Nerve growth factor suppresses apoptosis of murine neutrophils

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NERVE

GROWTH

FACTOR

SUPPRESSES APOPTOSIS NEUTROPHILS

1050-1056

OF MURINE

YukikoKamtanl, Kaori Usamil, Mitsuhiro Okadal, Shinya Shimizu2 andHiroshi Matsuda1* 1Departmentof Veterinary Surgery, Collegeof Agriculture, University of OsakaPrefecture, Sakai, Osaka593, Japan 2Laboratory of Immune Cytology, National Institute of Animal Health, TsukubaScienceCity, Ibamgi 305, Japan Received

June

23,

1992

SUMMARY: We investigated inhibitory activity of nerve growth factor (NGF) on apoptosisof murine peritoneal exudate neutrophils. During culture for 9 h, apoptotic cells were identified by morphological changesunder a light microscope:nuclearpyknosis and chromatin condensationwith or without cytoplasmic vacuolation. The apoptotic state was confirmed by DNA fragmentation indicating the endogenousendonucleaseactivation. When neutrophilswere incubated in the presence of NGF, the proportion of cells with the morphologicalchangeswas decreasedin a dosedependent manner,and the developmentof the characteristicDNA fragmentationwasrestricted. The apoptosissuppressingactivity of NGF wasabolishedby the addition of anti-NGF monoclonal antibody. These results suggest that NGF may suppressneutrophil apoptosis by preventing the endogenous endonucleaseactivation. 0 1992Academrc Press,1°C.

Neutrophils released from the bone marrow are the main cellular component rapidly emigratingfrom blood to areasof acuteinflammation;the major function of neutrophilsis regardedas phagocytosisof microorganismsand cellular debris [I]. The neutrophil movement is caused by various kinds of chemoattractantsfor neutrophilslargely producedat the inflammation sites. Blood neutrophilsconstitute two topologically distinct pools (circulating and marginatedpools) and their blood-half time is only several hours, Death of neutrophils occurs by the active process of programmedself-destruction termed apoptosis[2]. Apoptotic neutrophils are disposedby specific recognition and phagocytosisby macrophages[2,3]. However, little is known about the life spanof neutrophilsin blood vesselsand emigratedin tissuesunder inflammatory conditions. Nerve growth factor (NGF) is a neurotrophic polypeptide that is necessaryfor the survival, differentiation and function of basalforebrain cholinergic neuronsin the central nervous system, as well asperipheralsympatheticandembryonic sensoryneurons[4-71. The biological actions of NGF are mediatedthrough interaction with specific receptorsfound on normal or neoplastic target cells derived from the neuralcrest. In addition to its neurotrophicactivities, NGF has been shown to have broader biological effects to non-neuronaltissues,such asdegranulationof rat peritoneal mast cells *To whom correspondenceshouldbe addressed. Abbreviations used in this paper: NGF, nerve growth factor; GM-CSF, granulocytemacrophage colony-stimulating factor; G-CSF, granulocyte CSF, IL, interleukiq mAb, monoclonalantibody; MEM, minimumessentialmedium.

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[8], a shape change in platelets[9], and modulation of both T- and B-cell-mediated immune responses [ 10,l I]. Recently, Matsuda et al [ 121 also reported that NGF induced the phenotypic change from interleukin (IL) -3-dependent bone marrow-derived

cultured mast cells to connective tissue-type mast

cells. Granulocyte-macrophage

colony-stimulating

factor (GM-CSF) and granulocyte CSF (G-CSF)

not only regulate human neutrophil development and differentiation but also enhance its survival and functional properties [13-1.51. NGF also causes a significant stimulation of granulocyte differentiation from human peripheral blood and umbilical cord blood [16,17]. We recently demonstrated that NGF as well as rGM-CSF,

unlike IL-3, enhanced survival, phagocytosis

and

superoxide production of mature murine aeutrophils [18]. Since NGF synthesis is locally increased in various kinds of injuries to tissues containing neuronal cells [ 19,201, there is possibility that NGF may influence survival and functions of neutrophils in inflammatory

responses. In the present study,

we demonstrated that NGF suppressed apoptosis of murine peritoneal neutrophils, suggesting that NGF produced in response to a variety of inflammatory stimuli may enhance neutrophil survival by suppressing apoptosis.

MATERIALS

AND

METHODS

Cytokines and other reagents: Allchemicals used were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan), unless otherwise indicated. NGF isolated in the 2.5s form from murine submaxillary glands was provided by Drs. A. M. Stanisz and J. Bienenstock(McMaster University, Ontario, Canada) [16,18]. Neurotropic biological activity of the purified NGF preparations was measured in the dissociated cell assay, using neonatal mouse superior cervical ganglion neurons; half-maximal response was at 1 rig/ml. The NGF preparations have no endotoxin activity by limulus assay even at a high concentration (1 pg/ml) [ 181. Purified murine IL-3 and murine rGM-CSF were obtained from Genzyme (Boston, MA). Purified murine IgG monoclonal antibody (n&b) to 2.5s NGF (clone pl) was a kind gift from Dr. E. M. Shooter (Stanford University, Stanford, CA). Control murine IgG m.4b was obtained from Becton Dickinson Immunocytometry Systems (Mountain View, CA). Cell isolation and culture: Male C57BL/6NJcl mice (8 to 15 weeks of age) purchased from Clea Japan (Tokyo, Japan) were injected intraperitoneally with 2 ml of 0.2% sodium caseinate in phosphate-buffered saline. Three h later, peritoneal exudate cells were collected by lavage of the peritoneum of each mouse with a total volume of 10 ml of minimum essential medium (MEM, GIBCO Laboratories, Garand Island, NY) containing 10 U/ml heparin. The cells were washed with MEM and fractionated on Percoll (Pharmacia Fine Chemicals, Uppsala, Sweden) density gradients according to the method described by Hart et al [2 11. The final purity of neutrophils was mom 90%. The cells were resuspended at 2 X lo5 cells/ml in a-MEM (GIBCO Laboratories) containing 10% fetal calf serum with or without each cytokine. Cultures were performed using 96-well tissue culture plates (Nunc, Roskilde, Denmark). Each well contained 50 pl of the cell suspension. Plates were incubated at 37’C in a fully humidified atmosphere of 5% CO2 in air. At the designated time points, the viability of the cells was assessed by trypan blue exclusion. The remaining samples were immediately spun in a cytocentrifuge (Cytospin, Shandon Southern, Elliott, IL). The Cytospin preparations were fixed in methanol and stained with Giemsa, and then for assessment of the percentage of cells showing morphology of apoptosis, 200 cells/slide were examined. Electrophoresis of LLNA: Isolation and electrophomsis of neutrophil DNA were carried out by using a modification of the technique described by Smith et al [22]. A total of 106 neutrophils was incubated in 20 ,ul cell lysis buffer [lo mm01 EDTA, 50 mm01 Tris-HCl @H S.O), 0.5% sodium dodecyl sulfate and 0.5 mg/ml proteinase K] at 50°C for 1 h in a water-bath. After addition of 30 pl 40 mm01 Tris-HCl, 1 mm01 EDTA (TE buffer, pH 8.0), crude DNA preparations were extracted twice with an equal volume of phenol/chloroform. DNA in the isolated aqueous phase was 1051

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precipitated for 30 mm in 0.1 volume 3 mol NaOAc and 2.5 volume 100% ethanol at -8O’C. The DNA precipitates were recovered by centrifugation at 18,500 x g for 5 min at 4’C!, washed with 70% ethanol, dried in the vacuum for 5 mitt, and dissolved in 20 ~1 of TE buffer. RNase A (heat treated to inactivatecontaminant DNase activity) was added to a concentration of 0.2 mg/ml and incubation at 37°C was continued for 1 h. Samples were heated to 70°C for 10 mm, mixed with 5 pl 0.25% bromophenol blue and 40% sucrose, and then loaded into each well of a 2% agarose gel containing 0.5 &ml ethidium bromide. Electrophoresis was carried out in 40 mm01 Tris-acetate, 1 mm01 EDTA (pH 7.5) at 100 V, 80 mA, until the marker dye had migrated 3-4 cm.

RESULTS Apoptosis

of

neutrophils

isolated

from

the

peritoneal

cavity:

Marked, time-related

morphological changes were observed under a light microscope in murine peritoneal neutrophils aged in culture for 9 h. The aging neutrophils showed nuclear pyknosis or chromatin condensation with or without cytoplasmic vacuolation (Fig 1). Since cells undergoing apoptosis show DNA fragmentation wntaining

-2OO-bp multiple pattern exhibiting endogenous endonuclease activation [23-251, we

carried out agarose gel electrophoresis of DNA extracted from aging neutrophils. As shown in Fig 2, the ladder with -2OO-bp typical steps of endonuclease activation was observed in neutrophils aged for 6 h, but not in those before the culture. Thus, apoptotic state of aging neutrophils was demonstrated by the closely related morphological and biochemical changes [2].

Fii. 1. cells) were Cytocentrifuge

Morphological features

of aging

neutrophils.

Murine

peritoneal

neutrophils

(104

incubated in 50 yl of a-MEM containing 10% fetal calf serum at 37“C for 9 h. preparations were stained with Giemsa. Arrowheads show the cells with apoptosis.

J&&. Agamse gel electrophoresis of DNA isolated from aging neutrophils. Lane 2, DNA from 106 neutrophils before incubation; lane 3, DNA from 106 neutrophils after incubation for 6 h; lane 1, molecular size standards (#X174/HaeIII digest, Marker 4, Nippon Gene, Tokyo, Japan). Molecular sizes in bp are indicated on the left for the gel.

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lm

Viability

*

20

T

*

50

500

-

03 O 3

6 Time

g I2

04

0

0.5 NGF

(h)

5

(nglml)

&.J. Time course of apoptosis and viability (m) of neutrophils. Viability was checked for neutrophils aged in control medium by trypan blue exclusion. 0 , control medium; A, 50 U/ml K3; l ,50 rig/ml NGF, A, 50 U/ml rGM-CSF. Each point represents the mean of eight to seventeen experiments. w. Apoptosis-suppressing effect of NGF to murine neutrophils. When 104 neutrophils were incubated for 9 h with various concentrations of NGF, the proportion of cells with apoptosis was examined. Data represent the mean f SE of ten experiments. *Significantly lower than control medium at P < .OS by Student’s t test.

Effect of NGF, rGM-CSF, or IL.-3 on neutrophil incubated

for up to 9 h in medium

apoptosis: Peritonealneutrophilswere

alone or in the presence

of NGF, rGM-CSF

or K-3,

and the

percentageof cellsundergoingapoptosiswasexamined.In the culture mediumalone, the proportion of cells showing the typical featuresof apoptosiswas rapidly increasedand was a maximal level of 40% at 9 h in culture, whereasmorethan 92% of cells excluded trypan blue even at 9 h (Fig 3). On the other hand, the addition of 50 rig/ml NGF to the culture medium lowered the percentageof neutrophils with apoptosis,which was about half the value obtainedfrom incubation in the culture medium alone for 3 to 9 h (Fig 3). The addition of 50 U/ml rGM-CSF lowered the proportion of neutrophilswith apoptosisin a fashionroughly similarto 50 rig/ml NGF. In the presenceof 50 U/ml IL-3, however, the proportion of cells showing apoptosiswas comparableto that in the culture medium alone(Fig 3). Peritonealneutrophilswere incubatedwith various concentrations(0.5 to 500 rig/ml) of NGF, andthe proportion of cells with apoptosiswas examined at 9 h after the initiation of the culture. As shown in Fig 4, NGF induced a dose-dependentdecreasein the proportion of neutrophils with apoptosis, by less than half as compared with the culture medium alone when added at a concentrationof 500 rig/ml. Effect of anti-NGF mAb on apoptosis suppression: In the next experiment, the specificity of the apoptosis-suppressing effect of NGF was examined in morphological observation and in agarosegel electrophoresisof DNA, using anti-NGF tnAb (clone pl). When neutrophils were 1053

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Table 1 of the inhibitory effect of NGF on neutrophil apoptosis by anti-NGF mAb*

Cytokine None NGF NGF NGF NGF NGF rGM-CSF rGM-CSF

Apoptosis (%)t

mAb

40.9 19.6 19.7 25.8 32.7 39.2 19.4 18.8

None None Control (10 pg) Anti-NGF (2 pg) Anti-NGF (10 pg) Anti-NGF (20 pg) Control (10 ug) Anti-NGF (10 pg)

f 2.6 k 1.2$ + 0.9$ + 3.2* + 3.15 _+3.75 k 0.6$ ? 0.6f

* Neutrophils (104 cells) were incubated for 9 h in medium containing 50 rig/ml NGF or 50 U/ml rGM-CSF with various concentrations of anti-2.5s NGF mAb or control IgG mAb. t Values were expressed as the mean ? SE of eight to fifteen experiments. $ P < .002, when compared with medium alone. 0 P < .002, when compared with NGF alone, NGF + control mAb, rGM-CSF + control mAb, and rGM-CSF + anti-NGF mAb.

incubated for 9 h, the proportion of cells showing apoptosiswas examined in various culture conditions (Table 1). The proportion of neutophils with apoptosiswas decreasedin the culture supplementedwith 50 rig/ml NGF by lessthan half as comparedwith the culture medium alone. Neutrophils incubatedwith a fixed doseof NGF (50 ngjml) and increasingdosesof anti-NGF mAb showeda dose-dependentincreasein the proportion of cells with apoptosis.In contrast, the addition of 10 pg/ml control IgG mAb to 50 rig/ml NGF did not neutralize the effect of NGF. Neither antiNGF mAb nor control mAb abrogatedthe apoptosis-suppressing effect of rGM-CSF. DNA from neutrophils aged in culture for 6 h was loaded (Fig 5). The DNA fragmentation exhibiting endonucleaseactivation wasobservedin neutrophilsagedin the culture mediumalone, but not in the culture mediumwith 50 rig/ml NGF. The addition of anti-NGF mAb abolishedthe effect of NGF. rGM-CSF (50 U/ml) alsoinhibited developmentof the DNA fragmentation in neutrophils, but

12

3

4

5

6

Fie. Agarose gel electrophomsis of DNA isolated from 106 neutrophils aged for 6 h in five different culturemedia.Lane2, control medium;lane3, NGF (50 n&ml); lane4, NGF (50 rig/ml) + anti-NGF mAb (10 p&nl); lane 5, rGM-CSF (50 U/ml); lane 6, rGM-CSF (50 U/ml) + anti-NGF mAb (10 l&ml); lane 1, molecular size standards (oX174/HaeIII digest, Marker 4). Molecular sizes in bp are indicated on the left for the gel.

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the effect of rGM-CSF was not neutralized by the addition of anti-NGF mAb. Control IgG mAb did not influence the activity of NGF or rGM-CSF.

DISCUSSION Apoptosis essentially

is generally regarded as an active process of endogenous cell death which

contributes

to many physiological

responses, such as embryonic development and

selective depletion of some populations in immunocompetent cells [22-261. During the process of apoptosis,

cells exhibit

typical

morphological

changes, namely,

nuclear condensation

and

oligonucleosomal DNA fragmentation which are attributable to endogenous endonuclease activation through little known cellular signalling [27]. Short-term cultures of neutrophils obtained from the peritoneal cavity after stimulation with sodiumcaseinate led to the time-related nuclear morphological changes and DNA fragmentation representing the apoptotic state. The morphological and biochemical features were comparable with the result of human neutrophils reported by Savill et al [2]. Since murine neutrophils undergoing apoptosis could be identified by the closely related morphological and biochemical changes, we examined effect of NGF on apoptosis of murine peritoneal neutrophils. The results obtained, demonstrated that, under the condition tested, 2.5s NGF suppressed the apoptosis of murine neutrophils.

Our previous work showed that NGF prolonged the survival of murine

blood and peritoneal neutrophils over 5 days in culture [ 181. Thus, we consider that the viabilitysustaining activity of NGF is attributed to suppressing apoptosis. NGF is not detectable in the serum of normal adult mice [5], but aggressive behavior (fighting stress) rapidly increases serum NGF levels up to 300 rig/ml [28]. Exercise, excitement, infection, inflammation

and many types of stress are known to lead to neutrophilia. These give rise to a

possibility that neutrophils increased in the process of inflammation and stress are influenced by NGF in the bloodstream. In addition to neuronal tissues, synthesis of NGF was detected in various peripheral tissues including epithelial cells, smooth muscle cells, and fibroblasts [29]. Ling et al [30] showed that both the conditioned medium from a human fibroblast line (HF-15) CSF markedly

sustained the neutrophil survival. However,

sustaining activity in the fibroblast-conditioned

and human rGM-

they failed to abolish the viability-

medium by using anti-serum

to GM-CSF,

and

suggested the existence of some soluble factor sustaining the survival of human neutrophils distinct from GM-CSF.

NGF is spontaneously secreted from chick and murine fibroblast

Therefore, there is a possibility that fibroblast-derived

lines [L&31].

NGF might affect the survival of neutrophils

emigrated in local tissues.

ACKNOWLEDGMENTS: We would like to thank Drs. J. Bienenstock and A. M. Stan& of M&aster Univemity for providing ultrapurified NGF and Dr. E. M. Shooter of Stanford University for providing antr-2.5s NGF mAb. We also thank Drs. Y. Imai and M. Komori of Department of Molecular Biology for technical advice about electrophoresis of DNA and for use of their facilities. This work was supported by the Basic Research Core System, the Special Coordination Fund for Promoting Science and Technology in Japan. 1055

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REFERENCES 1.

2. 3. :. 6: 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 2 25: E: 28. 29. 3°F

Wilhelm, D. L. (1971), In Pathology: Inflammation and Healing (W. A. D. Anderson Ed.), p 14-67. The CV Mosby Company, St. Louis. Savill, J. S., Wyllie, A. J., Henson,J. E., Walport, M. J., Henson, P. M., and Haselett, C. (1989) J. Clin. Invest. 83,865-875. Duvall, E., Wyllie, A. H., and Morris, R. G. (1985) Immunology 56,351-358. Levi-Montalcini, R., and Angeletti, P. U. (1963) Dev. Biol. 7,653-659. Thoenen, H., and Barde, Y. A. (1980) Physiol. Rev. 60,1284-1335. Yankner, B. A., and Shooter, E. M. (1982) Ann. Rev. B&hem. 51,845-868. Korsching, S. (1986) Trends. Neurosci. 9,570-577. Pearce,F. L., and Thompson,H. L. (1986) I. Physiol. 372,379-393. Gudat, F., Laubscher, A., Otten, U., and Pletscher, A. (1981) Br. J. Pharmacol. 74,533538. Manning, P. T., Russell, J. H., Simmons, B., and Johnson, E. M. (1985) Brain Res. 340,61-69. Otten, U., Ehrhard, P., and Peck, R. (1989) Proc. Natl. Acad. Sci. USA 86,10059-10063. Mats&, H., Kammn, Y., Ushio, H., Kiso, Y., Kanemoto, T., Suzuki, H., and Kitamura, Y. (1991) J. Exp. Med. 174,7-14. Weisbart, R. H., Golde, D. W., Clark, S. C., Wang, G. G., and Gasson, J. C. (1985) Nature 314,361-363. Begley, C. G., Lopez, A. F., Nicola, N. A., Warren, D. J., Vadas, M. A., Sanderson,C. J., and Metcalf, D. (1986) Blood 68,162-166. Lopez, A. F., Nicola, N. A., Metcalf, D., Burgess, A. W., Battye, F., Swell, W., and Vadas, M. A. (1983) J. Immunol. 131,2983-2988. Mat&a, H., Coughlin, M. D., Bienenstock, J., and Denburg, J. A. (1988) Proc. Natl. Acad. Sci. USA 85,6508-6512. Mats&, H., Switzer, J., Coughlin, M. D., Bienenstock, J., and Denburg, J. A. (1988) Int. Arch.. Allergy Appl Immunol. 86,453-457. Kamran, Y., Ushio, H., Koyama, H., Oikawa, M., Yoshihara, T., Kaneko, M., and Matsuda, H. (1991) Blood 77,1320-1325. Windebank, A. L., and Poduslo,J. (1986) Brain Res. 385197-200. Raivich, G., and Kmutzberg, G. W. (1987) J. Neurocytol. 16,689-700. Hart, P. H., Spencer,L. K., Nulsen, M. F., McDonald, P. J., and Finlay-Jones, J. J. (1986) Infect. Immun. 51,936-941. Smith, C. A., Wrlhams, G. T., Kingstonm, R., Jenkinson, E. J., and Owen, J. T. (1989) Nature 337,181-184. Cohen, J. J., and Duke, R. C. (1984) J. Immunol. 132,38-42. Wyllie, A. H., and Morris, R. E. (1985) Am. J. Pathol. 109,78-87. Ucker, D. S. (1987) Nature 327,62-64. Wyllie, A. H., Kerr, J. F. R., and Currie, A. R. (1980) Iut. Rev. Cytol. 68,251-306. McConkey, D. J., Orrenius, S., and Jondal, M. (1990) Immunol. Today 11,120,-121. Aloe, L., Alleva, E., Bohm, A., and Levi-Montalcini, R. (1986) Proc. Natl. Acad Sci. USA 83,6184-6187. Brandtlow, C. E., Heumann,R., Schwab, M. E., and Thoenen, H. (1987) EMBO J. 6,891899. Ling, C. J., Owen, W. F. Jr, and Austen, K.F. (1990) J. Clin. Invest. 85,601-604. Young, M., Oger, J., Blanchard, M. H., Asdourian, H., Amos, H., and Amason, B. G. W. (1974) Science 187,361-362.

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