Immunology 1994 83 582-588

Inhibition of expression of delayed hypersensitivity by neutralizing monoclonalanti-T-cell fibronectin antibody

S. MANDY, Z. FENG, L. S. CANFIELD, K. MANDY, X. QUAN, R. A. ROWEHL,* M. Y. KHAN,ttS. K. AKIYAMA§ & H. P. GODFREY Departments ofExperimental Pathology and t Cell Biology and Anatomy, New

York Medical College, Valhalla, New York, *Department of Microbiology, State University ofNew York at Stony Brook, StonyBrook, New York and § Laboratory ofDevelopmental Biology, National Institute ofDental Research, National Institutes of Health,

Bethesda, Maryland, USA

SUMMARY

T-cell fibronectin (FN) is a unique cellular FN that is rapidly synthesized by memory T cells inresponse to antigen. Monoclonal anti-T-cell FN antibodies have been used to clarify the role of T-cell FN in the in vivo expression of delayed hypersensitivity. IgGl(K) mouse anti-human T-cell FNmonoclonal antibodies 231 and 248 recognized epitopes on the FN cell-binding domain, were

cross-reactive with plasma FN, and neutralized human and guinea-pig T-cell FN monocyteagglutinating activity. When injected intradermally together with tuberculin or 30 min beforetopical application of reactive sensitizer, antibody 231 significantly decreased macroscopicexpression of guinea-pig delayed hypersensitivity at 24 hr in a dose-dependent manner. Similardoses of antibody 248 caused a slight statistically non-significant enhancement of delayed-typehypersensitivity (DTH) expression. Inhibition of visible skin responses was not associated withqualitative or quantitative changes in cellular infiltrates at the reaction site. Antibody 231modulated expression of delayed hypersensitivity in a qualitatively and quantitatively similarmanner to the FN-binding mycobacterial antigen 85 proteins. This is consistent with anti-T-cellFN and antigen 85 acting on the same molecule in vivo.

INTRODUCTION

The fibronectins (FN) are a family of highly conserved, highmolecular weight glycoproteins present in plasma andtissues.'2 They are involved in cell adhesion and motility,embryonic development, regulation of cell morphology,phagocytic function, wound healing and inflammation. Mostcellular FN are produced constitutively. One, T-cell FN, issynthesized and secreted by T lymphocytes in response toantigenic stimulation, and was initially recognized as alymphokine associated with delayed-type hypersensitivity(DTH).3'4

Because the various FN are produced from a single gene byalternative splicing ofmRNA transcripts, much of the moleculeis constant regardless of the cell source, and individual

Received 28 April 1994; revised 10 August 1994; accepted 21 August1994.

Abbreviations: Ag85, antigen 85; DNTB, 1-thiocyano-2,4-dinitro-benzene; DTH, delayed-type hypersensitivity; FN, fibronectin; PBS,phosphate-buffered saline; PPD, purified protein derivative oftuberculin.

t Present address: Department of Biochemistry, North Eastern HillUniversity, Shillong, India.

Correspondence: Dr H. P. Godfrey, Department of ExperimentalPathology, New York Medical College, Basic Science Building,Valhalla, NY 10595, USA.

members of this family are antigenically and functionallysimilar. Although T-cell FN shares many properties with otherFN (both plasma and cellular), it is biochemically andfunctionally distinct from them.5 Its MW (420 000) is slightlysmaller than that of other cellular FN,5 and it has a smallermRNA (5600 bp) than the others do (Z. Feng & H. P. Godfrey,unpublished observations). It is soluble under physiologicalconditions, while most other cellular FN are not. 1,2 In addition,T-cell FN is markedly more potent in vitro than other FN. It isactive at femtomolar concentrations,3 rather than the nano-molar concentrations characteristic of other FN."12 T-cell FNagglutinates monocytes or peritoneal exudate cells by an activemetabolic process involving interaction with multiple classes ofcell-surface integrin protein receptors, and translocates mono-cytes and neutrophils through collagen matrices by a genericphysical process involving interaction with cell-surface heparin-like molecules.

Several lines of evidence point to the involvement of T-cellFN in DTH reactions in vivo. In addition to its rapid synthesisand secretion by memory T cells and T-cell clones in responseto antigen, there is a close correlation between the ability ofcloned CD4+ and CD8 + murine T-cell lines to transfer DTHand their ability to secrete T-cell FN after antigenic stimu-lation.5 Furthermore, antigen 85 (Ag85), a group of closelyrelated FN-binding proteins secreted by Mycobacteriumtuberculosis and other mycobacteria,6 binds to and neutralizes

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T-cellfibronectin and delayed hypersensitivity

T-cell FN in vitro and modulates expression ofDTH in guinea-pigs.7 We have now examined the effect of neutralizing anti-T-cell FN monoclonal antibodies on expression of DTH insensitized guinea-pigs in order to extend our analysis of the roleof T-cell FN in mediating these responses. Our results suggestthat T-cell FN is involved in the early events initiating DTHinflammatory reactions.

MATERIALS AND METHODS

Proteins and proteolytic fragmentsPurified protein derivative of tuberculin (PPD) was purchasedfrom Statens Seruminstitut (Copenhagen, Denmark; RT52).Purified plasma FN was purchased from the New York BloodCenter (New York, NY). Proteolytic fragments of plasma FNwere prepared by digestion with tosylphenyalanylchloromethylketone-trypsin (Worthington Biochemical, Freehold, NJ) at a

FN-enzyme molar ratio of 100:1.8 They were purified byDEAE-cellulose and affinity chromatography over heparinand gelatin, and were quantified spectrophotometrically.8 Thefollowing fragments were used (corresponding FN domains); 1229000MW N-terminal heparin-binding (heparin 1);45 000MW collagen-binding (collagen), 75 000MW cell-bind-ing (cell 1); 146 000MW C-terminal heparin-binding (cell I,heparin II, fibrin II); 113000MW C-terminal heparin-binding(cell I, heparin II); 34 000MW C-terminal fibrin-binding (fibrinII). Purity of intact plasma FN and fragments was confirmed bySDS-PAGE. T-cell FN was purified from concentrated culturesupernatants of mitogen-stimulated normal human peripheralblood mononuclear cells by gelatin-affinity chromatographyand gel-filtration high-performance liquid chromatography(HPLC) and quantified by immunoassay as described else-where.9 Purified fibroblast FN was a gift from Dr J. Peters.

Enzyme-linked immunoassays and immunoblottingDot immunobinding assays and immunoblotting have beendescribed previously in detail.9 Plate immunoassays were

performed on triplicate aliquots of FN or FN fragmentsadsorbed to the wells of round- or flat-bottomed immunoassayplates (Dynatech, from Fisher Scientific, Springfield, NJ).Plates were developed using horseradish peroxidase,1I alkalinephosphatasell or enzyme-amplified12 detection systems. Eachplate included positive and negative controls. Horseradishperoxidase or alkaline phosphatase reactions were stoppedafter 60 min at 230 and read at 405 nm; amplified reactions werestopped after 20 min at 230 and read at 490 nm. Reactions wereread in a temperature-controlled, vertical path spectrophoto-meter (ThermoMax, Molecular Devices, Menlo Park, CA).Results are reported as mean absorbance.

Monoclonal antibodiesAnti-T-cell FN was raised by intraperitoneal injection of micewith 100 ng purified human T-cell FN in 0-2 ml Freund'scomplete adjuvant containing 0-2mg heat-killed mixed strainsof human M. tuberculosis (Ministry of Food, Fisheries andAgriculture, Weybridge, UK). Mice were boosted four times atmonthly intervals by intraperitoneal injection of 100 ng T-cellFN in 0-2 ml Freund's incomplete adjuvant. Before each boost,mice were bled from the tail and serum anti-T-cell FNantibodies assayed by dot blotting. After the last boost, micewere rested for 2-4 months, then injected intravenously with

100 ng T-cell FN 84 hr before killing by CO2 overdose.Production of hybridomas was performed by a standardprocedure with the myeloma line P3X63-Ag8.653.13 Hybri-doma supernatants were screened by dot blotting against 1 ngpurified human T-cell, fibroblast and plasma FN. Hybridomasreacting preferentially with T-cell FN were selected, expanded,subcloned by limiting dilution, and isotyped (Pharmingen,San Diego, CA). Two IgGl(K) anti-T-cell FN monoclonalantibodies, clones 231.28 and 248.13, were selected for furtherstudy. Antibodies for these studies were purified from serum-free hybridoma culture supernatants by precipitation with(NH4)2SO4. Non-reduced SDS-PAGE of monoclonal anti-body preparations revealed only intact immunoglobulin and noother detectable proteins.

The following characterized monoclonal antibodies were

used: rat IgG2a anti-human plasma FN N-terminal heparin-binding domain, clone 304;14 rat IgG2a anti-human plasma FNcell-binding domain, clone 333;14 and mouse IgG1 anti-M.bovis Ag85, clone 233.15 Anti-M. bovis Ag85 clone 233 did notbind to human T-cell, fibroblast or plasma FN in dotimmunobinding assays.

Monocyte agglutination assay

Duplicate or triplicate aliquots of 60pg chromatographicallypurified human T-cell FN,9 or an equipotent amount of guinea-pig T-cell FN,16 were mixed with monoclonal antibody,incubated at 230 for 15min, and serially diluted in modifiedRPMI-1640 containing 10 fetal calf serum (FCS; lowestdilution 1/100, total volume 1-8ml). Controls were incubatedwith aliquots of sterile phosphate-buffered saline (PBS), pH 7-2,in place of monoclonal antibody. Human monocytes, 2-5-3 x 106 cells in 0-2 ml, were added to each assay tube as

indicator cells. After 4-6 hr incubation at 370, tubes were

gently swirled to dislodge loosely adherent clumps of macro-

phages and scored.9 Results are reported as geometric mean

titres.

Animals, sensitization and skin testingMale Hartley guinea-pigs (Elm Hill Farms, Chelmsford, MA)were sensitized with 1 ml Freund's complete adjuvant contain-ing 1 mg heat-killed mixed strains ofhuman M. tuberculosis and1 mg 1-thiocyano-2,4-dinitrobenzene (DNTB; ICN Biomedi-cals, Costa Mesa, CA), and boosted by topical applications ofDNTB in acetone.17 To determine the effect of anti-T-cell FNon DTH to PPD, PPD, monoclonal antibody and mixtures ofPPD and monoclonal antibody were injected intradermally in0-1 ml PBS into the flanks of sensitized guinea-pigs. Injectionswere coded and randomized, and were made without knowl-edge of the solutions' content. To determine the effect of anti-T-cell FN on DTH to DNTB, monoclonal antibody or 0 1 ml PBSwas injected intradermally into the flanks of sensitized guinea-pigs, and 5 y1 acetone or graded doses ofDNTB in 5 ,ul acetonetopically applied to the injection sites 30min later. Negativecontrols of PBS injections and topical acetone applicationswere given to all animals. The immunogenicity of anti-T-cellFN precluded injecting any animal with it more than once.

Injection sites and sites of topical testing were examined forerythema and oedema at 1, 2, 3, 4 and 18 hr after injection; theresponses were scored qualitatively and recorded. Reactionsizes of PPD responses were measured with calipers in twoperpendicular directions at 24 hr, the time of maximum

583

S. Mandy et al.

macroscopic response.'7 On several occasions, reaction sizeswere measured at 18-20hr as well. Reaction sizes of PPDresponses are reported as mean area in mm2, obtained bymultiplying reaction measurements, or as mean percentage ofthe PPD-positive control reaction. Contact sensitivity toDNTB was measured by scoring test sites for erythema at24 hr.'7 The smallest dose of DNTB in nmol that elicitederythema was designated the threshold dose; the size of thethreshold dose is inversely related to the degree ofcontact DTHexhibited by a sensitized animal.'8 Results of contact testing are

reported as geometric means of these threshold doses. Punchbiopsies (4 mm) ofPPD and DNTB reactions were taken fromketamine-anaesthetized animals, fixed in glutaraldehyde,embedded in paraffin wax, sectioned and stained withGiemsa.'7 Coded sections were examined for deep andsuperficial dermal cellular infiltrates by three independentobservers (KM, XQ, HPG). Deep dermal infiltrates were

evaluated qualitatively on a scale of 0 to 4 and numbers ofsuperficial dermal infiltrating cells were counted in 10 oil powerfields.7 Results are presented as mean qualitative assessment ofdeep dermal infiltrates and mean cell numbers/five oil fields ofsuperficial cell infiltrates.

0

CD

cucn.0

n

3

2

00.1 1.0

(b)

01 1.0

Fibronectin or fragment (pmol)

Figure 2. Domain specificity of mouse anti-human T-cell FNmonoclonal antibodies. Intact plasma FN (S) or purified proteolyticfragments [146000MW C-terminal heparin-binding fragment (A);75 000MW cell-binding fragment (V); 29 000MW N-terminal heparin-binding fragment (U); 113 000MW C-terminal heparin-binding frag-ment (A); 43000MW collagen-binding fragment (0); 34000MWC-terminal fibrin-binding fragment (V)] were adsorbed to immuno-assay microplates and developed with 4Yg/ml anti-human T-cell FN231 (a) or 248 (b) using alkaline phosphatase technology. Results are

shown as mean absorbance from three independent assays. Error barshave been omitted for clarity (coefficient of variation, 12%).

Statistical analysisSignificance of differences in means was determined by use ofStudent's t-test (single comparison) or analysis of variance andScheff6's test (multiple comparisons).'9 Significance of differ-ences in antibody inhibition of monocyte agglutination activityand in reactivity of monoclonal antibodies in plate immuno-assays was determined by parallel-line analysis.20

RESULTS

Characterization of monoclonal anti-human T-cell FN antibodies

Anti-human T-cell FN 231 and 248 reacted with T-cell FN andwere cross-reactive with plasma FN (Fig. 1). Parallel-lineanalysis indicated that antibodies 231 and 248 were 39% and47% cross-reactive with human plasma FN, respectively.Neither monoclonal antibody reacted with T-cell or plasma

1 5

E

c

a)0100)

CD

.0

.0

0.0

FN on Western blotting (data not shown). Epitope localizationstudies using proteolytic fragments of plasma FN indicatedthat at 4 Mg/ml both monoclonal antibodies reacted withthe 70000MW cell-binding, 146 000MW C-terminal heparin-binding (containing the primary cell-binding domain) and29 000MW N-terminal heparin-binding FN fragments(Fig. 2). Neither monoclonal reacted significantly with the113000MW C-terminal heparin-binding, collagen-binding or

C-terminal fibrin-binding fragments. These results are consis-tent with antibodies 231 and 248 recognizing epitopes on bothcell-binding and N-terminal heparin-binding FN domains.

IgG has been reported to bind non-specifically to the FNN-terminal heparin-binding domain.2' In order to determine

0-8

E

u,

c0

D0

c

coen0

I . . .

0.01 01

Fibronectin (pmol)

Figure 1. Cross-reactivity of mouse anti-human T-cell FN monoclonalantibodies with purified human plasma FN. T-cell FN (I, A) or

plasma FN (0. A) were adsorbed to ELISA microplates anddeveloped with 2.ug/ml anti-T-cell FN 231 (I, 0) or 248 (A, A),using an enzyme-amplified detection system. Results are shown as mean

absorbance from five independent assays. Error bars have been omittedfor clarity (coefficient of variation, 11%).

0 1 10Anti-fibronectin mAb (u/ml)

Figure 3. Reactivity of mouse IgGl anti-human FN monoclonalantibodies with plasma FN N-terminal heparin-binding fragment.Plasma FN (filled symbols), 0-44pmol, or its 29000MW N-terminalheparin-binding fragment (open symbols), 6-4pmol, were adsorbed toELISA microplates and developed with anti-plasma FN 304 (V, V)(N-terminal heparin-binding domain-specific), anti-plasma FN 333(, El) (cell-binding domain-specific), anti-T-cell FN 231 (-, 0), oranti-T-cell FN 248 (A, A) using horseradish peroxidase technology.Results are shown as mean absorbance from two independent assays.Error bars have been omitted for clarity (coefficient of variation, 15%).

584

T-cellfibronectin and delayed hypersensitivity

if the reactivity of monoclonal anti-T-cell FN with the FNN-terminal heparin-binding fragment was antigen-specific, thereactivity of these antibodies with this fragment was comparedto that of two domain-specific monoclonal anti-human plasmaFN antibodies, 304 (N-terminal heparin-binding domain) and333 (cell-binding domain) (Fig. 3). Anti-plasma FN 304reacted with the N-terminal heparin-binding fragment at0 4yg/ml. Anti-plasma FN 333 and anti-T-cell FN 231 onlyshowed significant reactivity with the N-terminal heparin-binding fragment at antibody concentrations > 3 yg/ml. Anti-T-cell FN 248 did not react with the N-terminal heparin-binding fragment at any tested concentration under these con-

ditions. These results suggest that the reactivity of monoclonalanti-T-cell FN with the FN N-terminal heparin-bindingfragment is not antigen-specific, and indicate that these anti-bodies recognize epitopes located on the FN cell-binding domain.

Inhibition of T-cell FN monocyte agglutination activity byanti-T-cell FN

Anti-human plasma FN antibodies specific for the primarycell-binding domain inhibit T-cell FN monocyte agglutinatingactivity.9" 122 We therefore examined the ability of monoclonalanti-human T-cell FN antibodies with similar domainspecificity to block T-cell FN activity. Both anti-T-cell FNmonoclonal antibodies significantly inhibited monocyte agglu-tination mediated by 60 pg T-cell FN in a dose-dependentmanner at doses under 0.1 ng (slightly less than a fivefold molarexcess) (P < 0 05, t-test) (Fig. 4). The inhibitory potencies ofantibodies 231 and 248 were statistically indistinguishable byparallel-line analysis (P > 0-05). The monoclonal antibodycontrol did not inhibit T-cell FN activity. Both anti-humanT-cell FN antibodies were cross-reactive for guinea-pig T-cellFN, and completely inhibited its activity at doses < 0 1 ng (datanot shown).

Inhibition of delayed skin reactivity by monoclonal anti-T-cellFN antibodies

The inhibitory action of monoclonal anti-T-cell FN on human

107

- 106c

105

:, 104

103

° 1020

101

Tv

I-i

0 001 0.1Antibody dose (ng)

Figure 4. Inhibition of T-cell FN monocyte agglutination activity byanti-T-cell FN monoclonal antibodies. T-cell FN (60 pg) was incubatedwith PBS (0), anti-T-cell FN231 (0), anti-T-cell FN 248 (-), or

anti-Ag85 233 (monoclonal antibody control) (V), and assayed formonocyte agglutination activity. Results are shown as geometric meantitre ± SE (4-10 determinations for each point). * Significant inhibitioncompared to untreated value (P < 0-05, t-test).

110

c 1(

.2

0co0c

C

.0

Ia)a:'

00 t_+

I~~~~~~~~

90 T

10~~~~~~~~~~~~80\

70

3 10 30 100 300Anti-fibronectin mAb (ng)

Figure 5. Inhibition of PPD-elicited DTH skin reactions by monoclonalanti-T-cell FN 231. Sensitized guinea-pigs (n = 46) were injectedintradermally with 500 ng PPD (dashed line), 500 ng PPD mixed withanti-T-cell FN 231 (0), 500 ng PPD mixed with anti-T-cell FN 248 (A),anti-T-cell FN 231 (0) and anti-T-cell FN 248 (A); reactions were

measured at 24 hr. Results are shown as mean skin reactions relative toPPD response (100%) ± SE. Each point is a mean of 9-40 animals.Mean PPD reaction size (+ SE): 145 ± 5mm2. * Significant inhibitionof response compared to PPD (P < 0 05, Scheffe multiple comparison

analysis of variance); ** significant inhibition of response compared toPPD (P < 0-01, Scheffe multiple comparison analysis of variance).

and guinea-pig T-cell FN prompted study of their ability tomodulate expression of in vivo DTH. Intradermal injections of500 ng PPD elicited no immediate or Arthus reactions ofoedema and erythema at 1-4 hr. Typical erythematous DTHreactions were first visible at 6-10 hr and were maximal at24 hr. In the absence of PPD, intradermal injections of PBS(data not shown) or monoclonal anti-T-cell FN did not elicitsignificant reactions at either 1-4 hr (data not shown) or 24 hr(Fig. 5). When injected together with PPD, neither monoclonalantibody caused erythematous reactions at 1-4 hr (data notshown). Anti-T-cell FN23 1, but not anti-T-cell FN 248,significantly inhibited PPD-elicited DTH reactions at 24 hr ina dose-dependent manner (Fig. 5). Maximal inhibition was

observed at the highest tested doses, 270-300 ng, and was

highly significant (P < 0-01, Scheffe multiple comparisonanalysis of variance). There was no significant differencebetween the inhibition observed at 18-20 hr and that at 24 hr(data not shown). Inhibited delayed reactions were not onlysmaller, but noticeably paler and less intense than reactionselicited by PPD alone.

Biopsies were taken from six animals showing stronginhibition of DTH expression, to determine if the inhibitionof visible DTH reactivity induced by anti-T-cell FN231 was

associated with changes in cellular infiltrates at the reactionsite. The deep and superficial cellular infiltrates of PPD-elicitedDTH reactions inhibited by 300 ng anti-T-cell FN 231 were

statistically indistinguishable from those elicited by PPD aloneor by PPD and anti-T-cell FN 248 (P > 0 05, Scheffe multiplecomparison analysis of variance) (Fig. 6). The enhancementof reactivity seen with antibody 248 was not statisticallysignificant (P > 0 05, Scheffe multiple comparison analysis ofvariance). Neither anti-T-cell FN monoclonal antibody elicitedany significant dermal infiltrate when injected alone, nor were

infiltrates elicited by injections of PBS (data not shown).An additional group of 12 guinea-pigs was sensitized to

study the effect of anti-T-cell FN on the expression of contact

585

1

S. Mandy et al.

mAb Skin response Dermal infiltrates

PPD Inhibition Deep Superficial(mm2) (%) (qualitative) (cell no.)

0 1 2 3 4 0 50 100150

- 167±21 -

231 126±21 24±11* P248 192±24 -17±6 _

Figure 6. Inhibition of expression of PPD-elicited DTH by anti-T-cellFN 231 is not associated with changes in cellular infiltrates. Sensitizedguinea-pigs (n = 6) were injected intradermally with 500ng PPD,500ng PPD mixed with 300ng anti-T-cell FN 231 and 500ng PPDmixed with 300 ng anti-T-cell FN 248. Reactions were measured at 24 hrand biopsied. Skin reaction and inhibition relative to PPD response

(100%) are shown as means ± SE. Histological evaluation of skinbiopsies is shown as mean qualitative scores ± SE for deep dermalcellular infiltrates (solid bars) and as mean cell number/five oilfields ± SE for superficial dermal infiltrates of mononuclear cells(shaded bars) and basophils (open bars). * Mean differs significantlyfrom value obtained with PPD alone (P < 0.05, ScheffE multiplecomparison analysis of variance).

DTH. Intradermal injections of 90 ng anti-T-cell FN 231significantly decreased expression of DTH to topical DNTB by3 6-fold, compared with responses in animals receiving PBSinjections (P < 0-05) (Fig. 7). The slight enhancement ofreactivity seen with antibody 248 was not statisticallysignificant. Despite the striking difference in reactivity to0-4nmol DNTB of skin sites receiving monoclonal antibody231 and monoclonal antibody 248 or PBS, there was no

significant qualitative or quantitative difference in cellularinfiltrates in biopsies from these sites (P > 0 05, Scheff6

ThresholdmAb skin response

DNTB Inhibition(mmol) (-fold) I

_ 043(016)

231 1-53*(0-12)

248 0-23(0 15)

Dermal infiltratesDeep Superficial

(qualitative) (cell no.)0 1 2 3 4 0 25 50

3-6-

-053

Figure 7. Inhibition of expression of DNTB-elicited contact DTH byanti-T-cell FN 231 is not associated with changes in cellular infiltrates.Sensitized guinea-pigs (n = 12) injected intradermally with 90 ng anti-T-cell FN 231, 90 ng anti-T-cell FN 248 and PBS, and contact reactionswere elicited at the site of injection by graded doses of topically appliedDNTB (0 1-2 nmol). Skin reactivity is shown as geometric mean

threshold reactivity (SE in log units). Skin biopsies were taken ofreactions elicited by 0-4 nmol DNTB. Histological evaluation of skinbiopsies is shown as mean qualitative scores ± SE for deep dermalcellular infiltrates (solid bars) and as mean cell number/five oilfields + SE for superficial dermal infiltrates of mononuclear cells(shaded bars) and basophils (open bars). * Mean differs significantlyfrom value obtained with DNTB over PBS-injected skin (P < 0 05,f-test).

0*3

_ it

O 00o._ .__- c

0.0

Ze 80

c

0

*= 60

c 40

o20

D 0

0 1 2 3 4 51/Fibronectin-binding protein (moa)x1012

10-1 100 101 102Fibronectin-binding protein (pmol)

Figure 8. Inhibition ofexpression ofDTH in guinea-pigs by anti-humanT-cell FN 231 (0) and mycobacterial Ag85 (A). Data using anti-human T-cell FN 231 from the present study and data using Ag85 fromreferences 7 and 23 are used. Inset shows double reciprocal plot of datapoints between 02 and 32 pmol and fitted line. Its y-intercept is-1-6 x 10"/mol.

multiple comparison test) (Fig. 7). Anti-T-cell FN 231 thusinhibited macroscopic erythema and not cellular infiltrates inboth PPD and contact DTH reactions.

Comparison of effects of anti-T-cell FN and Ag85 on expressionof DTH

Ag85, a group of closely related 30 000-32 000 MW FN-binding proteins secreted by M. tuberculosis and othermycobacteria,6 can modulate expression of DTH in vivo.723Like anti-T-cell FN 231, Ag85 binds to and inactivates T-cellFN and inhibits expression of guinea-pig DTH at 24 hr withoutaffecting cellular infiltrates. Dose-response plots of theinhibition of DTH responses induced by anti-T-cell FN 231and by Ag85 fell on the same log-normal dose response curve

(Fig. 8), and double reciprocal plots of these data were fitted byidentical straight lines (Fig. 8, inset). This is consistent withanti-T-cell FN and Ag85 acting on the same molecule in vivo.

DISCUSSION

The association of T-cell FN with DTH has been recognized formany years,3'4 but the role of T-cell FN in mediating in vivoexpression of DTH is still unclear. The present study has usedneutralizing monoclonal anti-T-cell FN antibodies specific forepitopes located on a 70 000MW cell-binding FN fragment toanalyse this role. These anti-T-cell FN antibodies cross-reactedwith human plasma FN and guinea-pig T-cell FN, a notunexpected finding in view of the high degree ofconservation ofFN structure within and across species,t and could therefore beused to examine T-cell FN action in sensitized guinea-pigs.Expression of DTH in vivo was inhibited by only one of theanti-T-cell FN antibodies (antibody 231). Inhibition of DTHexpression by this antibody was not associated with anysignificant change in cellular infiltrates at the reaction site

586

T-cellfibronectin and delayed hypersensitivity 587

compared to sites receiving PBS or the other anti-T-cell FNantibody (antibody 248). The localization of epitopes recog-nized by anti-T-cell FN antibodies to the same FN fragmentdoes not imply epitopic identity because ofthe largeMW of thisfragment. The difference in biological activity betweenantibodies 231 and 248 may reflect differences in epitopespecificity, affinity or both. Our laboratories are now engagedin analysing the epitope specificities and affinities of antibodies231 and 248 to answer this question.

Both anti-T-cell FN antibodies blocked T-cell FN-mediatedmonocyte agglutination, presumably by interfering withbinding of the T-cell FN cell-binding domain to cell-surfaceFN receptors in a manner similar to cell-binding domain-specific anti-FN antibodies such as 333.9,11,22 The antigenicallynon-specific interaction of these monoclonal anti-FN with theFN N-terminal heparin-binding domain is unlikely to play anyrole in their inhibitory activity in vitro, since we have previouslyshown that the much stronger antigen-specific interaction ofanti-plasma FN 304 with this domain does not inhibit T-cellFN-mediated monocyte agglutination.22

Inhibition of macroscopic skin reactions at 24 hr byantibody 231 was not associated with any significant changesin deep or superficial cellular infiltrates at the reaction site. Theapparent lack of effect on the intensity or composition ofcellular infiltrates was unexpected, especially since monocyteagglutination has been perceived as an in vitro correlate of theclumps of mononuclear cells present in biopsies of DTHreactions.3-5 While histological analysis might not be sensitiveenough to detect changes in cellular infiltrates associated with asignificant 15% inhibition of macroscopic response,23 it wassomewhat surprising that no histological changes were seen inbiopsies from reactions showing a significant 25% or 3-6-foldinhibition of macroscopic response, especially since partialinhibition of macroscopic delayed skin responses has beenreported in reactions showing significant decreases in cellularinfiltrates.24'25

The mechanism of inhibition of DTH expression bymonoclonal anti-T-cell FN in the absence of any obviouseffect on cellular infiltrates is unclear. T-cell FN is producedprimarily by T-helper type 1 (Thi) CD4+ and CD8+ T cells.5Activated cloned Thl and CD8+ T-cell lines secrete 15-75times more T-cell FN than do Th2 lines (Z.-H. Feng, S. Mandy,P. Taylor, B. A. Askonas, T. R. Mosmann & H. P. Godfrey,manuscript in preparation). Soon after elicitation and beforesignificant vasodilation occurs, the high MW of FN ensuresthat most, if not all, soluble FN at the tissue site is likely to belocally synthesized T-cell FN. This suggests that T-cell FNcould have a direct action on microvascular endothelial cells.We have recently obtained evidence for this hypothesis.Picomolar concentrations of T-cell FN can activate culturedhuman umbilical vein endothelial cells to express increasedlevels of adhesion molecules (E-selectin, vascular cell adhesionmolecule-1, intercellular adhesion molecule-1) and to increasetheir transport of macromolecules.26

In addition to, or instead of, any direct action onendothelial cells, T-cell FN could be involved in theextravascular localization and/or activation of small numbersof critically important cells necessary for initiation of thevascular components of DTH reactions via release ofinflammatory mediators or pro-inflammatory cytokines.These cells could be a particular subset of FN-binding

mononuclear phagocytes27 whose release of pro-inflammatorycytokines is modulated by adherence.28 FN-binding memoryT-effector cells are another, perhaps more likely, source forthese cells. Antigen-specific murine T cells able to mediate DTHbind to FN; their ability to transfer DTH is inhibited bytreatment of recipients with antibodies, peptides or peptidomi-metic agents that block FN binding to the PIl integrins very lateantigen-4 or very late antigen-5.293' In this connection, arecent report32 that tumour necrosis factor-a, a cytokineproduced by activated keratinocytes33 and T cells34 as well asby activated monocytes, can bind to the FN N-terminal domainand augment PI1-mediated adhesion of CD4+ T cells to FN,may be relevant.

Anti-T-cell FN 231 modulated DTH expression in aqualitatively and quantitatively similar manner to the myco-bacterial FN-binding Ag85 proteins. The volume over whichanti-T-cell FN and Ag85 exert their action in vivo is not known.Initially, this volume is the injection volume (0- 1 ml). It rapidlydecreases, as the injection equilibrates with dermal interstitialfluid over the next hour, to a fraction of this (typically 20% ofwet tissue weight), and then begins to increase as a result of theonset of DTH-associated vasodilation. In the present study,DTH reactions to PPD and contact reactions to DNTB haddiameters of 12-14mm. A 14-mm diameter circle of normalguinea-pig skin weighs about 180mg (our unpublished obser-vations). For this size skin site, 0-05 ml is a plausible mean valuefor the 'action volume' over the 0-3-hr period. Assuming noloss of inhibitor from the reaction site, this action volume andthe negative y-intercept of the double reciprocal plot,1-6 x 10'1/mol, yield a Kd of 1-3 x 10-7M for the interactionof anti-T-cell FN and Ag85 with T-cell FN. This is similar to Kdreported for the binding of soluble plasma FN to the FNreceptors of fibroblasts in suspension.35

In summary, a neutralizing anti-human T-cell FN mono-clonal antibody inhibits expression of DTH in sensitizedguinea-pigs without affecting cellular infiltration at thereaction site. This could indicate that T-cell FN acts directlyon endothelial cells in mediating in vivo DTH.

ACKNOWLEDGMENTS

We wish to thank Dr M. Inchiosa and Dr E. A. Goidl for many helpfulsuggestions and discussions, and Dr S. Levine for histologicalassistance. This work was supported by grant CA34141 from theNational Cancer Institute (H. P. Godfrey), US Department of Healthand Human Services and by the Intramural Research Program of theNational Institute of Dental Research (S. K. Akiyama). M. Y. Khan isVisiting Fellow of the Fogarty International Center.

REFERENCES1. HYNEs R.O. (1989) Fibronectins. Springer Verlag, New York.2. MOSHER D.F. (1989) Fibronectin. Academic Press, San Diego.3. LOLEKHA S., DRAY S. & GOTOFF S.P. (1970) Macrophage

aggregation in vitro. A correlate of delayed hypersensitivity. JImmunol 104, 296.

4. GODFREY H.P. (1976) Hapten-specific responses to contactsensitizers: use of fluorodinitrobenzene to elicit migration inhibi-tion and macrophage agglutination factors from lymph nodes ofcontact-sensitive guinea-pigs. Immunology 30, 685.

5. GODFREY H.P. (1990) T cell fibronectin: an unexpected inflamma-tory lymphokine. Lymphokine Res 9, 435.

588 S. Mandy et al.

6. WIKER H.G. & HARBOE M. (1992) The antigen 85 complex: a majorsecretion product of Mycobacterium tuberculosis. Microbiol Rev 56,648.

7. GODFREY H.P., FENG Z.-H., MANDY S. et al. (1992) Modulation ofexpression of delayed hypersensitivity by mycobacterial antigen 85fibronectin-binding proteins. Infect Immun 60, 2522.

8. KHAN M.Y., JAIKARIA N.S., FRENZ D.A., VILLANUEVA G. &NEWMAN S.A. (1988) Structural changes in the NH2-terminaldomain of fibronectin upon interaction with heparin. Relationshipto matrix-driven translocation. J Biol Chem 263, 11314.

9. GODFREY H.P., CANFIELD L.S., HAAK-FRENDSCHO M., MELANCON-KAPLAN J., BROWN E.J. & KAPLAN A.P. (1989) Relationship ofhuman macrophage agglutination factor to other fibronectins.Immunology 67, 321.

10. AKIYAMA S.K. & YAMADA K.M. (1985) Comparison of evolu-tionary distinct fibronectins: evidence for the origin of plasma andfibroblast cellular fibronectins from a single gene. J Cell Biochem27, 97.

11. GODFREY H.P., CANFIELD L.S., KINDLER H.L., ANGADI C.V.,TOMASEK J.J. & GOODMAN J.W. (1988) Production of a fibronectin-associated lymphokine by cloned mouse T cells. J Immunol 141,1508.

12. STANLEY C.J., JOHANNSSON A. & SELF C.H. (1985) Enzymeamplification can enhance both the speed and the sensitivity ofimmunoassays. J Immunol Meth 83, 89.

13. LIPSICH L.A., LEWIS A.J. & BRUGGE J. (1983) Isolation ofmonoclonal antibodies that recognize the transforming proteinsof avian sarcoma viruses. J Virol 48, 352.

14. NEWMAN S.A., FRENZ D.A., HASEGAWA E. & AKIYAMA S.K. (1987)Matrix-driven translocation: dependence on interaction of amino-terminal domain of fibronectin with heparin-like surface compo-nents of cells or particles. Proc Natl Acad Sci USA 84, 4791.

15. DROWART A., DE BRUYN J., HUYGEN K. et al. (1992) Isoelectriccharacterization of protein antigens present in mycobacterialculture filtrates and recognized by monoclonal antibodies directedagainst the Mycobacterium bovis BCG antigen 85 complex. ScandJImmunol 36, 697.

16. GODFREY H.P., CANFIELD L.S., ANGADI C.V., ZAGACHIN L.M.,KIELPINSKI G.G. & COLVIN R.B. (1990) Characterization oflymphokine fibronectin from guinea pig lymphoid cell culturesupernatants. Immunobiology 180, 109.

17. GODFREY H.P., PHILLIPS M.E. & ASKENASE P.W. (1983) Histo-pathology of delayed-onset hypersensitivities in contact sensitiveguinea pigs. Int Arch Allergy Appl Immunol 70, 50.

18. GODFREY H.P. & BAER H. (1971) The effect of physical andchemical properties of the sensitizing substance on the inductionand elicitation of delayed contact sensitivity. J Immunol 106, 431.

19. WALLENSTEIN S., ZUCKER C.L. & FLEISS J.L. (1980) Some statisticalmethods useful in circulation research. Circ Res 47, 1.

20. JESTY J. & GODFREY H.P. (1986) Parlin, a general microcomputerprogram for parallel-line analysis of bioassays. Am J Clin Pathol85, 485.

21. ROSTAGNO A.A., FRANGIONE B. & GOLD L.I. (1991) Biochemical

studies on the interaction of fibronectin with Ig. J Immunol 146,2687.

22. GODFREY H.P., FRENZ D.A., CANFIELD L.S., AKIYAMA S.K. &NEWMAN S.A. (1989) Non-chemotactic translocation of phagocyticcells mediated by a fibronectin-related human lymphokine. JImmunol 143, 3691.

23. GODFREY H.P. (1993) T cell fibronectin, delayed hypersensitivityand human disease. In: Biology ofSalmonella (eds F. C. Cabello &C. Hormaeche), p. 299. Plenum Press, New York.

24. BABA T., YOSHIDA T. & COHEN S. (1979) Suppression of cell-mediated immune reactions by monosaccharides. J Immunol 122,838.

25. SOBEL R.A., HANZAKOS J.A., BLANCHETrE B.W., WILLIAMS A.M.,DELLAPELLE P. & COLVIN R.B. (1987) Anti-T cell monoclonalantibodies in vivo. I. Inhibition of delayed hypersensitivity but notcutaneous basophil hypersensitivity reactions. J Immunol 138,2500.

26. QUAN X. & GODFREY H.P. (1994) Induction ofendothelial adhesionmolecules by T cell fibronectin (FN). FASEB J8, A135.

27. SZCZEPANIK M., BRYNIARSKI K., PRYJMA J. & PTAK W. (1993)Distinct populations of antigen-presenting macrophages arerequired for induction of effector and regulatory cells in contactsensitivity response in mice. J Leukocyte Biol 53, 320.

28. STANDIFORD T.J., KUNKEL S.L., KASAHARA K., MILIA M.J., ROLFEM.W. & STRIETER R.N. (1991) Interleukin-8 gene expression fromalveolar macrophages: the role of adherence. Am J Respir CellMolec Biol 5, 579.

29. FERGUSON T.A., MIZUTANI H. & KUPPER T.S. (1991) Two integrin-binding peptides abrogate T cell-mediated immune responses invivo. Proc Natl Acad Sci USA 88, 8072.

30. GREENSPOON N., HERSHKOVIZ R., ALON R., VARON D., SHENKMANB. & MARx G. (1993) Structural analysis of integrin recognitionand the inhibition of integrin-mediated cell functions by novelnonpeptide surrogates of the Arg-Gly-Asp sequence. Biochemistry32, 1001.

31. FERGUSON T.A. & KUPPER T.S. (1993) Antigen-independentprocesses in antigen-specific immunity. A role for alpha 4integrin. J Immunol 150, 1172.

32. ALON R., CAHALON L., HERSHKOVIZ R. et al. (1994) TNF-a binds tothe N-terminal domain of fibronectin and augments the #i-integrin-mediated adhesion of CD4 + T lymphocytes to theglycoprotein. J Immunol 152, 1304.

33. GRIFFITHS C.E.M., BARKER J.N.W.N., KUNKEL S. & NICKOLOFFB.J. (1991) Modulation of leucocyte adhesion molecules, a T-cellchemotaxin (IL-8) and a regulatory cytokine (TNF-alpha) inallergic contact dermatitis (rhus dermatitis). Br J Dermatol 124,519.

34. GOLDFELD A.E., MCCAFFREY P.G., STROMINGER J.L. & RAO A.(1993) Identification of a novel cyclosporin-sensitive element in thehuman tumor necrosis alpha gene promoter. J Exp Med 178, 1365.

35. AKIYAMA S.K. & YAMADA K.M. (1985) The interaction of plasmafibronectin with fibroblastic cells in suspension. J Biol Chem 269,4492.