[CANCER RESEARCH 43, 5857-5861, December 1983]

Stimulation of Rat Peritoneal Mast Cell Migration by Tumor-derivedPeptides1

Thomas J. Poole and Bruce R. Zetter2

Departmentsof Physiologyand Surgery, Children's Hospitaland Harvard Medical School, Boston,Massachusetts02115

ABSTRACT

The accumulation of mast cells is characteristic of a numberof pathological states. We demonstrate here the directionalmotility of mast cells in vitro in response to tumor-derived pep-

tides. Rat peritoneal mast cells were isolated on Percoli gradientsand maintained in serum-free medium containing transferrin,

albumin, soybean lipid, and cholesterol. The isolated mast cellsmigrated under agarose in response to medium conditioned byany of eight tumor cell lines but not to medium conditioned byany of a variety of nontumorigenic cell types. The tumor-derived

activity is dialyzable (cutoff, M, 3500), stable to trypsin treatmentand to heating at 56°,but destroyed by heating to 100°or by

treatment with Streptomyces griseus protease or carboxypepti-

dase A. Ultrafiltration suggests a molecular weight of 300 to 1000.Two tripeptides, glycylhistidyllysine and A/-formylmethionyl-leucylphenylalanine, were also found to be potent chemoattrac-tants for mast cells. A/-Formylmethionylphenylalanine and valyl-

glycylserylglutamic acid (eosinophil chemotactic Factor A) hadsignificantly less chemoattractant activity over the same rangeof concentrations. Several peptide analogues of glycylhistidyllysine were tested and found to have no activity. The growth ofcapillary blood vessels toward a growing tumor is generallypreceded by an accumulation of mast cells at the tumor site.Based on the results presented here and previous data from ourlaboratory on mast cell stimulation of capillary endothelial cellmigration, we propose an hypothesis that the chemoattractionof mast cells by tumor-derived peptides may be an important

early event in tumor neovascularization.

INTRODUCTION

The perivascular localization of mast cells, along with theirability to release vasoactive agents, has led to speculation thatmast cells may play a supportive role in the maintenance ofmature blood vessels and perhaps also in the growth of newblood vessels (13, 21, 25,26). Although the majority of researchin recent years has concentrated on the unique capacity of themast cell to package and selectively release a variety of potentbiochemicals including those mediating hypersensitivity and vascular permeability (15,18, 32, 38), it is clear that the regulationof the number and location of mast cells is also of great biologicalsignificance. Mast cells are found at increased densities at thesites of several pathological conditions that are associated withhigh rates of new capillary growth (neovascularization) and alsoin several other pathological conditions. These include psoriasis(16), urticaria pigmentosa (26), immune reactions (29), heman-

1Supported by Grant CA28540 from the USPHS, National Cancer Institute. A

preliminaryreport of these results was presented at the meeting of the AmericanSociety of Cell Biology, November 1981 (22).

2Glick-Franzheim cancer research scholar. To whom requests for reprintsshould be addressed.

ReceivedJanuary 28,1983; accepted August 10, 1983.

giomas (benign vascular tumors) (8), and various malignant tumors (3, 10,13, 28, 33, 36).

It is unclear presently whether the mast cells that accumulateat these sites do so as a result of (a) the migration of fullydifferentiated mast cells from nearby sites or (b) the differentiationof a precursor cell type already located at the site. To answerthis question, it is first necessary to demonstrate that mast cellswill migrate in response to relevant chemical stimuli.

Migration of blood basophils has been shown previously to bestimulated by the complement component C5a (12, 14) and bya partially purified factor derived from stimulated lymphocytes(14). Although mast cells and basophils are similar in their met-

achromasia and content of histamine and heparin, they havedistinct ultrastructural, enzymatic, and chemical properties (4,7).It has not yet been established whether chemoattractants forblood basophils will have similar activity for tissue mast cells.

In the present study, we have investigated the migratoryresponse of rat peritoneal mast cells to factors released from avariety of normal and tumor-derived cells. Using a modificationof the "chemotaxis under agarose" assay developed by Nelson

ef al. (17) for the study of leukocyte chemotaxis, we havedetermined that mast cells move toward chemoattractants produced by each of the 8 tumor cell types studied. Nontransformedcells failed to produce such factors. Our preliminary biochemicalcharacterization suggests that the tumor-derived chemoattractants for mast cells are small peptides.

MATERIALS AND METHODS

Mast Cell Isolation. Peritoneal cell suspensions were obtained from300- to 400-g male Sprague-Dawley rats by peritoneal lavage. Peritoneal

lavages (50 ml/rat) were obtained in 4-(2-hydroxyethyl)-1-piperazineeth-anesulfonic acid-buffered Medium 199 (Grand Island Biological Co.,Grand Island, N. Y.) supplemented with 0.1% crystalline BSA3 (Calbi-ochem-Behring, La Jolla, Calif.). The cell mixture was pelleted and

resuspended in 0.75 ml of the same medium, mixed with 3.5 ml ofisotonic Percoli (Pharmacia Fine Chemicals, Inc., Piscataway, N. J.)solution (1.11 g/ml), and overlaid with 0.5 ml of medium (5). Centrifugationto equilibrium (125 x g, 20 min) on such a one-step Percoli gradientroutinely yielded greater than 97% pure mast cell suspensions. Afterscreening several different media, we found that incubation in a serum-free medium containing transferrin, albumin, soybean lipid, and cholesterol (Synmed; Centaurus Biologicals Corp., Anaheim, Calif.) improvedthe subsequent survival of mast cells in culture, as demonstrated bytrypan blue exclusion and minimal degranulation over a 10-hr period.

Migration Assay. Mast cell migration was measured using a modification of the agarose technique designed by Nelson ef al. (17) for usewith human leukocytes. Falcon 35-mm tissue culture plastic dishes were

precoated by spreading 50 pi of a 1% aqueous solution of crystallineBSA onto the dish with a rubber policeman and drying in the air streamof a laminar flow tissue culture hood. In a similar manner, other substratecoatings were tested in preliminary studies. Undiluted calf serum (Flow

3The abbreviations used are: BSA, bovine serum albumin; N-fMet, N-formyl-methionyl;DME, Dulbecco's modified Eagle's medium.

DECEMBER 1983 5857

on July 21, 2021. © 1983 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

T. J. Poole and B. R. Zetter

Laboratories, Inc., Rockville, Md.) and collagen (Vitrogen; Flow Laboratories) were air dried on théculture dishes after spreading; gelatin (DifcoLaboratories, Inc., Detroit, Mich.) was dried as a 1.5% solution in calcium-and magnesium-free Dulbecco's phosphate-buffered saline (Grand Island

Biological Co.) and fibronectin (Clg; Collaborative Research, Waltham,Mass.) at a concentration of 50 Mg/ml in distilled water. Lytex agarose(2.4%, type HSA; Accurate Chemical and Scientific Co., Westbury,N. Y.) was mixed with an equal volume of 2x Medium 199 supplementedwith 0.2% BSA. Agarose solution (2.5 ml) was pipetted into each 35-mmdish, allowed to gel at room temperature, and then chilled to 4°.Three

holes, 3 mm in diameter and 3 mm apart, were punched with a templateand an Ouchteriony punch (Miles Laboratories, Inc., Elkhart, Ind.). Theplugs were removed with a hypodermic needle or by a vacuum lineattached to the punch. Inverted dishes were preincubated at 37°in a

5% COz-humidified incubator. Any accumulated moisture was removed

from the wells by vacuum aspirator prior to.use. The center of the middlewell was marked with a hypodermic needle at x30. Mast cells (2 to 4 x10*) were added to the center well in 10 ¿ilof Synmed. The other well

received the test solution, either tumor cell- or normal cell-conditioned

Synmed or a test substance dissolved in Synmed. Synthetic peptidestested for activity were purchased from the following companies: Gly-His-Lys, N-fMet-Leu-Phe, and N-fMet-Phe from Calbiochem-Behring;Gly-His-Gly from Serva Fine Biochemicals; His-Lys-HBr and His-Gly-Glyfrom Vega-Fox Biochemicals, Tucson, Ariz.; and Val-Gly-Ser-Glu (ECF-

A) from Miles-Yeda, Ltd.After 4 hr of incubation, the dishes were fixed in phosphate-buffered

10% formalin overnight, the agarose was removed, and the dishes wererinsed with distilled water and air dried. They were then stained for 3 minwith Bismarck-Brown dye (0.5% in 50% ethanol acidified with 0.2%

acetic acid). The migration pattern was quantified by measuring lineardistances at x30 with an ocular micrometer in a Wild stereomicroscopeor with a digital readout stage micrometer on a Nikon profile projector atx20.

Cell Culture. Mouse Sarcoma 180 cells, XC rat sarcoma cells, andprimary expiants of rat kidney fibroblasts and bladder epithelial cellswere grown in Falcon 60-mm dishes or in T-25 or T-75 tissue culture

flasks in DME (Grand Island Biological Co.) with 10% calf serum (FlowLaboratories). Walker rat carcinoma cells were grown in DME with 10%fetal calf serum (Flow Laboratories). C6 rat glioma and MH1C1 Morrisrat hepatomas were grown in Ham's F-12 medium supplemented with

15% horse serum (Grand Island Biological Co.) and 2.5% fetal calf serum.Conditioned medium was prepared by replacing the growth medium ofdense, subconfluent cultures (approximately 5x10" cells/sq cm) with

serum-free Synmed for 48 hr. The medium was removed and centrifugedto remove floating cells. This conditioned medium was frozen (-20°)

until ready for testing when it was thawed and passed through a 0.45-^m filter. All media were supplemented with glutamine plus penicillin-

streptomycin sulfate solution (Irvine Scientific, Irvine, Calif.).Characterization Studies. Two-mi aliquots of tumor-conditioned me

dium were subjected to heat or enzyme treatments. Heat treatment wascarried out at 56°for 30 min or 100°for 2 min. Immobilized enzymes

were used to test protease sensitivity. Insoluble trypsin-polyacrylamide(porcine pancreas), insoluble Streptomyces griseus protease-cellulose,or insoluble carboxypeptidase A-agarose (bovine pancreas) (all from

Sigma Chemical Co., St. Louis, Mo.) were incubated with aliquots ofconditioned medium for 1 hr at room temperature at a final concentrationof 1 unit enzyme per ml conditioned medium. All immobilized enzymeswere washed with serum-free DME extensively before use. After 1 hr ofincubation, the immobilized enzyme was removed by centrifugation.Trypsin activity was determined by hydrolysis of tosyl arginine methylester using a spectrophotometric assay (11).

Conventional dialysis was performed with Spectrapore tubing (cutoff,M, 3,500) for 24 hr at 4° against 2 changes of Synmed. AmiconUltrafiltration was performed at 4°using either Model 202 (200 ml) or

Model 52 chambers. Conditioned medium was passed sequentiallythrough Amicon UM-10 (cutoff, M, 10,000), UM-2 (cutoff, M, 1,000), and

UM-05 (cutoff, M, 500). The filtrates and reténtales were tested for

activity in the agarose assay.

RESULTS

We routinely obtained suspensions of mast cells of greaterthan 97% purity using a single one-step Percoli gradient procedure as described in "Materials and Methods." Approximately2.5 x 105 mast cells can be obtained from each 350-g rat. The

serum-free medium (Synmed) conditioned by mouse sarcoma

cells was tested using a modification of the agarose assay ofNelson ef a/. (17). The conditions of our assay of 1.2% agarose,3-mm wells placed 3 mm apart, and 4-hr incubation are similar

to those used by others for leukocytes (17,24). We do, however,routinely coat the tissue culture plastic of our assay dishes withBSA, which gave us greater migration distances than with un-

coated dishes. The maximum chemotactic index in 4 hr wasinfluenced by precoating dishes as follows: BSA > calf serum >fibronectin > gelatin > collagen. The maximum response in 4 hrseems to reflect the theoretical limit of maintenance of a chemicalgradient of diffusion in such a configuration (37,39). The measurefrom the edge of the well containing mast cells to the cell frontfacing the test well was termed the stimulated distance, whereasthe distance from the edge to the cell front facing the controlwell was called the unstimulated distance. The ratio of thestimulated distance to the unstimulated distance was defined asthe "chemotactic index."

Specificity of Activity. Chart 1 shows the effect of increasingconcentrations of mouse sarcoma-conditioned medium on the

directional migration of rat peritoneal mast cells. The chemotacticindex shows a dose response with a peak at 50% sarcoma-

conditioned medium of 5.1 [mean of 12 plates ±0.5 (S.E.)]. Theresponse dropped to 2.5 ±0.6 at 100% (n = 6). Chart 2 shows

the activity produced by a variety of cell types. All conditionedmedia were prepared without added serum or plasma. Conditioned media from 7 additional types of tumor cells all showedsignificant activity, whereas conditioned media from 7 normal(nontumorigenic) cell types failed to demonstrate activity. Tumors

X

a5-

o

0 25 50 75 100% SARCOMA CONDITIONED MEDIUM

Chart 1. Response of purified mast cells to mouse sarcoma-conditioned medium. Dense cultures (1O4cells/sq cm) of mouse Sarcoma 180 cells were incubated

with fresh Synmed for 48 hr. Increasing concentrations of the conditioned mediumwere made by dilution with fresh Synmed, and the mixtures were added to testwells in the agarose assay as described in "Materials and Methods." A chemotactic

index of 5.0 would correspond to an absolute movement of approximately 250 ^min the direction of the test well in comparison with a movement of approximately50 i/m in the direction of the control well. Points, mean of this ratio; oars, S.E.

5858 CANCER RESEARCH VOL. 43

on July 21, 2021. © 1983 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

10-

uo

816 Lewis WolKer Hepatomo XC C6 ProstateLung Sarcoma Glioma

MOCK

Chart 2. Ability of tumorigenic and nontumorigenic cell types to produce mastcell chemoattractants. All conditioned media were mixed with an equal amount offresh Synmed immediately before testing. Mast cell motility was observed inresponse to conditioned medium from 8 tumor cell types but not to medium fromany of 7 nontumorigenic cell lines or primary expiants of normal tissues. The tumorcell lines tested were the rat Walker 256 carcinoma, Morris MhdCi hepatoma, XCsarcoma, C6 glioma, and prostate carcinoma and the mouse B-16 melanoma,Lewis lung carcinoma, and Sarcoma 180. Nontumorigenic cells tested includedlate-passage MDCK epithelial cells, fourth-passage human foreskin fibroblasts(HFF), fifth-passage bovine aortic smooth muscle cells, primary expiants of ratbladder and kidney, and purified rat peritoneal mast cells. Bars, S.E.

found to produce mast cell chemoattractants included the ratMorris hepatoma, C6 glioma, XC sarcoma, and Walker carcinoma, and prostate carcinoma, along with the mouse Sarcoma180, Lewis lung carcinoma, and B16 melanoma. The nontumorigenic cell types tested included early passage human foreskinfibroblasts, bovine aortic endothelial and smooth muscle cells,primary expiants of rat bladder and kidney, and the canine MDCKepithelial cell line.

Preliminary Biochemical Characterization of the Activity.The stability of mast cell chemotactic activity from tumor-condi

tioned medium to a variety of treatments was tested in a preliminary attempt to characterize the chemoattractant(s) (Table 1).The activity was stable to heating to 56°for 30 min. Heating to100°for 2 min produced a precipitate; the supernatant did not

retain activity. The tumor-derived activity was stable to treatment

with immobilized trypsin but was greatly reduced by an immobilized protease from S. griseus. Incubation with immobilized car-

boxypeptidase A completely eliminated the activity. Activity waslost from conditioned medium dialyzed extensively against alarge volume of normal Synmed in dialysis tubing with a molecular-weight cutoff of 3500. Conditioned medium held at 4°for

the same amount of time retained 90% of its activity. Whendialysis was carried out overnight in an equivalent small volume(3 ml) of medium, activity could be detected in both the dialysate

Peptide-stimulated Mast Cell Migration

(chemotactic index = 2.6) and the retentate (chemotactic index= 3.0). Sequential Amicon filtration (Table 1) showed that theprincipal activity existed in a molecular species which passedthrough filters with molecular-weight cutoff greater than 1000but was retarded by a filter with an approximate molecular-

weight cutoff of 500.Chemoattractant Peptides. Small peptides such as the N-

formylmethionyl peptides have been shown previously to bechemotactic for neutrophils and leukocytes (27). We thereforetested a number of peptides for their ability to stimulate mastcell motility. Two peptides were found to be potent chemoattractants for rat mast cells: Gly-His-Lys and N-fMet-Leu-Phe. Thedose response to Gly-His-Lys for mast cells in the agarose assayis shown in Chart 3. The maximum chemotactic index of 4.8 ±

Table 1Characterization of a mast cell Chemoattractant in sarcoma-conditioned medium

Proteolytic digestions were performed as described in 'Materials and Methods."

Treated samples were mixed with an equal volume of fresh Synmed before testing.

TreatmentUntreated56°,

30min100°,2minImmobilized

trypsinImmobilizedS. griseusproteaseImmobilizedcarboxypeptidaseADialysis

(cutoff, M,3500)UM-10filtrateUM

2filtrateUM-05filtrateChemotactic

index85.25.31.15.12.30.81.45.05.21.0

* Mean of duplicate samples from 3 separate experiments (6 assays).

XLJO

O

Ü

LuIO

3-

2-

100 200 300 400 500

Chart 3. Mast cell migration in response to the synthetic peptide Gly-His-Lys(GHL). Maximum chemotactic index was obtained when the stimulus was addedto the test well at a concentration of 200 /¿g/ml.Bars, S.E.

DECEMBER 1983 5859

on July 21, 2021. © 1983 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

T. J. Paole and B. R. Zetter

X

io

lo

yIu

GLY - HIS-LYS GLY-HB-GLY HIS-LYS HS-GLY-GLY FMET-LEU-PHE FMËT-PHE VW.-GLY-SCR-GLU

Chart 4. Response of mast cells to several synthetic peptides. Peptides weretested at doses ranging from 0.2 to 500 >ig/ml. Representative data are shown foreach peptide at 200 fig/ml. Bars, S.E.

0.5 (S.E.) at 200 pg/m\ is approximately the same as the maximum response to tumor-conditioned medium. Higher concentra

tions of peptide gave lower chemotactic indices in a patternsimilar to that seen with tumor-conditioned medium. Severalpeptide analogues of Gly-His-Lys had little or no activity at 200¿ig/ml:chemotactic indices = 1.7 for Gly-His-Gly (S.E. = 0.7);1.6 for His-Lys (S.E. = 0.6); and 1.5 for His-Gly-Gly (S.E. = 0.5)(see Chart 4). Whereas the chemotactic index for 200 ^g of N-fMet-Leu-Phe per ml was 4.0, that for N-fMet-Phe was 1.7 at

the same concentration. All peptides were tested at 0.2, 2.0,20,50, and 500 fig/mi, and in no case did any other concentrationgive a higher chemotactic index than that shown for 200 /¿gofthe same peptide. The eosinophil chemotactic peptide Val-Gly-Ser-Glu was tested and found to possess a moderate amountof activity that was consistently lower (chemotactic index = 3.0±0.8) than that seen with either Gly-His-Lys or N-fMet-Leu-Phe.

DISCUSSION

The data presented here demonstrate that tumor cells producea substance (or substances) that can induce the directionalmigration of rat peritoneal mast cells. This activity was found inculture medium conditioned by 8 different tumor cell types butnot in medium conditioned by human foreskin fibroblasts, WI-38human lung fibroblasts, MDCK epithelial cells, bovine aorticsmooth muscle or endothelial cells, primary expiants of rat kidneyfibroblasts, rat bladder epithelium, or mast cells themselves.Characterization and partial purification suggest that the tumor-derived activity resides in a small peptide. The active material isstable to trypsin treatment and to heating to 56°but is destroyedby a broad-range bacterial protease and by the exopeptidase,

carboxypeptidase A. Dialysis and ultrafiltration demonstrate amolecular weight of approximately 300 to 1000.

In addition, we have found that the naturally occurring tripep-tide Gly-His-Lys and the synthetic peptide N-fMet-Leu-Phe are

potent chemoattractants for rat peritoneal mast cells. Four otherpeptides we tested did not stimulate migration in the agaroseassay. N-fMet-Leu-Phe is the much-studied peptide capable ofstimulating chemotaxis of a wide variety of cell types (30). Gly-His-Lys has been shown previously to stimulate the growth of

liver cells in culture and to promote the survival of macrophages,mast cells, and eosinophils in vitro (19, 20) but has not beenshown previously to be a chemoattractant.

The experiments reported above provide evidence that mastcells are motile and can move in the direction of a gradient of

tumor-derived peptides as well as toward synthetic peptides.

The chemotactic index as originally defined by Nelson ef al. (17)probably reflects both stimulation of random migration (chemo-kinesis) and directed migration (chemotaxis). As the cells moveacross the relatively long distance (3 mm) between the wells,they encounter a relatively broad gradient with a large differencebetween the first and last concentrations encountered (39). Inthe course of traversing this gradient, the cells may be exposedto some concentrations with optimal chemotactic activity andothers with optimal chemokinetic activity, or both phenomenamay take place simultaneously. This is in contrast to assayswhich use 2 fluid-filled chambers separated by a thin membrane

where the gradient is sharper and more easily manipulated todetermine differences between random and directed movements(40). Unfortunately, we have been unable to successfully usefilter assays with mast cells and have therefore had to rely solelyon the agarose assay in this study. It would appear probable,however, that the gradients encountered by mast cells movingthrough connective tissues toward a tumor would more closelyresemble the broad gradients formed in the agarose assay thanthe sharp step gradients formed in the filter assays and that bothchemotaxis and chemokinesis are likely to play a role in these invivo movements.

The present findings may have relevance to the sequence ofevents in tumor-induced neovascularization (tumor angiogene-

sis). Previous results from this laboratory have demonstratedthat mast cell accumulation at a tumor site precedes the growthof new capillaries toward a tumor (13) and that heparin releasedfrom mast cells can stimulate the migration of cultured capillaryendothelial cells (2). Ausprunk and Folkman (1) had reportedearlier that capillary endothelial cell migration is an essentialcomponent of tumor angiogenesis. As a working model, wepropose that mast cells chemically attracted to tumor nodulesmay secrete heparin within the collagenous stroma leaving atrack or path behind them. This mast cell track could then serveto stimulate and direct the migration of capillary sprouts towardthe tumor. Such a model requires that mast cells be capable ofdirectional migration toward a tumor stimulus, and such evidenceis presented here. The model also requires that mast cells depositheparin as they move between the host blood vessels and thetumor nodule. This could be accomplished by degranulation ofsome mast cells along the path of migration or by the gradualrelease of heparin from individual mast cells as they migrate.Recently, it has been shown that other constituents of mast cellgranules can be released in a slow, graded manner (18, 35). Arole for heparin in neovascularization is further substantiated bythe ability of protamine, a heparin antagonist, to inhibit angiogenesis in vivo (34).

The experiments reported above provide evidence that mastcells are motile and can migrate toward tumor-derived peptidesas well as the synthetic peptides Gly-His-Lys and W-fMet-Leu-

Phe. If mast cells do play a role in modulating neovascularization,then the mast cell migration assay described here may proveuseful in identifying enhancers and inhibitors of angiogenesis thatoperate at the level of the mast cell. In this regard, it is ofparticular interest that a complex of Gly-His-Lys and copper has

been reported recently to promote neovascularization in vivo(23). The ability of other modulators of mast cell motility tostimulate or inhibit the growth and maintenance of blood vesselsis under investigation currently.

5860 CANCER RESEARCH VOL. 43

on July 21, 2021. © 1983 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Peptide-stimulated Mast Cell Migration

ACKNOWLEDGMENTS

We thank Dr. J. Fdkman, Dr. J. Glowacki, Dr. M. Klagsbrun, Dr. P. D'Amore,

and Dr. J. Stone for their helpful discussions and Pauline Breen and Mary JoCanavan for their help in preparing the manuscript. Animals used in this study weremaintained in accordance with the guidelines of the Committee on Animals of theHarvard Medical School and those prepared by the Committee on Care and Useof Laboratory Animals of the Institute of Laboratory Animal Resources, NationalResearch Council [Department of Health, Education, and Welfare Publication (NIH)78-23, revised in 1978],

REFERENCES

1. Ausprunk, D., and Folkrnan. J. Migration and proliferation of endothelial cellsin preformed and newly formed blood vessels during tumor angiogenesis.Microvasc. Res., 14: 53-65, 1977.

2. Azizkhan, R. G., Azizkhan, J. C., Zetter, B. R., and Folkman, J. Mast cellheparin stimulates migration of capillary endothelial cells in vitro. J. Exp. Med.,752:931-944,1980.

3. Baroni, C. On the relationship of mast cells to various soft tissue tumours. Br.J. Cancer, 18: 686-691,1964.

4. Cline, M. J. The White Cell. Cambridge, Mass.: Harvard University Press, 1975.5. Enerback, L., and Svensson, J. Isolation of rat peritoneal mast cells by

centrifugation on density gradients of Percoli. J. Immunol. Methods, 39: ISSUS, 1980.

6. Folkman, J., and Cotran, R. Relation of vascular proliferation to tumor growth.Int. Rev. Exp. Pathol., 76: 207-248,1976.

7. Galli, S. J., and Dvorak, H. F. Basophils and mast cells: structure, function,and role in hypersensitivity. In: S. Gupta and R. A. Good (eds.), Cellular,Molecular, and Clinical Aspects of Allergic Diseases, pp. 1-53. New York:Plenum Publishing Corp., 1979.

8. Glowacki, J., and Mulliken, J. B. Mast cells in hemangiomas and vascularmalformations. Pediatrics, 70: 48-51,1982.

9. Goetzl, E. J., and Austen, K. F. Purification and synthesis of eosinophilotactictetrapeptides of human lung tissue: identification as eosinophil chemotacticfactor of anaphylaxis. Proc. Nati. Acad. Sci. U. S. A., 72: 4123-4127,1975.

10. Greggio, H. Les cellules granuleuses (mastzellen) dans les tissus normaux etdans certains maladies chirurgicales. Arch. Med. Exp., 23: 323-375,1911.

11. Hummel, B. C. W. A modified spectrophotometric determination of chymotryp-sin, trypsin, and thrombin. Can. J. Biochem. Physid., 37:1393-1399,1959.

12. Kay, A. B., and Austen, K. F. Chemotaxis of human basophil leukocytes. Clin.Exp. Immunol., 11: 557-563,1972.

13. Kessler, D. A., Langer, R. S., Pless, N. A., and Folkman, J. Mast cells andtumor angiogenesis. Int. J. Cancer, 78: 703-709,1976.

14. Lett-Brown, M. A., Boetcher, D. A., and Leonard, E. J. Chemotactic responsesof normal human basophils to C5a and to lymphocyte-derived chemotacticfactor. J. Immunol., 777: 246-252,1976.

15. Lewis. R. A., and Austen, K. F. Mediation of local homeostasis and inflammationby leukotrienes and other mast cell-dependent compounds. Nature (Lond.),293:103-108,1981.

16. Mottaz, J. H., Zelickson, A. S., Thome, F. G., and Wachs, G. Blood vesselchanges in psoriatic skin. Acta Derm-Venereol., 53:195-198,1973.

17. Nelson, R. D., Quie, P. G., and Simmons, R. L. Chemotaxis under agarose: anew and simple method for measuring Chemotaxis and spontaneous migrationof human polymorphonuclear leukocytes and monocytes. J. Immunol., 775:1650-1656,1975.

18. Oertel, H. L., and Kaliner, M. The biological activity of mast cell granules. III.Purification of inflammatory factors of anaphylaxis (ICF-A) responsible forcausing late-phase reactions. J. Immunol., 727:1398-1402,1981.

19. Pickart, L„Freedman, J. H., Loker, W. J., Peisack, J., Perkins, C. M.,Steinkamp, R. E., and Weinstein, B. Growth-modulating plasma tripeptide mayfunction by facilitating copper intake into cells. Nature (Lond.), 288: 715-717,1980.

20. Pickart. L., and Thaler, M. M. Growth-modulating plasma tripeptide: relationship hetween molecular structure and DNA synthesis in hepatoma cells. FEBSLett., 704:119-122,1979.

21. Plonsky, L. D., and Boyles, J. Vascular associations of mast cells in thediaphragm. Microvasc. Res., 22: 127-142,1981.

22. Poole, T. J., and Zetter, B. R. Mast cell Chemotaxis to tumor-derived factors.

J. Cell Bid., 97:77a,1981.23. Raju, K. S., Alessandri, G., Ziehe, M., and Cullino, P. M. Cerutoplasmin, copper,

ions, and angiogenesis. J. Nati. Cancer Inst., 69:1183-1188,1982.24. Repo, H. Leukocyte migration agarose test for the assessment of human

neutrophil Chemotaxis. I. Effect of environmental factors on neutrophil migration under agarose. Scand. J. Immunol., 6: 203-218,1977.

25. Riley, J. F. The relationship of the tissue mast cells to the blood vessels in therat. J. Pathol. Bacteriol., 65: 461-469,1953.

26. Ryan, T. J. Factors influencing the growth of vascular endothelium in the skin.Br. J. Dermatol., 82: 99-111,1970.

27. Schiffman, E., Corcoran, B. A., and Wahl, S. M. N-Formylmethionyl peptidesare chemotactic for leukocytes. Proc. Nati. Acad. Sei. U. S. A., 72: 1059-1062, 1975.

28. Simu, G., and Csaba, G. Mast cells in tumour-bearing patients. Acta Morphol.Acad. Sci. Hung., 20. 327-338,1972.

29. Smith, S. S., and Basu, P. K. Mast cells in corneal immune reaction. Can. J.Ophthalmol., 5:175-182,1970.

30. Snyderman, R., and Goetzl, E. J. Molecular and cellular mechanisms ofleukocyte Chemotaxis. Science (Wash. D. C.), 273: 830-837,1981.

31. Staemmler, M. Utersuhcung übervorkommen und bedeutung der histigenmastzellen im menschlichen Korper unter normalen und pathologischen Verhältnissen.Frankf. Z. Pathol., 25: 391-435,1921.

32. Sullivan, T., Parker, K. L., Stenson, W., and Parker, C. W. Modulation of cyclicAMP in purified rat mast cells. I. Responses to pharmacologie, metabolic, andphysical stimuli. J. Immunol., 744:1473-1479,1975.

33. Svennevig, J.-L. In situ identification of inflammatory cells in malignant, non-lymphoid human tumours. Arch. Pathd. Microbiol. Scand. Sect. A, 88: 387-392, 1980.

34. Taylor, S., and Folkman, J. Protamine is an angiogenesis inhibitor. Nature(Lond.), 297: 307-312, 1982.

35. Theoharides, T. C., Bondy, P. K., Tsakalos, N. D., and Askenase, P. W.Differential release of serotonin and histamine from mast cells. Nature (Lond.),297:229-231,1981.

36. Thorensen, S., Tangen, M., and Hartvett, I. Mast cells in the axillary nodes ofbreast cancer patients. Diagn. Histopathol., 5: 65-67,1982.

37. Vicker, M. G. Ideal and non-ideal concentration gradient propagation in Chemotaxis studies. Exp. Cell Res., 736:91-100,1981.

38. Wasserman, S. I. The mast cell: its diversity of chemical mediators. Int. J.Dermatd., 79:7-17,1980.

39. Zigmond, S. H. Gradients of chemotactic factors in various assay systems. In:R. van Furth (ed.) Mononuclear Phagocytes, pp. 461-473. Boston: MartinusNijhoff Publishers, 1980.

40. Zigmond, S. H., and Hirsh, J. G. Leukocyte locomotion and Chemotaxis—newmethods for evaluation and demonstration of a cell-derived chemotactic factor.J. Exp. Med., 737: 387-410, 1973.

DECEMBER 1983 5861

on July 21, 2021. © 1983 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

1983;43:5857-5861. Cancer Res   Thomas J. Poole and Bruce R. Zetter  Tumor-derived PeptidesStimulation of Rat Peritoneal Mast Cell Migration by

  Updated version

  http://cancerres.aacrjournals.org/content/43/12_Part_1/5857

Access the most recent version of this article at:

   

   

   

  E-mail alerts related to this article or journal.Sign up to receive free email-alerts

  Subscriptions

Reprints and

  .pubs@aacr.orgDepartment at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

  Permissions

  Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/43/12_Part_1/5857To request permission to re-use all or part of this article, use this link

on July 21, 2021. © 1983 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from