Clinical and laboratory studies

Detection of undegraded fibrin and tumor necrosisfactor-(X in venous leg ulcersAlain L. Claudy, MD,a Massoud Mirshahi, MD,b Claudine Soria, PhD,c andJeannette Soria, PhDC St. Priest en Jarez and Paris, France

The pathogenesis of venous leg ulcers is based on the leakage of fibrinogen leading to peri­capillary fibrin cuff and plugging of capillaries by white blood cells. Eight patients withvenous leg ulcers have been studied with a panel of antibodies reactive for fibrinogen, fibrin,fibrin degradation products, and various cell-associatedmarkers for polymorphonuclear cells,monocytes, and Band T lymphocytes. Our results showed that pericapillary fibrin cuff wasmainly composed of undegraded fibrin and that, in the granulation tissue, tumor necrosisfactor-a and elastase activities were detectable in monocytes and polymorphonuclear cells,respectively. Only few activated lymphocytes were present. On the basis of these results, itis assumed that inflammation generated by activated white blood cells that accumulate un­der unrelieved pressure is the key event. Tumor necrosis factor-a synthesized by activatedmonocytes may therefore induce the formation of pericapillary fibrin cuffs. Pericapillary fi­brin cuffs and toxic metabolites released by polymorphonuclear cells may explain the absenceof wound repair. (J AM ACAD DERMATOL 1991;25:623-7.)

The pathogenesis of venous ulcerations is un­known. Several hypotheses have been advanced.Most postulate leakage of fibrinogen and the devel­opment ofpericapillary fibrin cuffs (PFCs) that leadto a diffusion barrier to oxygen and nonhealingulceration.1-4 PFCs occur predominantly near theulcer surface,5 It is also known that other mecha­nisms in addition to high venous pressure take place,such as distension of endothelial pores, decreased fi­brinolytic activity, increased platelet adhesion todamaged endothelium, fibroblast proliferation, newcollagen deposition, and angiogenesis. I, 6 The pur­pose of our study was to provide a better under­standing of the pathogenesis of venous leg ulcers bydetecting and localizing immunoreagents, fibrin andfibrinogen degradation products,

PATIENTS

We studied eight female volunteers (mean age 66years).Theinclusioncriterion was chronic "clean"venousulceration of the leg regardless of duration, without clin­ical signs of infection, and without arterial obstruction as

From the Service de Dermatologie, Hopital Nord, St Priest en Jarez":lnstitut National de la Sante et de la Recherche Medicalcb; and theLaboratoire d'Hematologie, Hotel Dieu, Paris.c

Accepted for publication April 18, 1991.Reprint requests: A. L.CJaudy, Servicede DermatoJogie, Hopital Nord,

42277 St Priest en Jarez, France.

16/1/30247

confirmed by Doppler ultrasonography, Allpatients hada history ofdeepvenous thrombosis proved by impedanceplethysmography and Doppler blood flow measurements.The skin changes were typical of chronic stasis dermati~

tis.

METHODSTissue samples

Three 3 mm punch biopsy specimens were obtainedfrom the middle of the ulceration. One specl1l1en wasstained with hematoxylin-eosin-saffron. One specimenwas snap-frozen and cut in 4~m sections, One specimenwas processed according to the AMEX method.7The tis­sue was fixed in acetoneat -20° C overnight, dehydratedin acetone at 4° C for 15 minutes and at room temper­aturefor 15 minutes, consecutivelycleared in methylben­zoate for 30 minutes and in xylene for 30 minutes, andthen penetrated with paraffin at 60° C for 2 hours inavacuum evaporating embedder,

ImmunoreagentsDetails ofthe antibody panels, specificities and sources

are summarized in Tables I and II. Monoclonal anti­bodies directed against fibrinogen derivatives are asfollows:8- 12 F2C5 is specific against plasmin-fibrinogendegradation products. The epitope recognized by F2C5 ishidden intheundegraded molecule. ThereforeF2CS doesnot react with intact fibrinogen. E8 is specific for unde­graded fibrin and reacts with an epitope located at theN-terminalportion of fibrin {J chain,12 Ithas no cross-re­activity with intact fibrinogen or fibrin degradation prod-

623

624 Claudy et aI.

Joumal of theAmerican Academy of

Dermatology

Table I. Antibodies used on paraffin-embeddedtissue sections

Table III. Results of immunolabeling studies ineight patients with chronic venous leg ulcers

Antibody Specificity ISource Antibody I Labeling result

BD, Becton Dickinson (Sunnyvale, Calif.); ee, Coulter Clone (Hia­leah, Fla.); OD, Ortho Diagnostic Systems (Raritan, N.J.).

BG, Biogenex (San Ramon, Calif.); GZ, GenzymeLab (Boston, Mass.);OL, our laboratory (C. and J. Soria).*Polyclonal.

ucts. F1E1 recognizes immobilized fibrinogen and onlypoorly recognized fibrinogen in solution.9, II Monoclonalantibody directed against leukocyte elastase was providedby our laboratory (unpublished data). Monoclonal anti­body directed against surface membrane antigens andcytoplasmic antigens were diluted 1:100; incubation timewas 30 minutes. Goat antimouse Ig antiserum was usedas a second-step reagent. Rabbit antihuman tumor ne­crosis factor (TNF) was the neutralizing antibody used ata concentration of 5 p,g/mi. Anti-S-100 protein antibodywas diluted 1:20.

Staining procedures

Paraffin sections. Standard staining (hematoxylin,eosin, saffron) and toluidine blue staining were performedon each biopsy specimen. The acetone-methyl benzoate­xylene (AMEX)-fixed sections were deparaffinized withsuccessive incubations with toluene for 45 minutes, 90%ethanol for 10 minutes, 75% ethanol for 10 minutes. Af­ter washing with phosphate-buffered saline, the sectionswere incubated for 45 minutes at room temperature withthe various monoclonal or polyclonal antibodies. Theslides were washed and incubated for 45 minutes at room

F2C5FIEIE8 +TNF-a +Vimentin +Anti-S-l00 ±Leu-4 ±Leu-3aOKT6Leu-2a ±Leu-M3 +B4 ±Bl ±IL-2RHLA-DR +

temperature with peroxidase-labeled avidin-biotin com­plex. The chromogenic reaction was then performed with0.05% diamino benzidine tetrahydrochloride (DAB) and0.01% hydrogen peroxide diluted in acetate buffer (pH5.0).

Frozen sections. All monoclonal antibodies were usedwith an immunoperoxidase method (HistoGen peroxi­dase-antiperoxidase immunostaining system, BioGenexLaboratory, San Ramon, Calif.). The incubation stepswere as follows: primary antibody, peroxidase-conjugatedgoat-antimouse Ig antibody, peroxidase-mouse-antiper­oxidase (PAP complex). After washing, the sections werestained with 3-amino-9-ethylcarbazole (ABC) and hy­drogen peroxide diluted in acetate buffer (pH 5.0). Nu­clear counterstaining was performed with hematoxylin.

RESULTS

Results are shown in Table III. Routine staining(Fig. 1) showed a loose matrix of collagen, fibro­blasts, neovascularization, and granulation tissuethat consisted of macrophages and a few polymor­phonuclear (PMN) cells in perivascular areas. Adense matrix of collagen without a cell infiltrate waspresent under the granulation tissue. Sparse lym­phocytes and mastocytes were detectable. Vimen­tin-positive cells were abundant amid the granula­tion tissue-staining fibroblasts. Few S-100-positivecells were present.

Positive staining was found for undegraded fibrin(E8) in the vicinity of vessels (Fig. 2). Fibrin-degra­dation products and immobilized fibrinogen werenot detected by F2C5 and FIEI, respectively.

-, Absence oflabeling; +, labeling ofnumerous cells; ± ,labeling ofonlya few cells.

OLOLOLGZOLBGBG

BDBDODBDBDCCCCBDBD

ISource

Fibrin degradation productsImmobilized fibrinogenFibrin {3 chainTNFLeukocyte elastaseFibroblast, endothelial cellLangerhans cell, melanocyte

Leu-4Leu-3aOKT6Leu-2aLeu-M3B4BlIL-2RHLA-DR

F2C5FIElE8Anti-TNF*Anti-elastaseAnti-vimentinAnti-S-100*

Table II. Antibodies used on frozen-tissuesections

_A_n_tibod_Y--l~ Specificity

3 Pan-T lymphocyte4 Helper T lymphocyte6 Langerhans cell8 Suppressor T lymphocyte

14 Monocyte, macrophage19 B lymphocyte20 B lymphocyte25 Activated T lymphocyte

Activated T lymphocyte

Volume 25Number 4October 1991 Venous leg ulcers 625

2Fig. 1. Photomicrograph ofbiopsy specimen ofvenous leg ulcer. PFCs occur predominantlyaround dilated capillaries near the ulcer surface. (Hematoxylin-eosin-saffron stain; X40.)Fig. 2. AMEX-fixed, paraffin-embedded tissue section stained with E8. Perivascular areasshow strong immunoreactivity. (Immunoperoxidase stain; X250.)

TNF-a localized in intracapillary monocytes (Fig.3). Elastase activity was present in the polymorpho­nuclear cells near the vessels. Monoclonal antibod­ies directed against T and B lymphocyte surfacemembrane antigens (Leu-4, Leu-3a, Bl, B4, IL-2R)stained only a few scattered cells predominantly inthe vicinity of blood. vessels. Anti-CD14 (Leu-M3)antibody stained a small proportion of cells through­out the tissue sections. DC6+ cells were not found.DR+ cells (monocytes, macrophages, B cells, andactivated T cells) were noted mostly in and aroundvessel walls.

DISCUSSION

Granulation tissue in the base of chronic nonin­fected venous leg ulcers consists of macrophages,PMN cells in perivascular areas, fibroblasts, neovas­culature embedded in a loose matrix of collagen, fi­bronectin, and hyaluronic acid.3, 5 Lymphocytes andmastocytes are poorly represented, as shown in ourstudy. PFC and plugging of capillaries by whiteblood cells are thought to be the two main mech­anisms that results in formation of venous legulcers. 1-4

PFCs result from a conversion of extravasatedtissue fibrinogen to fibrin. The detection of fibrin

.. .

Fig. 3. AMEX-fixed, paraffin-embedded tissue sectionstained with anti-TNF-a antibody. (Immunoperoxidasestain; X250.)

usually relies on reagents that cannot distinguishfibrinogen from fibrin or from their degradationproducts.8 Polyclonal antisera cross-react with bothmolecules and therefore do not allow a specific iden­tification in tissues. The positive labeling with E8that reacts with the N-terminal part of the f3 chainof fibrin demonstrated that pericapillary cutIs arecomposed mainly of undegraded fibrin. This anti­body does not stain fibrinogen or fibrinogen degra-

626 Claudy et al.

dation products.12 Furthennore, negative resultswere obtained with F2C5, which is specific for fibrindegradation products, and FIEI, which recognizesimmobilized fibrinogen. We have presented addi­tional evidence that PFCs are mainly composed ofundegraded fibrin. Excessive fibrin deposition maycause sustained inflammatory response and forincreased fibroblast migration, a prerequisite forgranulation tissue formation in wounds.

Furthermore, accumulation of undegraded :fibrinmay be explained by decreased fibrinolytic activityin superficial dilated capillaries.2, 13 We assume thathypercoagulability may be dependent on TNF.

In our study, the presence of TNF-a immunore­activity was mainly found in intracapillary mono­cytes. TNF-a may be an important signal at the siteof neovascularization during nonnal repair pro­cesses because it represents a potent stimulus to newvessel fonnation. 14.16 Decreased oxygen tension, aphenomenon that occurs in venous leg ulcers, isknown to cause cultured monocytes to produceTNF-a in vitro.l6 TNF-a released by monocytesand macrophages has a potent mitogenic effect onfibroblasts, induces collagenase synthesis, increasesthe production of interleukin 1 (IL-l), IL-6, granu­locyte/macrophage colony-stimulating factor, andtransfonning growth factor-Ii TNF-a also actsthrough the upregulation of intracellular adhesionmolecules (ICAM-!). It thus leads to leukocyte se­questration, which is an early step in chronicinflammation.17. 18 TNF-a also activates the coagu­lation pathway via a marked increase in cell-associ­ated tissue factor activity and inhibits fibrinolysis bypromoting the release ofPAI-I, an inhibitor of plas­min fonnation. 16. 19 TNF-a-induced decreased fi­brinolytic activity may therefore be the main causeof PFC formation. 13, 20 The role of PMN in woundrepair has been emphasized.21 PMN cells, when ac­tivated, release toxic metabolites of oxygen, prote­olyticenzymes, and lipid products, and Increase theirelastase activity. All these factors contribute to en­dothelial damage, increased vessel permeability, anddeposition of fibrinogen and fibrin. The role of elas­tin degradation in tissue remodeling and in woundhealing is important and depends on elastase, whichis one of the lysosomal constituents released fromPMN cells. Elastase, a sensitive marker of activatedPMN cells,22 is also detectable in dennal fibro­blasts.23 Its activity is stimulated by IL-l.24 Wefound its presence mainly in PMN cells and not infibroblasts.

Journal of theAmerican Academy of

Dermatology

We have shown that Band T lymphocytes are notthe dominant cell type in the granulation tissue of legulcers (Table III). In cutaneous wound repair, lym­phocytes playa minor role.21 A cooperation betweenmacrophages and lymphocytes may take place ifabacteria-activated macrophage initiates a cell-me­diated type of hypersensitivity reaction. Such an op­portunity may frequently occur in chronicvenous legulcers. However, in our study, only sparse B and Tlymphocytes were found in the vicinity ofthe vessels.Whether lymphocyte infiltration represents a factorthat delays wound healing remains doubtful.

We hypothesize that inflammation generated byactivated white blood cells that accumulate withunrelieved venous pressure is the first event inchronic leg ulcers. TNF, a key mediator of inflam­mation that decreases fibrinolytic activity, may sec­ondarily induce the fonnation ofPFCs. PFCs, toxicmetabolites released by PMN cells and the lack ofsustained release of growth factors may explain theabsence of wound repair.

REFERENCES1. Browse NL, Bumand KG. The cause ofvenous ulceration.

Lancet 1982;2:243-5.2. Coleridge Smith PD, Thomas P, Scurr JH, et aI. Causes of

venous ulceration: a new hypothesis. Br Med J 1988;296:1726-7.

3. Falanga V, Moosa HH, Nemeth AJ, et al. Dermal peri­capillary fibrin in venous disease and venous ulceration.Arch Dermatol 1987;123:620-3.

4. Thomas PRS, Nash GB. Dormandy JA. White cell accu­mulation in dependent legs of patients with venous hyper­tension: a possible mechanism for trophic changes in theskin. Br Med J 1988;296:1693-5.

5. Falanga V, Kruskal J, Franks J1. Fibrin- and fibrinogen­related antigens in patients with venous disease and venousulceration. Arch Dermatol 1991;127:75-8.

6. Vanscheidt W, LaaffH, Wokalek H, et aI. Pericapillary fi­brin cuff: a histological sign of venous ulceration. J CutanPathol 1990;17:266·8.

7. Sato V. Mukai K, Watanabe S, et al. The AMEX method:a simplified technique of tissue processing and paraffin em­bedding with improved preservation of antigens for immu­nostaining. Am J PathoI1986;125:431-5.

8. Soria J, Soria C, Boucheix C, et aI. Monoclonal antibodiesthat react preferentially with fibrinogen degradation prod­ucts or with cross-linked fibrin split products. Ann NYAcad Sci 1983;408:665-6.

9. Soria J. Soria C. Mirshahi M, et aI. Conformational changein fibrinogen induced by absorption to a surface. Journal ofColloid Interface Science 1985;107:204.

10. Mirshahi M, Soria J, Soria C, et aI. A latex immuno-assayof fibrin/ogen degradation products in plasma using amonoclonal antibody. Thromb Res 1986;44:715.

11. Soria C, Soria J, Mirshahi M, et al. Platelet-associated fi·brinogen: conformational change of fibrinogen induced byits adsorption to platelet surface. In: Lane D, Henschen A,

Volume 25Number 4October 1991

Janasi HK, et ai, eds. Fibrinogen, fibrin formation and fi­brinolysis. New York: Walter de Gruyter 1986;4:185.

12. Soria J, Mirshahi MC, Mirshahi S, et a1. Soluble fibrin inplasma using a specific fibrin-monoclonal antibody directedagainst the N-terminal part ofthe beta chain. In: MatsudaM, Iwanaga S, Takada A, et ai, eds. Fibrinogen 4. Currentbasis and clinical aspects. Amsterdam: Excerpta Medica,1990:187-94.

13. Lotti T, Fabbri P, Panconesi E. The pathogenesis ofvenousleg ulcers [Letter]. J AM ACADDERMATOL 1987;16:877-9.

14. Ksander GA, Pratt BM, Desiletsavis P, et al. Inhibition ofconnective tissue formation in dermal wounds covered withsynthetic, moisture vapor permeable dressings and itsreversal by transforming growth factor (3. J Invest Derma­toI1990;95:195-201.

15. Ksander GA, Sawamura SJ, Ogawa Y, et al. The effect ofplatelet releasate on wound healing in animal models. JAMACAD DERMATOL 1990;22:781-91.

16. Barath P, Fishbein MC, Cao J, et a1. Detection and local­ization of tumor necrosis factor in human atheroma. Am JCardioI1990;65:297-302.

17. Schirren ca, Scharffetter K, Hein R, et a1. Tumor necro­sis factor a induces invasiveness of human skin fibroblastsin vitro. J Invest Dermatol 1990;94:706-10.

18. Polak B, Duperray A, Troesch A, et al. Biogenesis of the

Venous leg ulcers

vitronectin receptor inhumanendothelial cell: evidence thatthe vitronectin receptor and GPIIb-I1Ia are synthesized bya common mechanism. Blood 1989;73:1519-24.

19. Bevilacqua MD, Pober lS, Majeau GR, et al. RecombinantTNF-induces procoagulant activity in cultured humanvascular endothelium. Characterization and comparisonwith actions of interleukin-l, Proc Natl Acad Sci USA1986;83:4533-7.

20. Hebda PA, Eaglestein WH. The stimulatory effect oftransforming growth factor-beta and epidermal growthfactor on epidermal migration [Abstract]. Clin Res1986;34:755A.

21. Clark RAF. Cutaneous tissue repair: basic biologic consid­erations. I. J AM ACAD DERMATOL 1985;13:701-25.

22. Lammers AM, Van de KerkhofPCM, Schalwijk JS, et al.Elastase, a marker for neutrophils in skin infiltrates. Br JDermatoI1986;115:181-6.

23. Homsy R, Pelletier-Lebon P, TOOer JM, et al. Character­ization ofhuman skin fibroblasts' elastase activity. JInvestDermatoI1988;91:472-7.

24. Croute F, Delaporte E, Gaucherand M, et al. Keratinocyte­fibroblast interactions: 11-1 and keratinocyte soluble fac­tors enhance elastase activity of dermal fibroblasts [Ab­stract]. J Invest DermatolI990;94:392-3.

Detection of cytokine-induced protein ')I-immuneprotein-IO (')I-IPIO) in atypical melanocyticproliferationsBruce R. Smoller, MD,a, b and James Krueger, MDb Stanford, California, andNew York, New York

'Y-Immune protein-lO Cr-!PI0) is a cytokine whose expression has been shown to be inducedby interferon-'Y. Itis a member of a group ofclosely related cytokines (e.g., intedeukin 8 andplatelet factor 4) with chemotactic properties. 'Y-IPIO has been detected in keratinocytes,lymphocytes, monocytes, and endothelial cells in immunologically mediated processes, suchas positive tuberculin skin tests, and in growth-activated keratinocytes, such as in psoriasis.Keratinocytes in normal epidermis do not produce 'Y-IPIO. We tested the hypothesis that ke­ratinocytes adjacent to dysplasticnevi andmelanomas would produce 'Y-IPI0, perhaps as partof an immune response to a tumor, and that this response would not be seen in ordinary mel­anocytic nevi. We used an affinity-purified, polycional rabbit anti-')'-IPI0 antibody to exam­ine 10 nevi with moderate to severe histologic dysplasia, one superficial spreading melanoma,and 10 compound melanocytic nevi with no features ofdysplasia. As predicted, keratinocytessurrounding allof the cytologically atypical melanocytic lesions displayedstrong staining with'Y-IP10. There was no staining of keratinocytes adjacent to ordinary melanocytic nevi. Theobserved keratinocyte staining with 'Y-IPI0 may be related to a host immune response to an­tigenically abnormal cells. (J AM ACAD DERMATOL 1991;25:627-31.)

From tile Departments of Dermatology and Pathology, Stanford Uni­versity Medical Center"; and the Departments of Pathology andMedicine, Division of Dermatology, New York Hospital-CornellUniversity Medical Center.b

Accepted for publication June 5,1991,Reprint requests: B. Smaller, MD, Department of Pathology, Stanford

University Medical Center, Stanford CA 94305.16/1/31409

627