. Neutrophils [ 1 x 10') were reacted tion (ATCC, Rockville, MD). This cell line was cultured in CM (31) with either 4 x lo6(O),2 x 10' (0). or 1 x lo6(A)activated T lymphocytes. and wasactivated and fixed a s described for peripheral blood T cells. mAb antibodies to TNF. mAb (TNF-E)a t 1.2 mg/ml with a neutralization capacityof >5 X 1O5 neutralizing U/ml and a murine IgGl mAb (LTX-9) raised against human rTNF-P a t 3.5 pg/ml (-100 neutralizing U/ng) were kindly provided by Dr. A.Adolf (ErnstBoehringer Institute, Vienna). Statlstical analyses. Results were analyzed by the two-tailed ttest.
T
RESULTS
Effects of activated T cells on neutrophils.T lymphocytes cultured in the presence of PHA-PMA when added to neutrophils induced an oxygen-dependent respiratory burst shown as an increased production of lucigenindependent chemiluminescence ( p < 0.01) (Fig. 1).T lymphocytes not activated with PHA-PMA had no effect. Effect of paraforma1dehyde;fixed activated Tcells on neutrophil chemiluminescence. Because of the difficulties in distinguishing between effects of membrane-associated mol&ules and those released 13Y the T cells
No IMLP
ILMP
Figure 3. The effects of paraformaldehyde-fixedT lymphocytes onthe neutrophil chemiluminescence response. Neutrophils were treated with nonactivated T cells (EA] or activated T cells ( ) and then examined for ability to respond toFMLP. The results are presented as mean SEM of three experiments. The values represent the stimulation index = peak rate of chemiluminescenceproduced by neutrophils in the presence of T cells/the chemiluminescenceof neutrophils in the absence of T cells. 'p < 0.05. when comparingeffects of nonstimulated with stimulated T cells.
*
.."
T'
W
5
3
No fMLP
HBSS
non-activated activated T cells T cells
Figure 1. Stimulation of a neutrophil chemiluminescenceresponse by activated T lymphocytes. Neutrophils were treated with either medium (B], unstimulated T lymphocytes ( 1, or PHA-PMA-activatedT lymphocytes (m). The results are presented as mean f SEM of three experiments. *pco.o1.
ILMP
Figure4. Theeffect of the T celllineHUT-78 (paraformaldehydefixed) on the human neutrophil chemiluminescence response. Neutrophils were treated with nonactivated T cells [EA) or activated T cells ( and then examined for abilitytorespondto FMLP. The results are presented as mean f SEMof four experiments and values represent stimulation indexes (as for Fig. 3)." p < 0.05, when comparing effects of nonstimulated with stimulated HUT-78 cells.
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NEUTROPHIL ACTIVATION BY T LYMPHOCYTES
4, the neutrophil for a response to FMLP. The fixed-activatedT cell linewhensonicatedlosttheirabilityto activate neutrophils. The activated Tcell line induced a chemiluminescence responseof 31.6 f 0.8 mV as intact cellsbutonly of 8.6 k 1.4 mV when the cellswere sonicated. Neutrophils treated with nonactivated T cells showed a response of 8.4 k 0.4 mV which was similar to that shown by neutrophils incubated with medium only, 9.8 f 2.8 mV. Effect of activated T lymphocytes on neutrophil superoxide production.Further support for the stimulation of a neutrophil respiratory burstby activated T cells was TIME OFT CELL CULTURE(h) shown by the ability of fixed activated lymphocytes but not the nonstimulated cells to cause significant stimula- Figure 7. Culture incubation time dependency of the development of tion of superoxide production (Fig. 5) and also by the neutrophil stimulating activityby PHA-PMA treated T lymphocytes.Neutrophils were either treated with T cell suspending medium(0)or treated finding that activated T lymphocytes and FMLP induced with "nonactivated" (H] or PHA-PMA activated T cells [ELI). The results a synergistic response (Fig. 6). The results presented in represent meanf SEM of four experiments.' p < 0.05 compared with the (Fig. 7) show that ability the of T lymphocytes to stimulateeffect of nonactivated T cells neutrophils was culture time dependent with the maxi1 mal and significant ( p < 0.05) stimulation developing after 3 days of culture. The effect of separating T cells and neutrophils by membrane filters. By phase contrast microscopy, the T cells were seen tobe closely associated with neutrophils. While activated T cells induced a significant neutrophil W response ( p < 0.001), activated T cells separate from neutrophils by membrane filters (with Costar Transwell 8W ' n cell culture chamber, 3.0 pm), failed to respond to the 3 v) activated T cells(Fig. 8).This suggests thatcell-cell con0 HBSS Na-T a-T a-T + mf tact is required for T cells to stimulate neutrophils. T*
B
Figure 8. The ability of membrane filter [mf)separated paraformaldehyde-fixed T lymphocytes to stimulate neutrophil superoxide production.Neutrophilsweretreatedwithmediumonly (B), nonactivatedT 1, activated T lymphocytes ( a - T )(a),and activated T lymphocytes separated from the neutrophils by membrane filters .).1 The results are the mean f SEM of three cultures. * p < 0.001.
Effect of antitumor necrosis factor antibodies. The direct effects of activated T lymphocytes on the basal neutrophil response is similar to that seen with TNF-a and TNF-P (10, 13, 15).We therefore examined the effects of treating T lymphocytes with mAB which neutralize v) HBSS non-actlvated acttvated TNF-a and TNF-P activity. The paraformaldehyde-fixed, T cells T cells activated T cells were treated for 1 5 min with 500 neuFigure5 The effect of peripheral blood T cells (paraformaldehydetralizing U of TNF-a or TNF-(3 antibody/106 T cells and fixed) on neutrophil superoxide production. Neutrophils were treated with HBSS [a). nonactivated T cells (@, or activated T cells [@]. The results then thesewere mixed with neutrophils for examination represent the mean SEM of three experiments " p < 0.02, compared for a superoxide response. The results showed that actiwith unstimulated T cells. vated T cells treated withmAb to either TNF-a or TNF-P were just aseffective as nontreated Tcells in stimulating neutrophils. In the superoxide assay (four experiments) neutrophils treated with activated T cellsproduced 9.60 f 3.27 nmol of superoxide compared with only 1.55 f 0.77 nmol by neutrophils treated with nonactivated T cells. The response of neutrophils (14.03 f 5.34 nmol) treated with activated T cells pretreated with anti-TNF-a mAb was not significantly different from that of activated T cells not treated with the antibody. Similar results were observed with mAb to TNF-P. The neutrophil v) fMLP activated fMLP + activated T cells T cells superoxide response (4.31f 0.23 nmol) was not affected Figure 6. "Priming" effects of activated T cells (paraformaldehydeby addition of nonactivated T lymphocytes (3.35f 0.31 fixed] for the FMLP neutrophil superoxide response. Neutrophils were nmol) but was significantly increasedby the addition of pretreated or not treated with activated T cells and then examined for a n as mean f SEM of three FMLP response. The results are presented activated T cells (13.01f 0.05 nmol). When activated T experiments. Key: (D)FMLP: [B)activated T cells, or (8)activated T cells cells were pretreated withmAb the response was essenplus FMLP. * p < 0.001. compared with FMLP only; p < 0.001, compared with activated T cells only. tially the same (13.915 0.69 nmol). _+
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NEUTROPHIL ACTIVATION BY T LYMPHOCYTES
Acknowledgment. We are grateful to Marie-Therese Kaufmann for excellent technical assistance. The data shows that activated T cells stimulate neutrophil oxygen-dependent respiratory burst by close cell-cell REFERENCES contact. Paraformaldehyde-fixed, activated T cells were 1. Campbell, P. A. 1990. The neutrophil, a professional killer of bacalso able to stimulate this response in neutrophils. There teria. may be controlled by T cells.Clin. E x p . Immunol. 79: 141. was marked donor neutrophil variation in their responses 2. Palmer. D.G., N. Hogg, and P. A. Revell. 1986. Lymphocytes polymorphonuclear leukocytes, macrophages and platelets in synovium to the T cells. Because the cells were fixed it is unlikely involved by rheumatoid arthritis. A study with monoclonal antibodthat the effects observed were due to release of mediators ies. Pathology 18:431. by the activated T cells especially sincethe fixation time 3. Czuprynski, C. J., P. M. Henson. and P. A. Campbell. 1985. 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