Abstract
The HT-1080 human fibrosarcoma cell line exhibited a plasminogen-dependent ability to inactivate recombinant anaphylatoxin C5a or zymosan-activated serum. The inactivation was obtained at physiological levels of both plasminogen (2 microM) and C5a (1-5 nM). Inactivated C5a and zymosan-activated serum were no longer able to induce chemotaxis and degranulation of neutrophils. Inactivation of C5a paralleled the emergence of plasmin activity, assayed by cleavage of the synthetic substrate H-D-valyl-L-leucyl-L-lysine-p-nitroanilide (S-2251). Both C5a inactivation and S-2251 cleavage were inhibited by the plasmin inhibitor alpha 2-antiplasmin, the urokinase inhibitor amiloride, and by anti-urokinase antibodies. In a cell-free system, inactivation of C5a was shown to depend on the simultaneous presence of urokinase and plasminogen and was inhibited by alpha 2-antiplasmin and by anti-urokinase antibodies. SDS-polyacrylamide electrophoresis demonstrated the cleavage of C5a by the plasminogen activation system and inhibition of the cleavage by amiloride. Amino acid sequencing of the band corresponding to the C5a degradation product revealed that C5a was cleaved at positions Lys14-His15 and Arg40-Ile41; cleavage at position Arg40-Ile41 seemed to be responsible for the loss of activity. Since neoplastic cells extensively produce and exhibit plasminogen activator activity, the present observations suggest that plasminogen activation may, by inactivation of C5a, reduce the anti-tumor immune response and support the immunological escape phenomenon of tumors.
Highlights
The HT-1080 human fibrosarcomcaell line exhibited atake place, invasive cells have t o express proteolytic activity for plasminogen-dependentabilitytoinactivaterecombi- cellular detachment, breakdown of the components of interstinantanaphylatoxin C5aor zymosan-activatedserum. tial matrices, basement membrane reattachment, and subse
The proteolytic activity of the both plasminogen (2 p ~ a)ndC5a (1-5 MI).Inactivated plasminogen activation system plays a major role in the inva
A role for plasminogen activators in these to induce chemotaxis and degranulatioofnneutrophils. events is supported by studies on plasminogen activators in Inactivation of C5a paralleled the emergencoef plasmin tumors, which demonstrated higherplasminogen activator acactivity, assayedby cleavage of the synthetic substrate tivity in malignant tissuecso, mpared t o benign tumors [11,12]. hHtCchei5-belaDli-tp-ifonvrlraaeaselcymatslim-yviLnasi-tltoleiienomruhnidc,iybeianl,in-taLodacr-ntl5yidav-s,2ai-nab2tein5yoqt1ni-panocnliflatetrsiCao-muva5arinanogiw,lkeidatihnwse(eas5ehsr-ueo2erw2oa5innkn1hti)tnoB.iibbadoosiIettedn-heidiaens-b. ypWcerolTiltdshhupetchreoieddteieusxacsceuetehpu-atrtiotyoknpnieenoopfaplmslaease.smltaiicnnootmugmeanoarnasdcctniovenauttraooirnb(l1aa3ns-tt1oi6-mi1n,afmlcaemolslmts twauthmoircoyhr pendonthesimultaneouspresence of urokinaseand activity which reduces the immune defenses of the host has plasminogen and was inhibited by a,antiplasmin and been suggested for many years [17, 18]
Summary
The HT-1080 human fibrosarcomcaell line exhibited atake place, invasive cells have t o express proteolytic activity for plasminogen-dependentabilitytoinactivaterecombi- cellular detachment, breakdown of the components of interstinantanaphylatoxin C5aor zymosan-activatedserum. tial matrices, basement membrane reattachment, and subse-. The HT-1080 human fibrosarcomcaell line exhibited atake place, invasive cells have t o express proteolytic activity for plasminogen-dependentabilitytoinactivaterecombi- cellular detachment, breakdown of the components of interstinantanaphylatoxin C5aor zymosan-activatedserum. The proteolytic activity of the both plasminogen (2 p ~ a)ndC5a (1-5 MI).Inactivated plasminogen activation system plays a major role in the inva-. SDS-polyacrylamideelec- The purpose of the present study was to examine whether trophoresis demonstrated the cleavage of C5aby the the plasminogen activator system present ineoplastic cells is plasminogen activation system and inhibition of the involved in the mechanism allowing these cells to escape the cleavage by amiloride.Aminoacidsequencing of the immunological system of the hostby acting asanti-inflammaband corresponding to thCe5a degradation product re- tory agents. Arg"Ile4'; cleavage at position Arg40-ne4l seemed to be present on neoplastic cells (HT-1080) on the activity of the responsible for the lossof activity
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