Immunology 1995 84 440-445

Effects of fibronectin and group B streptococci on tumour necrosis factor-aproduction by human culture-derived macrophages

E. B. PEAT,* N. H. AUGUSTINE, W. K. DRUMMOND, J. F. BOHNSACK & H. R. HILL Divisions of

Clinical Immunology and Allergy and Clinical Pathology, Departments of Pathology, Medicine and Pediatrics, University of UtahSchool of Medicine, Salt Lake City, Utah, USA and *London School of Hygiene and Tropical Medicine, London, UK

SUMMARY

Group B streptococci (GBS) are an important cause of sepsis and shock in the new-born. We havepreviously reported that GBS induce the production of tumour necrosis factor-c (TNF-ca) byhuman monocytes and culture-derived macrophages. We have also shown that fibronectin (FN)promotes interaction between GBS and human phagocytes. In the present study, we investigatedthe effect of FN and GBS on the production of TNF-a by adult and neonatal culture-derivedmacrophages. We report that soluble FN alone was a strong stimulus for the production ofTNF-aiby culture-derived macrophages (FN 50 ,g/ml = 623 33 ± 47 pg/ml TNF, versus media alone3 ± 1 5 pg/ml; P < 0-0001). While GBS also induce the production of TNF-a by macrophages, theaddition of FN to GBS had more than an additive effect on TNF-a levels. FN-mediated TNF-caproduction by macrophages was inhibited by both soluble arginine-glycine-aspartic acid (RGD)peptide (71%; P<000001) and anti-,f3-integrin monoclonal antibody 7G2 (54%; P<0-0001).Neonatal culture-derived macrophages produced significantly more TNF-ac in response to GBS(356 4 pg/ml + 27-7) than adult cells did (222-0 pg/ml ± 21 -0; P = 0-037), and dramatically more inresponse to FN alone (neonatal 193 1-0 pg/ml 23-0 versus adult 463-5 ± 43.5 pg/ml; P < 0-000 1).FN may contribute to the high levels of TNF-a production implicated in the pathophysiology ofGBS sepsis and shock.

INTRODUCTION

Group B streptococci (GBS) are an important cause of sepsisand shock in new-born humans, with 15-30% mortality." 2Early onset disease, characterized by respiratory distress,hypoxaemia and progressive systemic hypotension, has beenlinked to Gram-negative septic shock.3'4 It has been postulatedthat the pro-inflammatory cytokines tumour necrosis factor-a(TNF-ci), interleukin (IL-1) and IL-6, produced by antigen-presenting cells in response to Gram-negative endotoxin, play amajor role in the pathophysiological response leading to theseptic shock syndrome.5-"1

Cytokines function as intercellular messengers in anautocrine or paracrine manner.'2 Physiological effects of thepro-inflammatory cytokines, which alter vascular permeability,cell migration and adhesiveness, are beneficial in promotingan effective local inflammatory response.'2 Deleterious effectsmay result from 'systemic spillover' of TNF-a. Intravenousinfusion in animals causes cardiac depression, altered vascularpermeability, progressive hypotension, lung injury and hypox-aemia.6 l"14 Elevated TNE-x concentrations in both children

Received 30 June 1994; revised 8 September 1994; revised 1November 1994.

Correspondence: Dr H. R. Hill, Department of Pathology,University of Utah School of Medicine, Salt Lake City, UT 84132, USA.

and adults have been associated with increased mortality fromseptic shock. 5-17

The role of TNF-a in Gram-positive sepsis is increasinglybeing recognized.'8 We have previously reported that GBSinduce the production of TNF-a by human monocytes andculture-derived macrophages.'9 The detection of TNF-a in theserum, urine and cerebrospinal fluid of patients with neonatalGBS sepsis,'9 and the finding that anti-TNF-a antibodiesenhance early survival in a rat model of neonatal group Bsepsis,20 suggest an important role for TNF-a in thepathophysiology of GBS sepsis and shock.

Fibronectin (FN) is a high molecular weight dimeric protein(440 000-550 000 MW) that exists as a number of alternativesplicing products of a single gene.21'22 Soluble plasma, insolublecell-associated and extracellular matrix FN play important rolesin cell adhesion and migration, as well as cell differentiation andgrowth.23 Adult plasma levels of FN average 300 pg/ml, incomparison with neonatal levels of approximately 100 g/ml.24

There is a growing appreciation of the importance of FN inthe acute inflammatory response.25'26 In contrast to the singlereceptor described on neutrophils, Brown & Goodwin27reported the presence of at least two types of macrophagereceptors for FN. One of these recognizes small syntheticpeptides containing the Arg-Gly-Asp-Ser (RGDS) sequence,while the other recognizes a larger FN fragment, including

440

Effects offibronectin and group B streptococci on TNF-a production

RGDS, derived from chymotrypsin digestion of the molecule.27The observation was also made that, in response to FN, restingmonocytes enhance phagocytosis without the need for anyapparent additional stimulus as required by neutrophils.27More recently, it has been shown that both neutrophils andmonocytes/macrophages express a FN receptor similar to, butdistinct from, the platelet #3-integrin IIb/IIIa,28 and thatmonocytes/macrophages express the classical FN receptorvery late activation antigen-5 (VLA-5).26'29-32 In addition tothese RGD-responsive receptors, macrophages have beenshown to express VLA-4,2932 which recognizes the sequenceEILDV (minimum essential sequence LDV) found in thealternatively spliced CS-1 variable region of FN.33

These are the first studies to explore the effects of thecombination of a bacterial pathogen and FN on the inductionof TNF production by human culture-derived macrophages.

MATERIALS AND METHODS

Preparation ofmacrophagesPeripheral blood samples were obtained from adult volunteersafter informed consent, and collected into heparin-containingsyringes (10pg/ml). Umbilical cord blood, obtained afternormal-term deliveries, was placed in acid-citrate-dextroseanticoagulant solution (Becton Dickinson Co., Rutherford,NJ). Whole blood was diluted with an equal volume ofendotoxin-free Hank's balanced salt solution (HBSS;Whittaker Bioproducts, Walkersville, MD). The samples were

then layered on a separation medium (Ficoll-Paque; PharmaciaInc., Piscataway, NJ) at a ratio of 2: 1 and centrifuged at 1000gfor 30min. The interface mononuclear cell layer containingmonocytes and lymphocytes was aspirated and washed twicewith HBSS. The cells were then resuspended in endotoxin-freeRPMI-1640 culture medium (Whittaker Bioproducts) withL-glutamine (2 mM; Whittaker Bioproducts), gentamicin (50 Yg/ml), HEPES buffer (N-hydroxyethylpiperazine-N-ethane sul-phonic acid; 10 mmol/l; Whittaker Bioproducts) and 10%pooled normal human serum at a concentration of 2-0 x 106mononuclear cells/ml.

Macrophages were prepared by culturing mononuclear cellsin 6-well plastic Multiwell Tissue Culture Plates (BectonDickinson, Lincoln Park, NJ) for 6-7 days. Two millilitres ofcell suspension was added to each well and incubated in a 5%CO2 incubator at 370 for 2 hr, to allow the cells to adhere. Thewells were then washed with HBSS to remove non-adherentcells, and reincubated at 37° with 2ml RPMI-1640 culturemedium, modified as described above. After 24hr, the cellswere again washed as above and reincubated with culturemedium as described for a further 5-6 days. Previous studieshave shown that human peripheral blood monocytes in culturedifferentiate into cells morphologically and functionally similarto macrophages.34 The cells were then washed with HBSS andstimulation experiments were performed. An additional platewas set up to count cells and confirm non-specific esterase-positive macrophages as myeloperoxidase-negative (mean yield1[2 x 105 macrophages/well).

Preparation of organismsA human blood isolate of encapsulated type III GBS (COHI)was cultured in Todd-Hewitt broth (Difco, Detroit, MI) at 370for 16-18 hr. The organisms were then washed three times in

HBSS and resuspended in HBSS at a standard concentration of5 x 108 colony-forming units (CFU)/ml, as determined by anoptical density (OD) of 0 9 at 620 nm (Spectronic 20; Bausch &Lomb, Rochester, NY). Bacterial suspensions were thenaliquoted, heat killed at 56° for 1 hr and stored at -70° forlater use in the experiments.

Stimulation experimentsFN (Gibco BRL, Grand Island, NY) was purified from plasmaby affinity chromatography on gelatin-sepharose; furtherpurification was carried out by ion-exchange chromatographyto remove glycosaminoglycans. Polyacrylamide gel electro-phoresis (PAGE) revealed a single band at approximately440 000MW, indicating intact FN. The arginine-glycine-asparticacid (RGD)- and arginine-glycine-glutamic acid (RGE)-containing peptides, GRGDSP (Gly-Arg-Gly-Asp-Ser-Pro)and GRGESP (Gly-Arg-Gly-Glu-Ser-Pro), were also obtainedfrom Gibco BRL. The murine monoclonal antibodies 7G2,which by Western blot recognizes the fl-chain of the #3-integrinimportant in RGD binding,27 and 3D9 against the complementreceptor-1, were kindly provided by Dr E. Brown (WashingtonUniversity, St Louis, MO). Cell cultures were incubated in a5% CO2 incubator at 370 for 6 hr. Supernatants were thenaspirated and centrifuged at 1000g for 10min before beingstored at - 700 for TNF-a assay later.

TNF-a assay

TNF-a concentrations were measured in culture supernatantsby an enzyme-linked immunosorbent assay (ELISA; R&D,Minneapolis, MI). All determinations were made in duplicate.

EndotoxinAll buffers, reagents and culture media were assayed for thepresence of endotoxin with the limulus amoebocyte lysatereaction (Endotect; ICN Biomedicals Inc., Costa Mesa, CA),which is sensitive to 0-1 ng endotoxin/ml.

StatisticsResults are expressed as mean ± 1 SE, with triplicate experi-ments performed with duplicate assays per experiment unlessotherwise indicated. Statistical analysis was carried out on allexperiments using the unpaired Student's t-test.

RESULTS

FN-stimulation of TNF-a production by culture-derivedmacrophages

Soluble FN markedly stimulated the production of TNF-a byculture-derived macrophages (Fig. 1). In contrast, solubleRGD peptides failed to enhance TNF-cx production. As shown,FN induced production of TNF-ax in a dose-dependent manner

(Fig. 2), with the peak response at 200 yg/ml FN, at which pointthe response reached a plateau. The concentration ofendotoxinin the FN, as determined by the limulus ameobocyte lysatereaction, was less than 0-1 ng/ml, a concentration below thatrecognized as causing macrophage activation. Addition ofpolymixin B (1 ug/ml) had no effect on FN-induced TNF-aproduction but markedly inhibited that due to Escherichia coliendotoxin (data not shown).

In previous studies, we have shown that GBS also induce

441

E. B. Peat et al.

800-

- 600-

c).2L 400'LL

z'- 200-

804I Mean ± SEn = 3

Media 25 50 25 50FN RGD

(pg/ml) (pg/ml)

Figure 1. Production of TNF-a by culture-derived macrophages fromadult control blood in response to soluble FN and soluble RGDpeptide. The data are from triplicate experiments, with three assays foreach experiment.

production of TNF-oa.19 As shown in Fig. 3, when FN was

added to GBS in increasing concentrations, the responses were

markedly increased compared to GBS or FN alone. The effectwas substantial even at very low doses of FN (5 pg/ml).

TNF-ax production in response to FN was also timedependent (Fig. 4). In the present investigations, GBS aloneinduced TNF-cx production up to concentrations of 600 pg/ml,with peak activity occurring at 12 hr. FN alone dramaticallyincreased production of TNF-x to concentrations as high as

5500 pg/ml, with peak activity at 6 hr. The combination ofGBSplus FN resulted in peak concentrations of TNF-a that, at6800 pg/ml were slightly more than additive when compared toGBS or FN alone. The peak was at the 6 hr time point, whichwas much earlier than the peak observed with GBS alone.

Inhibition of FN-induced TNF-a production

FN binds to many cells through a RGD sequence in its cell-binding domain. While substrate-bound RGD may provide a

stimulus mimicking intact FN, soluble RGD peptides mightbe expected to competitively inhibit the binding and effectsof FN. In contrast to the control peptide GRGESP, which hadno effect, the RGD-containing peptide GRGDSP showed some

inhibitory effect on macrophage stimulation by GBS and FN at50 pg/ml, and significant but incomplete inhibition at 100 pg/mland 200pg/ml GRGDSP (P < 0 0001; Fig. 5). GRGDSP, butnot GRGESP, partially blocked TNF-oc production by FNalone but had no effect on that due to GBS (data not shown).

The murine anti-human monoclonal antibody, 7G2, bindsto members of the #3-integrin family through their commonf-chain, as seen by Western blot analysis. As shown, the

- 604

'C 401LL

zH-' 20'

0 E]FN aloneEl FN + GBS (106/ml) z

- I mean±SE

0-

Media GBS 05 5 125 25 50(1 06/ml)

FN (pg/ml)

Figure 3. Production of TNF-a by culture-derived macrophages inresponse to GBS, 106 CFU/ml, and the effect of addition of FN. Thedata are from triplicate experiments, with triplicate assays for eachexperiment.

monoclonal antibody 7G2 had no effect on TNF-a productioninduced by GBS (Fig. 6a). In contrast, this anti-f33-integrinantibody caused significant but incomplete inhibition of TNF-acproduction induced by both FN alone and FN together withGBS (Fig. 6a). The control monoclonal antibody, 3D9, whichrecognizes the complement receptor-i, did not inhibit FN-induced TNF-Lx production (Fig. 6b). At a concentration of50 pg/ml, the antibody 3D9 had a stimulatory effect on TNF-Lxproduction, which may relate to direct binding to the com-

plement receptor (CR)-1 receptor.

Effect of FN on TNF-a production by neonatal culture-derivedmacrophages

Neonatal culture-derived macrophages produced significantlymore TNF-a in response to GBS than did control adult cells(Fig. 7; P = 0 037). The production of TNF-a induced inneonatal cells by FN alone or together with GBS was markedlygreater than with GBS alone, and significantly higher than thatinduced in adult cells by either FN alone (P < 0 0001) or FNtogether with GBS (P < 0-0001). These experiments were

repeated on five occasions and the results were essentiallyidentical.

DISCUSSION

Soluble FN is a strong stimulus for the production ofTNF-a byculture-derived macrophages adherent to plastic. While GBSalso induce the production of TNF-oa by macrophages, theaddition ofFN to GBS has slightly more than an additive effect

8000

6000 -

5000-

E 4000-

3000'Z 2000

1000

I Mean SEn= 2

- 6000-

.L 4000-LLz

2000-

50 100 200 300

FN (pg/ml)

Figure 2. Production of TNF-oc by culture-derived macrophages fromadult controls in response to increasing doses of soluble FN. These datarepresent duplicate experiments with two assays for each experiment.

o MediaA GBSA FN* GBS + FNI Mean ± SEn= 2

12Time (hr)

Figure 4. Effect of time on TNF-a production by culture-derivedmacrophages from adult controls in response to GBS, 106 CFU/ml,FN, 50pg/ml, or GBS plus FN. The data represent duplicatedeterminations, with duplicate assays for each experiment.

442

Effects offibronectin and group B streptococci on TNF-a production

1000- Mean ± SE

800T *P < 0-0001T

n= 3

E600

LL~ 400z

200

0

Media 0 10 25 50 100 200

RGD (g/ml)

Figure 5. Effect of soluble RGD peptide on TNF-a production byculture-derived macrophages from adult controls stimulated with GBS,106 CFU/ml, and FN, 50yg/ml. The data are from three experimentswith duplicate assays for each experiment.

on TNF-a levels. Both soluble RGD peptide and the anti-#3-integrin, 7G2, inhibit FN-mediated TNF-a production bymacrophages. Neither soluble RGD peptide nor 7G2 was

able to completely inhibit TNF-a production induced byFN. Neonatal culture-derived macrophages produced more

TNF-x in response to GBS than did adult cells, anddramatically more in response to FN alone or to FN with GBS.

Brown & Goodwin27 made the initial observation thatresting monocytes were capable of enhancing phagocytosis inresponse to FN without any additional stimulus. In our study,soluble FN alone was sufficient to induce adherent culture-derived macrophages to secrete TNF-a. The possibility thatendotoxin contamination is contributing to macrophageactivation must be addressed. All media and reagents usedwere endotoxin free and cells were cultured with antibiotic-containing medium. Endotoxin was detected in the soluble FN

gc)

z2500 -

2000-

1500-

1000-

500 -

(a)* P< 0-00001I Mean±SE* Without 7G2

] With 7G2n = 3

1*

(b)

I Mean±SE

Without 3D9

Fg With 3D9

Media GBS FN GBS(106/ml) (50pg/ml) +

FN

Figure 6. (a) Effect of the anti-f13-integrin antibody 7G2 on productionof TNF-a by culture-derived macrophages from adult controls inresponse to GBS, 106 CFU/ml, FN, 50 ug/ml, or GBS plus FN. Thedata represent three experiments with duplicate assays for eachexperiment. (b) Effect of the control antibody 3D9 on production ofTNF-a by culture-derived macrophages from adult control blood inresponse to GBS, 106 CFU/ml, FN, 50pg/ml, or GBS plus FN. Thedata represent three experiments with duplicate assays for eachexperiment.

3000 -

^ 2000-E

z1000 -

11

Control

* Neonate *I MeantSE* P= 0-037** P< 0-0001

~rIMedia GBS FN

(106/ml) (50pg/ml)GBS+

FN

Figure 7. Production of TNF-a by neonatal and adult control culture-derived macrophages in response to GBS, 106 CFU/ml, FN 50 pg/ml, or

GBS plus FN, adjusted for cell count (1 2 x 105 cells). The results depicta representative experiment.

at < 0 1 ng/ml, a concentration which is below that recognizedto cause macrophage activation.35 Moreover, addition ofpolymixin B at 1 pg/ml had no effect on TNF-x induced byFN or GBS but inhibited that due to E. coli lipopolysaccharide.

The intact FN molecule is a high molecular weight dimer(440 000-550 000 MW) and is, therefore, potentially capable ofinteracting with macrophages at multiple sites, either throughdifferent binding sites on the molecule or through multiple FNreceptors of the same type on the macrophage. At least threeFN receptors, VLA-4, VLA-5 and a Ilb/Illa-like #3-integrinreceptor, are known to be present on macrophages.2632 Inaddition, macrophages have been reported to interact withT-cell FN (macrophage agglutinin) through a fucose recep-

tor,36 and the FN heparin-binding site is known to interact withcell-surface proteoglycans.37 The monoclonal antibody 7G2binds to #3-integrins. In our study, 7G2 significantly but iscompletely blocked the TNF-a-stimulating activity of wholeFN. This suggests that, in addition to the Ila/IlIb-like receptor,the stimulatory activity of FN is mediated by other non-#3-integrin receptors. Soluble RGD peptides are known to blockFN binding to RGD-binding receptors.38'39 In these experi-ments, RGD peptides caused incomplete inhibition of TNF-aproduction. This suggests that not all of the FN-receptorinteractions resulting in TNF-a production were RGDmediated. There may be, therefore, a possible role for VLA-4interaction with an EILDV sequence. We have attempted toblock this with the monoclonal antibody 13, but unfortunately,the antibody was contaminated with endotoxin, making thedata uninterpretable. Further experiments are planned toexplore the fibronectin-macrophage receptor interactions ingreater detail.

There is increasing evidence to suggest that systemic TNF-acontributes significantly to the pathophysiology and poor

outcome in septic shock.5-17 The clinical picture of early onsetGBS sepsis closely resembles that of Gram-negative septicae-mia and shock.3'4 In this and previous studies,'9 we have shownthat GBS induce macrophage production of TNF-ca. We have,in addition, shown that soluble FN dramatically enhancesmacrophage production of TNF-a. FN plays a role in theactivation of macrophages in the acute inflammatory response

that is beneficial for host defences. It has been shown40 thattreatment of neonatal rats with FN and opsonic antibodyimproves survival in experimental GBS infection. Moreover,

443

444 E. B. Peat et al.

Alexander et al.41 have reported that pretreatment of neonatalrats with TNF-a improves survival in Gram-negative infection.Thus, the kinetics of local release of TNF-a by macrophagesmay profoundly affect outcome. In contrast, during laterinvasive infection, massive release of TNF-a induced bybacterial products plus FN may contribute to the harmfulpathophysiological effects. We believe that the interaction ofthe invading organism with FN and macrophages may be animportant determinant of both the kinetics and magnitude ofcytokine release, thereby profoundly affecting the local andsystemic inflammatory response.

Enhanced susceptibility to septic shock due to GBS ischaracteristic in the neonatal period. We have previouslyshown that mixed mononuclear cells from neonates producesignificantly more TNF-a than do cells from adults in responseto GBS.19 In this study, we have shown that neonatal culture-derived macrophages are also exquisitely sensitive to FN andGBS stimulation and produce dramatically more TNF-a thando adult control macrophages in response to these stimuli. It ispossible, therefore, that this markedly enhanced capability ofthe neonatal monocyte/macrophage to produce TNF-a maycontribute to the pathogenesis of GBS shock in the neonate.

The interactions between macrophages and FN are complexand incompletely understood. Not only does FN play a role inmacrophage adhesion to the extracellular matrix,23 it alsoaffects the physiological activity of the cell both in enhancingphagocytosis42 and, as we have shown, inducing macrophagecytokine secretion. The interaction may depend on the type ofFN (soluble plasma, insoluble cell-associated or extracellularmatrix) encountered, the cell type (circulating monocyte ortissue macrophage) involved and the local microenvironment.Vercellotti et al.43 have indicated that FN is altered whenexposed to phagocytic cell supernatants containing proteinases,and becomes 'inflamed' FN that is then capable of amplifyinginflammation. This inflamed FN may then be capable ofstimulating cytokine release. We are pursuing this possibilityemploying proteinase inhibitors. Beezhold & Personius" havealso reported that FN stimulates peripheral blood monocyteTNF secretion. The extravasation of 'inflamed' FN togetherwith plasma in the acute inflammatory response may contributeto the amplification of the response with beneficial local effectson host defences. Alternatively, in the lung, GBS, FN andmacrophages may interact, resulting in the massive release ofTNF that may, in turn, lead to endothelial cell and phagocyteactivation, leucocyte sequestration and vascular leak. This maycontribute to harmful systemic pathophysiology in neonatalGBS pneumonia and septic shock.

ACKNOWLEDGMENTS

This work was supported by US Public Health Service Grants AI- 13 150and AI-26733. We wish to thank Dr Eric Brown (WashingtonUniversity, St Louis, MO) for the monoclonal antibodies 7G2 and3D9. We also thank Don Morse for his assistance with the illustrationsand Jeannette Rejali for secretarial aid.

REFERENCES

1. HILL H.R. (1990) Group B streptococcal infections. In: SexuallyTransmitted Diseases (eds K. K. Holmes, P. A. Mardh, P. F.Sparling et al.), p. 851. McGraw-Hill, New York.

2. MCCRACKEN G.H. (1973) Group B streptococci: the new challengein paediatric infections. J Pediatr 82, 703.

3. FENTON L.J. & STRUNK R.C. (1977) Complement activation andgroup B streptococcal infection in the newborn: similarities to

endotoxin shock. Pediatrics 60, 901.4. SCHLIEVERT P.M., VARNER M.W. & GALASK R.P. (1991) Endotoxin

enhancement as a possible aetiology of early onset group B

fl-haemolytic streptococcal sepsis in the newborn. Obstet Gynecol61, 588.

5. TRACEY K.J., LOWRY S.F. & CERAMI A. (1988) Cachectin/TNF-a inseptic shock and septic adult respiratory syndrome (editorial). AmRev Resp Dis 138, 377.

6. HESSE D.G., TRACEY K.J., FONG, Y. et al. (1988) Cytokineappearance in human endotoxaemia and primate bacteraemia.Surg Gynecol Obstet 166, 147.

7. OKUSAWA S., GELFAND I.A. & IKEJAMA T. (1988) Interleukin-linduces a shock-like state in rabbits. Synergism with tumor necrosisfactor and the effects of cyclo-oxygenase inhibition. J Clin Invest81, 1162.

8. FONG Y., TRACEY K.J., MOLDWER L.L. et al. (1989) Antibodies tocachectin/tumor necrosis factor reduce interleukin-1 13 andinterleukin-6 appearance during lethal bacteraemia. J Exp Med170, 1627.

9. WAAGE A., BRANDTKAEG P., HALSTENSEN A., KIERULF P. & ESPEVIKT. (1989) The complex pattern of cytokines in serum from patientswith meningococcal septic shock: association between interleukin 1and interleukin 6 and fatal outcome. J Exp Med 169, 133.

10. ODIO C.M., FAINGEZICHT I., PARIS M. et al. (1991) The beneficialeffects of early dexamethasone administration in infants andchildren with bacterial meningitis. N Engl J Med 324, 1525.

11. DINARELLO C.A. (1991) The proinflammatory cytokines interleu-kin- 1 and tumor necrosis factor and the treatment of septic shocksyndrome. J Infect Dis 163, 1177.

12. ARAI K., LEE F., MIYAJIMA A., MIYATAKE S., ARAI N. & YOKOTA T.(1990) Cytokines: co-ordinators of immune and inflammatoryresponses. Annu Rev Biochem 59, 783.

13. TRACEY K.J., BEUTLER B. & CERAMI A. (1987) Cachectin/tumornecrosis factor induces lethal shock and stress hormone responses

in the dog. Surg Gynecol Obstet 164, 415.14. JOHNSON J., MEYNCK B., JESMOK G. & BRIGHAM K.L. (1989) Human

recombinant TNF-alpha infusion mimics endotoxaemia in awakesheep. J Appl Physiol 66, 1448.

15. OFFNER F., PHILIPPE J., VOGLAERS D. et al. (1990) Serum tumornecrosis factor levels in patients with infectious disease and septicshock. J Lab Clin Med 116, 100.

16. DAMAS P., REUTER A., GYSEN P., DEMONTY J., LAMY M. &FRANCHIMONT P. (1989) Tumor necrosis factor and interleukin-1serum levels during severe sepsis in humans. Crit Care Med 17, 975.

17. SULLIVAN J.S., KILPATRICK L., COSTANNO A.T., LEE S.C. & HARRISM.C. (1992) Correlation of plasma cytokine elevations withmortality rates in children with sepsis. J Pediatr 120, 510.

18. FAST D.J., SCHLIEVERT D.J. & NELSON R.D. (1989) Toxic shocksyndrome-associated staphylococcal and streptococcal pyogenicexotoxins are potent inducers of tumor necrosis factor production.Infect Immun 57, 291.

19. WILLIAMS P.A., BOHNSACK J.F., AUGUSTINE N.H., DRUMMONDW.K., RUBENS C.E. & HILL H.R. (1993) Production of tumornecrosis factor by human cells in vitro and in vivo, induced by groupB steptococci. J Pediatr 123, 292.

20. TETI G., MANCUSO G. & TOMASELLO F. (1993) Cytokine appearanceand effects of anti-tumor necrosis factor alpha antibodies in a

neonatal rat model of group B streptococcal infection. InfectImmun 61, 227.

21. HYNES R. (1985) Molecular biology of fibronectin. Annu Rev CellBiol 1, 67.

22. KORNJBLIHTT A.R., UMEZAWA K., VIBE-PEDERSON K.P. & BARALLEF. (1985) Primary structure of human plasma fibronectin:differential splicing may generate at least 10 polypeptides from a

single gene. EMBO J 4, 1755.

Effects offibronectin and group B streptococci on TNF-a production 445

23. McKEowN-LONGO P.J. (1987) Fibronectin cell surface interactions.Rev Infect Dis 9 (suppl.), S322.

24. MCCAFFERTY M.H., LEPOW M., SABA T.M., CHO E., MEUWISSEN H.,WHITE J. & ZUCKERBROD S.F. (1983) Normal fibronectin levels as afunction of age in the paediatric population. Pediatr Res 17, 482.

25. MOSHER D.F., PROCTOR R.A. & GROSSMAN J.E. (1981) Fibronectin:role in inflammation. Adv Inflamm Res (ed. G. Weissman), Vol. 2,p. 187. Raven Press, New York.

26. HOLERS V.M., RUFF T.G., PARKs D.L., McDoNALD J.A., BALLARDL.L. & BROWN E.J. (1989) Molecular cloning of a murinefibronectin receptor and its expression during inflammation. JExp Med 16, 1589.

27. BROWN E.J. & GOODWIN J.L. (1988) Fibronectin receptors ofphagocytes: characterization of the Arg-Gly-Asp binding proteinsof human monocytes and polymorphonuclear leukocytes. J ExpMed 167, 777.

28. GRESHAM H.D., GOODWIN J.L., ALLEN P.M., ANDERSON D.C. &BROWN E.J. (1989) A novel member of the integrin receptor familymediates Arg-Gly-Asp stimulated neutrophil phagocytosis. J CellBiol 108, 935.

29. KLINGEMANN H.G. & DEDHAR S. (1989) Distribution of integrinson human peripheral blood mononuclear cells. Blood 74, 1348.

30. HEMLER M.E. (1990) VLA proteins in the integrin family:structures, functions and their role on leukocytes. Annu RevImmunol 8, 365.

31. SPRINGER T.A. (1990) Adhesion receptors of the immune system.Nature 346, 425.

32. RUOSLAHTI E. (1991) Integrins. J Clin Invest 87, 1.33. WAYNER E.A., GARCIA P.A., HUMPHRIES M.J., McDONALD J.A. &

CARTER W.G. (1989) Identification and characterization of thelymphocyte adhesion receptor for an alternative cell attachmentdomain in plasma fibronectin. J Cell Biol 109, 1321.

34. ZUCKERMAN S.H., ACKERMAN S.K. & DOUGLAS S.D. (1979) Long

term human peripheral blood monocyte cultures: establishment,metabolism and morphology of primary human monocyte-macrophage cultures. Immunology 38, 401.

35. PRPIC V., WEIEL J.E., SOMERS S.D. et al. (1987) Effects of bacteriallipopolysaccharde on the hydrolysis of phosphatidylinositol-4-5-biphosphate in murine peritoneal macrophages. J Immunol 139,526.

36. GODFREY H.P. & DONSON J. (1990) Response of human macro-phages to macrophage agglutination factor (T cell fibronectin)involves a monocyte fucose receptor. FASEB J 4, A1757.

37. RUOSLAHTi E. (1988) Fibronectin and its receptors. Annu RevBiochem 57, 375.

38. WRIGHT S.D. & MEYER B.C. (1985) Fibronectin receptor of humanmacrophages recognizes the sequence Arg-Gly-Asp-Ser. JExp Med162, 762.

39. RuOSLAHTi E. & PIERSCHBACHER M.D. (1986) Arg-Gly-Asp: aversatile cell recognition signal. Cell 44, 517.

40. HILL H.R., SHIGEOKA A.O., AUGUSTINE N.H., PRITCHARD D.,LUNDBLAD J.L. & SCHWARTZ R.S. (1984) Fibronectin enhances theopsonic and protective activity of monoclonal and polyclonalantibody against group B streptococci. J Exp Med 159, 1618.

41. ALEXANDER H.R., SHEPPARD B.C., JENSEN J.C. et al. (1991)Treatment with recombinant human tumor necrosis factor-protects rats against the lethality, hypotension, and hypothermiaof gram negative sepsis. J Clin Invest 188, 34.

42. PROCTOR R.A. (1987) Fibronectin: an enhancer of phagocytefunction. Rev Infect Dis 9 (suppl.), S412.

43. VERCELLOTTI G.M., MCCARTHY J., FURCHT L.T., JACOB H.S. &MOLDOW C.F. (1983) Inflamed fibronectin: an altered fibronectinenhances neutrophil adhesion. Blood 62, 1063.

44. BEEZHOLD D.H. & PERSONIUS C. (1992) Fibronectin fragmentsstimulate tumor necrosis factor secretion by human monocytes.JLeukBiol51, 59.