Method of preventing and treating bacterial infection of sutures and prosthetic devices, and promoting ingress of leukocytes into tumor foci

An agent capable of inhibiting signalling mediated by a .beta..sub.1 integrin cell surface receptor of leukocyte cells will treat a bacterial infection associated with a surface of a foreign body over and around which fibrin has been deposited, or a malignant tumor over and around which tenascin has been deposited. In addition, coating a foreign body with a fibrinolytic agent will prevent chronic bacterial infection associated with the surface of the foreign body. Furthermore, an agent capable of stimulating signalling mediated by a .beta..sub.1 integrin cell surface receptor of leukocyte cells will treat chronic inflammation.

Throughout this application, various references are referred to within 
parentheses. Disclosures of these publications in their entireties are 
hereby incorporated by reference into this application to more fully 
describe the state of the art to which this invention pertains. Full 
bibliographic citation for these references may be found at the end of 
this application, preceding the sequence listing and the claims. 
BACKGROUND OF THE INVENTION 
Soluble or cell bound chemoattractants (1,2), stimulate polymorphonuclear 
leukocytes (PMN) to emigrate from the vasculature and migrate toward sites 
of injury, infection, and inflammation. PMNs express unique plasma 
membrane receptors for many different chemoattractants and cytokines 
e.g., IL-8, leukotriene B4 (LTB4), formyl-methionyl-leucyl-phenylalanine 
(fMLP) and TNF-.alpha.! (3). Interactions between these receptors and 
soluble or surface-bound chemoattractants or cytokines signal PMNs to 
alter their expression and/or activity of selectins and integrins (4,5), 
and regulate PMN spatial orientation and movements. (6). 
Tenascin, also referred to as cytotactin, hexabrachion, and 
glioma-mesenchymal extracellular matrix protein (41,43,44), forms a 
disulfide linked multimeric six-armed structure called a hexabrachion 
(43). Tenascin is expressed in many tissues during embryonic development, 
and is thought to play an important role in the development of muscles and 
tendons, mammary glands, hair follicles, teeth, kidney, bone and cartilage 
(43,50,51). In adults, tenascin is expressed in T-cell dependent regions 
of lymphoid tissues (40), in areas of cellular injury, and in malignant 
but not benign tumors (41,43). In areas of injury, tenascin is present in 
granulation tissue (41,55), in association with proliferating and 
migrating epidermal cells (42,49), and in arteries whose endothelial cells 
have been damaged (57). In malignant neoplasms, tenascin is produced by 
the tumor cells (63) and deposited in the stroma of gliomas, mammary 
carcinomas, colon cancers, Wilm's tumor, basal cell carcinomas, melanomas, 
and squamous cell carcinomas (41,64). 
There is little information regarding the physiological or 
patho-physiological role(s) of tenascin at sites of tissue injury, 
malignancy or atherosclerotic lesions. In extracellular matrices, tenascin 
promotes the adhesion of endothelial cells and bone marrow cells (38,48). 
Tenascin also blocks the attachment of several other cell types to 
fibronectin-coated surfaces in vitro (47,52), and the migration of neural 
crest cells (20,43,62). 
SUMMARY OF THE INVENTION 
The present invention provides a method of treating an infection caused by 
bacterial cells located on a surface of a foreign body over and around 
which fibrin has been deposited, the foreign body being present in a 
subject, which comprises administering to the subject an agent capable of 
inhibiting signalling mediated by a .beta..sub.1 integrin cell surface 
receptor of leukocyte cells in an amount effective to enhance the 
migration of leukocyte cells into or through the fibrin so as to permit 
the leukocyte cells to reach and kill the bacterial cells and thereby 
treat the infection. 
The present invention also provides a method of preventing a chronic 
infection from occurring due to the presence of bacterial cells on a 
surface of a foreign body in a subject, which comprises coating the 
foreign body before placing it in the subject with a fibrinolytic agent 
capable of preventing the accumulation of fibrin on the surface of the 
foreign body so as to permit leukocyte cells to reach and kill any 
bacterial cells present on the surface of the foreign body and thereby 
prevent the chronic infection. 
The present invention further provides a method of treating a malignant 
tumor comprising of malignant tumor cells over and around which tenascin 
has been deposited, the malignant tumor being present in a subject, which 
comprises administering to the subject an agent capable of inhibiting 
signalling mediated by a .beta..sub.1 integrin cell surface receptor of 
leukocyte cells in an amount effective to enhance the migration of 
leukocyte cells through the tenascin so as to permit the leukocyte cells 
to reach and kill the malignant tumor cells and thereby treat the 
malignant tumor. 
The present invention also provides a method of treating a chronic 
inflammation in a subject caused by an increase in the number of leukocyte 
cells present at the site of the chronic inflammation which comprises 
administering to the subject an agent capable of stimulating signalling 
mediated by a .beta..sub.1 integrin cell surface receptor of leukocyte 
cells in an amount effective to inhibit the migration of leukocyte cells 
toward the site of the chronic inflammation so as to reduce the number of 
leukocyte cells present at the site and thereby treat the chronic 
inflammation.

This invention will be better understood from the Experimental Details 
which follow. However, one skilled in the art will readily appreciate that 
the specific methods and results discussed are merely illustrative of the 
invention as described more fully in the claims which follow thereafter. 
EXPERIMENTAL DETAILS 
EXAMPLE 1 
The evolution of many chemically distinct chemoattractants and receptors 
suggests that in addition to promoting adhesion and directing PMN 
locomotion these molecules might regulate the strength of PMN adhesion to 
specific matrix proteins. The findings that TNF stimulates PMNs to adhere 
to fibrinogen-coated surfaces via CD11c/CD18 (7,8), while phorbol 
dibutyrate stimulates PMNs to adhere to these surfaces via CD11b/CD18 
(8,9), prompted the examination of the effects of different 
chemoattractants on PMN migration through three dimensional matrices 
composed of fibrin, collagen IV or Matrigel, and through gels formed by 
thrombin treatment of cell-free plasma. 
EXPERIMENTAL PROCEDURES 
Reagents 
Human monocyte recombinant IL-8 (Ser-IL-8).sub.72 and TNF-.alpha. were from 
Upstate Biotechnology Incorporated (Lake Placid, N.Y.). LTB4, fMLP and 
Ficoll-Hypaque were from Sigma (St. Louis, Mo.). Rhodamine conjugated 
polyethylene glycol was prepared as described (10). 
Preparation of Boyden-type chemotaxis chambers 
Becton-Dickinson cell culture inserts (pore sizes 3 or 8 .mu.m: Franklin 
Lakes, N.J.) were overlaid with the following proteins: 
Fibrin gels: 1 Unit of thrombin (a gift from Dr. John Fenton, Albany 
Medical College, Albany, N.Y.), in 5 .mu.l of PBS was added first to each 
insert. 0.1 ml phosphate buffered saline supplemented with Ca.sup.2+ and 
Mg.sup.2+ (PBS) containing 100 .mu.g commercial grade fibrinogen 
(Calbiochem. Inc., San Diego, Calif.) or purified fibrinogen a gift from 
Dr. Jeffery Weitz, (MacMaster University, Hamilton, On.)! was then placed 
into each 8.2 mm diameter insert on top of the thrombin. The mixture was 
incubated at 37.degree. C. for 5 min to allow fibrin gel formation 
(determined by visual inspection). 1 Unit PK (Calbiochem Inc., San 
Diego, Calif.; 10.sup.-5 M final concentration), in 100 .mu.l medium was 
added to each insert to inhibit thrombin, and gels were washed with 250 
.mu.l PBS to remove inactivated thrombin. The fibrin gels formed were 
about 1 mm thick as measured under a dissecting microscope. 
Collagen type IV and Matrigel matrices: 0.1 ml PBS containing 100 .mu.g 
human placental collagen IV (Fluka Chemical Corp., Ronkonkoma, N.Y.), or 
80 .mu.g of reconstituted basement membrane proteins (Matrigel, 
Collaborative Research, Bedford, Mass.), was placed into each insert and 
allowed to gel at room temperature for 24 hrs. 
Clotted Plasma: Whole blood was collected and the cellular components were 
removed by centrifugation. The resulting plasma was mixed with an equal 
volume of PBS, and 100 .mu.l of this mixture was placed into each insert 
containing thrombin and allowed to clot as described above. One unit of 
PK in 100 .mu.l PBS then was added and the inserts were washed with 250 
.mu.l of PBS. 
Fibrinogen or fibronectin: 0.1 ml of PBS containing 100 .mu.g/ml fibrinogen 
or fibronectin (New York Blood Center, New York, N.Y.), was placed into 
each insert (pore size 3 .mu.m). Inserts were incubated at 37.degree. C. 
for 60 min and washed with 250 .mu.l of PBS. Filters coated with 
fibrinogen or fibronectin were diffusely fluorescent as visualized by 
epifluorescent microscopy when incubated with the corresponding antibody 
fluorescein-labeled anti-fibrinogen, or anti-fibronectin monoclonal 
antibodies (Cappel, Malvern, Pa.)!, while uncoated filters, or filters 
incubated with fluorescein-labeled antibody of the opposite specificity, 
were not. 
PMN migration 
PMNs were prepared from fresh heparinized blood from healthy adult 
volunteers by sedimentation on Ficoll-Hypaque gradients. Contaminating red 
blood cells were removed by hypotonic lysis, as described (7). The purity 
of PMN isolated by this method is &gt;95% as determined by Wright-Giemsa 
staining (7). 10.sup.6 PMNs in 250 .mu.l of PBS supplemented with 5.5 mM 
glucose and 0.1% human serum albumin (PBSG-HSA), were placed in the upper 
compartment of each insert and incubated for 0-6 hrs at 37.degree. C. in a 
humidified atmosphere containing 95% air/5% CO.sub.2. At the times and 
concentrations specified, chemoattractants/cytokines were added to the top 
or bottom compartment in 250 .mu.l of PBSG-HSA. At the end of the 
incubation, the chambers were shaken to dislodge PMNs from the lower 
surface of the inserts. The medium in each lower compartment was collected 
and its content of PMNs was determined using either a Coulter Counter or a 
hemocytometer. Both methods gave similar results. Counts are expressed as 
the average number of PMNs that migrated into the lower compartment. 
Unless otherwise indicated, all values reported are the average of six 
data points from at least 3 independent experiments. 
Confocal Microscopy 
PMNs were suspended in medium containing 10 .mu.M calcein/acetoxymethyl 
ester (Molecular Probes, Eugene, Oreg.), 0.02% (w/v) pluronic F-127 
(Molecular Probes, Eugene, Oreg.), 2% heat inactivated calf serum 
(HyClone, Logan, Utah), and 0.2% DMSO, and mixed gently for 40 min at room 
temperature. Cells loaded with dye under these conditions exhibited no 
changes in motility (Mandeville and Maxfield, unpublished observations). 
The calcein-loaded cells were rinsed in PBSG-HSA and added to inserts 
containing fibrin gels in the presence or absence of TNF, fMLP, LTB4 or 
IL-8. Following incubation with PMNs, fibrin-coated filters were gently 
cut from their inserts using a razor blade, transferred to a glass slide, 
immersed in PBSG-HSA and covered with a glass coverslip. Migration of 
calcein-loaded PMNs through fibrin was analyzed using a Dialux 20 X 
microscope (Leitz) fitted with a K2 Bio confocal scanning optical 
attachment using a Nipkow spinning disk. The microscope was equipped with 
an image intensifier, charge coupled device camera and video frame 
averager. The surface of the fibrin gel was identified using reflection 
interference contrast microscopy. Cells were imaged with a Plan-neofluor 
25 X fluorescence objective (NA=0.8) using fluorescein optics (490 nm 
excitation, 525 emission) and a spinning disk with pinhole apertures. 
Serial confocal optical sections were acquired at 1 .mu.m intervals, 
digitized using the VolCon program (a PC-based image processing package, 
Indec Systems, Capitola, Calif.,). Three dimensional images were volume 
rendered using Microvoxel software (Index Systems), after passing data 
through a 3.times.3.times.3 Gaussian convolution filter. Each experiment 
was repeated at least twice using duplicate samples. 
PMN adhesion to fibrin-coated surfaces 
Fibrin coated Terasaki tissue culture plates were prepared as described 
(10). 5 .mu.l of PBSG-HSA, containing PMNs (10.sup.6 /ml) and the 
indicated chemoattractant, was added to each well of the plate. Plates 
were incubated at 4.degree. for 30 min to allow PMNs to settle to the 
bottom of the wells and warmed to 37.degree. for 15 min to allow PMNs to 
adhere. Non-adherent cells were removed as described (7), and 2.5% 
glutaraldehyde in PBS added to fix the adherent PMNs. PMNs adherent to 
each well were enumerated using a phase-contrast microscope. Values 
reported are the mean number of PMNs adherent to six wells from a 
representative experiment (n=3). 
Exclusion of rhodamine-labeled polyethylene glycol from zones of adhesion 
of PMNs to protein-coated surfaces 
10 kDa rhodamine labeled polyethylene glycol (Rh-PEG), prepared and used as 
described previously (10), does not bind to untreated glass, tissue 
culture plastic, or to cell membranes. Rh-PEG binds avidly to 
protein-coated surfaces, and can be detected easily by its fluorescence. 
Individual wells on glass microslides (Carlson Scientific, Peotone, Ill.) 
were coated with either fibrin, Matrigel or collagen IV in a manner 
similar to that for coating cell culture inserts except that 20 .mu.l of 
the various solutions were used per well. 20 .mu.l of PMNs (10.sup.6 
cells/ml in PBSG-HSA) were added to each well and PMNs were allowed to 
adhere for 15 mins at 37.degree.. The cells were washed in PBS, fixed with 
3.7% paraformaldehyde in PBS for 10 min, washed again with PBS, and 
further incubated with 10 kDa Rh-PEG at room temperature for 60 min. The 
preparation then was washed with PBS and immediately observed by phase and 
fluorescent microscopy at 400.times. magnification. Average values from 
three different experiments are reported as the percentage of PMNs that 
excluded Rh-PEG from zones of adherence between the cells and the 
underlying matrix. 
Degradation of .sup.125 I-labeled fibrin gels 
1.0 ml PBS containing 1 mg human fibrinogen, 1 mCi of Na.sup.125 I (NEN, 
Boston, Mass.), and 1 Iodobead (Pierce, Rockford, Ill.) was incubated for 
15 mins on ice. .sup.125 I-fibrinogen was separated from .sup.125 I by gel 
filtration over a Speedy Desalting Column (Pierce). &gt;97% of the .sup.125 I 
recovered in the fibrinogen-containing fractions was precipitable with 20% 
trichloracetic acid. 5 .mu.l PBS containing 1 Unit of thrombin, followed 
by 0.1 ml PBS containing 10.sup.6 cpm of .sup.125 I-fibrinogen (.about.10 
.mu.g) and 100 .mu.g unlabeled fibrinogen were added to each insert, as 
described above. The resulting .sup.125 I-labeled fibrin gels were treated 
with PK, washed, and incubated with 10.sup.6 PMNs as described in the 
text. At various times after PMN addition the medium was removed from the 
upper and lower compartments, and added to 0.1 ml of PBS containing 10 
mg/ml bovine serum albumin (BSA). Ice-cold trichloracetic acid was added 
to a final concentration of 20% and samples were centrifuged to sediment 
acid insoluble materials. TCA soluble and insoluble materials were 
separated by centrifugation and .sup.125 I in each fraction was determined 
using an LKB minigamma counter. 
Results 
IL-8 and LTB4, but not TNF or fMLP, promote the migration of PMNS through 
fibrin gels 
PMNs were placed into the upper compartment of inserts containing fibrin 
gels. IL-8, LTB4, fMLP, or TNF was placed in the medium in the lower 
compartment and the chambers were incubated at 37.degree. C. for 6 hrs. 
IL-8 or LTB4 stimulated 12-25% of PMNs to migrate through the fibrin gels 
and into the lower compartment. In the absence of a chemoattractant or in 
response to various concentrations of TNF (10.sup.-9 -5.times.10.sup.-6 M) 
or fMLP (10.sup.-10 -10.sup.-6 M), fewer than 0.3% of the PMNs migrated 
through fibrin gels into the lower compartments (FIGS. 1A and 2). 
Moreover, PMNs did not migrate through fibrin in response to the addition 
of 2-10% zymosan-activated human plasma (C5a) in the lower compartment. 
PMNs stimulated by IL-8 or LTB4, but not by TNF or fMLP, migrated through 
fibrin gels formed by thrombin treatment of commercial-grade fibrinogen 
(FIG. 1A) or of purified fibrinogen, or through plasma gels formed by 
thrombin treatment of human plasma (FIG. 1B). Moreover, the presence of 
20% human serum in the medium in both upper and lower compartments did not 
alter PMN migration through fibrin gels in response to IL-8, nor did the 
presence of serum promote PMN migration through fibrin gels in response to 
TNF or fMLP. That PMNs migrate through fibrin gels in the presence of 
human serum, and through gels formed from whole human plasma, indicates 
that IL-8 promotes PMN migration through fibrin gels containing the 
complex mixture of plasma proteins found under physiological conditions. 
The percent of PMNs that migrated through fibrin gels varied with the 
concentration of IL-8 or LTB4 placed in the bottom compartment (FIG. 2B). 
Maximal PMN migration occurred with 0.7.times.10.sup.-7 M IL-8 or 
0.2.times.10.sup.-7 M LTB4 (FIG. 2B). PMN migration decreased dramatically 
when IL-8 was used at concentrations &gt;10.sup.-7 M, consistent with the 
report of Smith et. al. (11) that high concentrations of IL-8 desensitize 
PMNs. In contrast, there was no indication of PMN desensitization in 
response to supra-optimal concentrations of LTB4 (FIG. 2B). 
To determine whether IL-8 and LTB4 promote PMN migration through fibrin 
gels by stimulating chemotaxis or chemokinesis we performed a 
checkerboard-type analysis (12). Few PMNs migrated through fibrin gels 
when IL-8 or LTB4 was placed in the upper compartment, or when the upper 
and lower compartments contained equal concentrations of IL-8 or LTB4 
(FIGS. 1A and 1B). As the difference in IL-8 or LTB4 concentrations 
between the upper and lower compartments decreased, the number of PMNs 
that migrated through the fibrin gels also decreased (FIGS. 3A and 3B). 
These results indicate that PMN migration through fibrin gels in response 
to IL-8 or LTB4 reflects chemotaxis, not chemokinesis. 
Between 25-50% more PMNs migrated through fibrin in response to LTB4 than 
to IL-8. It is unlikely that this difference reflects the response of 
different PMN subpopulations to LTB4 vs IL-8 since the same percentage of 
PMNs traversed fibrin gels in response to optimal concentrations of both 
LTB4 and IL-8 in the lower compartment as to LTB4 alone. Other 
investigators have reported that only 20-50% of PMNs migrate through 
filters (13), natural matrices, and cellular barriers (14), when 
stimulated by these chemoattractants. Since virtually all PMNs orient and 
crawl on surfaces when exposed to the chemoattractants (3), it is evident 
that all PMNs responded to them. The reason(s) why only a fraction of PMNs 
migrate through artificial or natural barriers in response to 
chemoattractants is unknown. 
PMNs migrated through fibrin gels more rapidly in response to an optimal 
concentration of LTB4 than to an optimal concentration of IL-8 (FIG. 4). 
Ten percent of LTB4-stimulated PMNs migrated through fibrin gels within 2 
hrs while fewer than 0.5% of IL-8-stimulated PMNs migrated through these 
gels in this time period (FIG. 4). By 6 hrs, maximal numbers of PMNs had 
migrated through fibrin gels in response to either IL-8 or LTB4. 
To visualize the interactions of chemoattractant-stimulated PMNs with 
fibrin gels, PMNs prelabeled with calcein (15), were added to the upper 
compartment of inserts containing fibrin gels. Chemoattractants were added 
to the medium in the lower compartment, the chambers were incubated at 
37.degree. C., and at the times indicated the fibrin-coated filters were 
removed and examined by confocal microscopy. After a 1 or 4 hr incubation 
with fMLP or TNF, almost all the cells remained on the gel's surface; 
fewer than 5% of TNF- or fMLP-stimulated PMNs penetrated a short distance 
into the fibrin gels; (FIGS. 5A, 5B, 5C, 5D, 5E, and 5F). In contrast, 
greater than 80% of IL-8-stimulated PMNs migrated deeply into the fibrin 
gels after a 4 hr incubation (FIGS. 5A, 5B, 5C, 5D, 5E, and 5F). Greater 
than 80% of LTB4-stimulated PMNs began to migrate into the fibrin gel 
after 1 hr, while few IL-8 stimulated PMNs penetrated the fibrin at this 
time. These results show that TNF and fMLP do not promote PMN invasion of 
fibrin, and that LTB4 stimulates PMNs to enter fibrin gels more rapidly 
than IL-8. The latter finding is consistent with the more rapid transit of 
fibrin gels by LTB4- than IL-8-stimulated PMNs described in FIG. 4. 
To further examine whether proteolysis of fibrin accounted for the 
selective ability of IL-8- or LTB4-stimulated PMNs to traverse these gels, 
the release of .sup.125 1I-labeled products was measured from 125I-fibrin 
incubated with PMNs for 6 hrs at 37.degree. C. in the presence or absence 
of each of these chemoattractants. The rate and extent of release of 
.sup.125 I-labeled acid soluble and acid precipitable products was similar 
for all four chemoattractants (FIG. 6). Even in the presence of 20% serum, 
chemoattractant-stimulated PMNs released no more .sup.125 I-labeled 
products than unstimulated PMNs. These results suggest that fibrin 
degradation does not account for the selective ability of LTB4- and 
IL-8-stimulated PMNs to traverse fibrin gels. 
LTB4, IL-8, TNF AND fMLP promote pmn migration through gels formed of 
matrigel and collagen IV 
To confirm that the inability of TNF-, fMLP-, or zymosan-activated human 
plasma-stimulated PMNs to migrate through fibrin gels reflected an effect 
of the interaction between the fibrin matrix and 
chemoattractant-stimulated PMNs, and not a general effect of any three 
dimensional matrix on PMNs stimulated with these chemoattractants, the 
ability of TNF, zymosan activated human plasma and fMLP to promote PMN 
migration through gels composed of basement membrane proteins (Matrigel) 
or collagen IV was examined (FIGS. 7A and 7B). TNF, fMLP, IL-8, 
zymosan-activated human plasma, or LTB4 added to the bottom chamber 
stimulated PMN migration through these gels (FIG. 7A). To determine 
whether fibrin affected PMN migration through collagen matrices, inserts 
coated with collagen IV gels were incubated with fibrinogen and thrombin 
to form fibrin sandwich on top of the collagen gels, and washed with 
PK-containing buffer. PMNs were added to the upper compartment and TNF 
to the lower compartment. The presence of fibrin prevented PMN migration 
through the collagen gels in response to TNF by about 75% (FIG. 7A). These 
results confirm that the effect of fibrin is selective and that it affects 
PMN migration in response to a specific subset of chemoattractants. 
To determine whether protein monolayers had the same effects on PMN 
migration as gels, inserts were coated with fibrinogen or fibronectin. The 
adsorption of these proteins to the filters that form the floor of the 
inserts was confirmed by immunofluorescence microscopy, as described in 
Experimental Procedures. fMLP, TNF, IL-8 and LTB4 all promoted PMN 
migration through filters to which fibrinogen or fibronectin had been 
adsorbed (FIG. 7B). 
PMNs adhere more closely to fibrin in response to fMLP or TNF than to LTB4 
or IL-8 
Is there a relationship between the ability of a chemoattractant to 
stimulate PMN migration through fibrin and its ability to promote close 
apposition of PMNs to fibrin? PMNs were incubated on fibrin-coated 
surfaces in the presence or absence of a chemoattractant for 15 min at 
37.degree. C. As expected, TNF, fMLP, LTB4 and IL-8 were equally effective 
in stimulating PMN adherence to fibrin (&gt;200 chemoattractant-stimulated 
PMNs vs .about.10 unstimulated PMNs adhered per mm.sup.2). The closeness 
of PMN adhesion to fibrin was evaluated by the ability of 10 kDa Rh-PEG 
(8,10), to penetrate into the zones of adhesion between 
chemoattractant-stimulated PMNs and fibrin. By this measure 70-80% of 
adherent TNF- or fMLP-stimulated PMNs excluded Rh-PEG from their zones of 
contact with the fibrin (FIG. 8). In contrast, only about 15% of adherent 
LTB4- or IL-8-stimulated PMNs formed adhesive zones that excluded Rh-PEG 
(FIG. 8). Furthermore, the adhesive zones formed by this 15% of 
IL-8-stimulated PMNs were at least 50% smaller in area than those formed 
by TNF- or fMLP-stimulated PMNs as judged by the area from which Rh-PEG 
was excluded. 
Previous studies (10) showed that the exclusion of fluorescein-conjugated 
F(ab).sub.2 anti-fibrinogen from zones of contact between ADP-stimulated 
platelets and fibrinogen-coated surfaces is a useful measure of the 
closeness of apposition between platelet membranes and the substrate. 
Therefore, the exclusion of fluorescein-conjugated F(ab).sub.2 anti-fibrin 
from zones of contact between LTB4- or IL-8-stimulated PMNs and fibrin was 
used as a measure of the interaction of these cells with fibrin-coated 
surfaces. About 50% of fibrin-adherent LTB4- or IL-8-stimulated PMNs 
formed adhesive zones that excluded this high molecular weight (100 kDa.) 
probe. As expected from studies with Rh-PEG (FIG. 8), &gt;99% of 
fibrin-adherent TNF- or fMLP-stimulated PMNs excluded 
fluorescein-conjugated F(ab).sub.2 anti-fibrin from their zones of contact 
with fibrin (data not shown). Thus, LTB4- or IL-8-stimulated PMNs adhere 
more closely to fibrin than unstimulated PMNs, even though these 
chemoattractants do not promote the very close apposition characteristic 
of fMLP- or TNF-stimulated PMNs. 
The effect of combinations of chemoattractants on migration of PMNS through 
fibrin gels 
The inability of fMLP- or TNF-stimulated PMNs to migrate through fibrin can 
be interpreted in at least two ways. First, fibrin blocks the capacity of 
PMNs to respond to fMLP or TNF. This seems unlikely since fMLP and TNF 
promote close apposition between PMNs and fibrin coated surfaces (FIG. 8). 
Second, fMLP or TNF signal PMNs to become sessile when they interact with 
fibrin. To examine the second possibility, the effects of combinations of 
chemoattractants on PMN migration through fibrin gels were monitored 
(FIGS. 9A and 9B). The presence of fMLP in the bottom compartment of the 
inserts reduced PMN migration through fibrin gels in response to IL-8 or 
LTB4 in a concentration dependent fashion. Higher concentrations of fMLP 
were required to effect equal inhibition of migration of LTB4-stimulated 
PMNs vs IL-8 stimulated PMNs (FIG. 9B). TNF had a small, reproducible, but 
statistically insignificant inhibitory effect on the migration of PMNs in 
response to IL-8 and no measurable effect on PMN migration in response to 
LTB4 (FIG. 9A). Thus, fMLP selectively reduced PMN migration through 
fibrin gels in response to IL-8 or LTB4 (FIG. 9B). 
The effects of combinations of chemoattractants on formation of close 
apposition of adhesion of PMNs with fibrin were also examined. fMLP in 
combination with IL-8 or LTB4 induced about 50% of PMNs to form zones of 
adhesion that excluded 10 kDa. Rh-PEG (FIG. 8). In contrast, only 15% of 
PMNs stimulated with IL-8 or LTB4 alone formed close zones of adhesion 
(FIG. 8). Thus, the capacity of PMNs to form close zones of adhesion on 
fibrin was inversely associated with the capacity of PMNs to migrate 
through fibrin gels under conditions where PMNs were stimulated with TNF, 
fMLP, IL-8 or LTB4 given alone or with fMLP in combination with IL-8 or 
LTB4. 
To determine the mechanism by which fibrin blocks PMN migration in response 
to fMLP, the effects of anti-integrin antibodies were tested (FIGS. 10A 
and 10B). PMNs incubated with anti-.beta..sub.1 integrin antibodies (2 
.mu.g/ml) or the peptide GRGDSP (SEQUENCE ID NO. 1) (1 mg/ml), but not 
GRGESP (SEQUENCE ID NO. 2), migrated through fibrin gels in response to 
fMLP. Control experiments showed that anti-.beta..sub.1 antibodies did not 
affect LTB4-stimulated PMN migration through fibrin. These studies show 
that interactions between PMN .beta..sub.1 integrins and matrix-associated 
ligands regulate PMN migration. They suggest that fMLP, but not LTB4 
signals binding of .beta..sub.1 integrins to .beta..sub.1 ligands (e.g. 
RDG) on fibrin. These studies also suggest that ligation of .beta..sub.1 
integrins signals fMLP-stimulated PMN to become sessile, and that by 
blocking .beta..sub.1 integrins with antibodies or peptides, PMNs are able 
to migrate into tissue sites containing fibrin, from which PMN would 
otherwise be excluded. 
Discussion 
Matrix proteins modulate cellular responses to hormones, cytokines, and 
growth factors 
Matrix proteins exert profound effects on adhesion, differentiation, 
migration, and/or secretion of epithelial cells (16,17), endothelial cells 
(18), neurons (19,20) and leukocytes (7,9,21-27). Matrix proteins also 
affect the ability of many types of cells to respond to hormones, growth 
factors, and cytokines (28,29). The findings that some chemoattractants 
(e.g., fMLP, TNF, C5a), promote PMN migration in the context of two types 
of extracellular matrix proteins (e.g., matrigel and collagens IV) (FIGS. 
5A, 5B, 5C, 5D, 5E, 5F, 7a, and 7B), and PMN immobilization in the context 
of another (e.g., fibrin) (FIGS. 1A, 1B, 2A, 5A, 5B, 5C, 5D, 5E, and 5F), 
are the first to show that specific matrix proteins regulate leukocyte 
chemotaxis. They show that fibrin gels, fibrin-impregnated collagen gels, 
and fibrin-containing plasma clots present selective barriers to the 
migration of fMLP- or TNF-stimulated PMNs and that chemotaxis of PMNs 
through three dimensional matrices is regulated by both the specific 
chemoattractant and the protein composition of the matrix with which the 
cells are in contact. 
Matrix proteins regulate PMN adhesion, phagocytosis and secretion 
Previous studies (7,9,21) have shown that TNF or phorbol ester stimulated 
PMNs adhere to fibrinogen-coated surfaces via different beta-2 integrins 
(CD11b/CD18 vs CD11c/CD18, respectively) and Lundgren-Akerlund et al. 
(22), and Thompson and Matsushima (23), have reported that fMLP stimulated 
PMNs adhere to protein coated surfaces with different efficiencies 
depending on the matrix protein used to coat these surfaces. With respect 
to phagocytosis, Pommier et al. (24), and Wright et al., (25) showed that 
the interaction of fibronectin with its .beta..sub.1 integrin activates 
complement receptors (CD11b/CD18) on monocytes and PMNs to phagocytose 
C3bi-coated particles. With respect to secretion, Monboisse et al., 
(26,27) reported that the interaction of unstimulated or 
chemoattractant-stimulated PMNs with collagen I-coated surfaces induces 
the secretion of proteolytic enzymes and O.sub.2.sup.-. In contrast, 
preincubation of PMNs with collagen IV blocks the ability of collagen I 
and of fMLP to stimulate resting PMNs to secrete these products (26,27). 
Similarly, adhesion of TNF-stimulated PMNs to extracellular matrix 
proteins that express Arg-Gly-Asp motifs enhances PMN secretion (30). The 
findings reported here add chemotaxis to the list of leukocyte functions 
modulated by their contact with matrix proteins. 
Relationship between strength of adhesion, closeness of PMN apposition to 
the substrate, and PMN migration 
DiMilla at al (31), have explored the relationship between strength of cell 
adhesion to a substrate and cell migration by following the spontaneous 
migration of human smooth muscle cells on surfaces that had absorbed 
varying concentrations of fibronectin or collagen IV. Under the conditions 
of their experiments, the rate of cell migration was maximal at an 
intermediate level of cell-substratum adhesiveness. Goodman et al. (32), 
found a similar biphasic relationship between the movement of murine 
skeletal myoblasts and the absorbed concentration of laminin on the 
substrate. 
While the strength of PMN adhesion to fibrin was not measured directly, the 
"closeness" of apposition between PMNs' matrix-adherent surfaces and 
matrices containing different proteins was examined by measuring the 
permeability of zones of contact between PMNs and the underlying matrix to 
macromolecular probes. "Close" apposition is defined as the exclusion of 
10 kDa Rh-PEG from zones of contact between the PMNs' substrate-adherent 
membranes and the matrix, and "loose" apposition as permeation of 10 kDa 
Rh-PEG into these zones. These results show that chemoattractants, such as 
IL-8 and LTB4, elicit "loose" apposition between PMNs and fibrin gels and 
promote PMN migration through these gels. Chemoattractants, such as FMLP 
and TNF, that signal "close" apposition between PMNs and fibrin gels do 
not promote PMN migration through these gels. This correlation was further 
supported by the findings that PMNs stimulated by any of these 
chemoattractants formed loose zones of adhesion (e.g., permeable to 10 kDa 
Rh-PEG) on collagen IV or Matrigel (FIG. 8), and migrated through these 
matrices (FIG. 7A); and that fMLP induced LTB4 or IL-8 stimulated PMNs to 
form close zones of apposition to fibrin and cease migration (FIGS. 8 and 
9). Thus, there is an inverse association between close PMN interaction 
with a matrix protein and the ability of PMNs to migrate though gels 
containing it. These findings suggest that "close" and "loose" apposition 
between PMNs and matrix proteins as defined here, are functionally 
equivalent to very strong and intermediate adhesion between cells and 
matrix, respectively, as defined by DiMilla et al (31). 
Fibrin degradation is not required for PMN chemotaxis 
The zones of close apposition formed between fMLP- or TNF-stimulated PMNs 
and fibrin gels are impermeant to molecules of &gt;10 kDa thereby excluding 
virtually all plasma protease inhibitors such as alpha.sub.1 anti-plasmin 
and alpha.sub.2 macroglobulin. Therefore, leukocyte proteases secreted 
into these zones function virtually uninhibited (33). In contrast, IL-8- 
or LTB4-stimulated PMNs adhere more loosely to fibrin-gels. Under these 
conditions, plasma protease inhibitors should have ready access to zones 
of contact with the substrate and inhibit the action of leukocyte 
proteases. Thus, fMLP- or TNF-stimulated PMNs might be expected to digest 
fibrin gels more efficiently than IL-8- or LTB4-stimulated PMNs. This was 
not observed (FIG. 6). There were no significant differences in the amount 
of radiolabel released from .sup.125 I-labeled fibrin by migrating LTB4- 
or IL-8-stimulated PMNs vs sessile fMLP- or TNF-stimulated PMNs, even in 
the presence of 20% serum. These findings suggest that PMNs that migrate 
through fibrin gels in response to IL-8 and LTB4 do so by mechanisms other 
than proteolyzing these gels. Lanir et al., (34) came to a similar 
conclusion in their studies of guinea pig macrophage migration through 
fibrin gels. 
That fMLP and TNF promote PMN migration through fibrinogen-coated filters 
(FIGS. 7A and 7B), is probably related to the observations that 
fMLP-stimulated PMNs efficiently degrade substrate-adherent proteins 
including fibrinogen (33), and fibronectin (35), thereby removing these 
proteins from the substrate and facilitating PMN movement. 
How do matrix proteins regulate leukocyte chemotaxis? 
The results shown in Example 1 indicate the following: Different 
chemoattractants activate different subsets of PMN integrins to bind to 
ligands on matrix proteins (7-9). The interaction of each type of 
activated PMN integrin with its cognate ligand on a matrix protein 
specifies a distinct set of cellular migratory or sessile responses. These 
responses may result from direct interaction of a matrix protein with the 
activated integrin or by signals sent by the activated integrin to other 
integrins on the same cell. There are several instances where ligation of 
one type of integrin by matrix proteins modulates the activity of another 
type of integrin. As described above, Pommier et al., (24) and Wright et 
al. (25) showed that ligation of .beta..sub.1 integrins by fibronectin 
activates the .beta..sub.2 integrin CD11b/CD18, (complement receptor 3) on 
monocytes and PMNs to phagocytose C3bi-coated particles. It was previously 
shown that ligation of .alpha.5.beta.1 on platelets by fibronectin 
stimulates platelets to form close zones of apposition with fibrinogen 
(10). Hutalia et al, (36) reported that ligation of .alpha..sub.5 
.beta..sub.1 integrin by RGD peptides induces the expression of matrix 
metalloproteinases by fibroblasts, whereas ligation .alpha..sub.4 
.beta..sub.1 integrin by intact fibronectin suppresses matrix 
metalloproteinase expression. 
In vivo inflammatory stimuli elicit the generation of multiple 
chemoattractants/cytokines. The present findings show that a hierarchy of 
cellular responses is generated when different combinations of 
chemoattractant receptors are stimulated simultaneously. Signals generated 
by fMLP receptors appear to override signals produced by LTB4 or IL-8 
receptors, thereby blocking the ability of LTB4 or IL-8 to stimulate PMN 
migration through fibrin gels (FIG. 8). In contrast, signals generated by 
TNF receptors have no effect on LTB4-stimulated PMN migration through 
fibrin gels, and a very weak inhibitory effect on IL-8-stimulated PMN 
migration through these gels (FIG. 8). 
PMN chemotaxis through three dimensional lattices composed of extracellular 
matrix proteins is regulated both by signals initiated by a specific 
chemoattractant, and by signals generated when specific PMN receptors 
interact with their cognate ligands on extracellular matrix proteins. 
Viewed from this perspective each of the many different chemoattractants 
provides PMNs both with general instructions to crawl, and with specific 
instructions to become sessile when specific receptors on these cells 
contact their cognate ligands on matrix proteins. Thus, chemoattractants 
provide tissue localization instructions for PMNs. It seems likely that 
chemoattractants also provide such instructions to other types of 
leukocytes as well. 
PMNs stimulated with zymosan-activated human plasma (C5a) did not migrate 
across fibrin-coated inserts but did migrate across matrigel-coated 
inserts. Thus, C5a, like fMLP and TNF, signals PMNs to stop migrating when 
they contact fibrin. 
EXAMPLE 2 
The inhibitory effects of extracellular matrix proteins on chemotaxis of 
leukocytes (7,8,9,65) prompted the examination the effects of tenascin on 
these processes. The present findings show that tenascin blocks chemotaxis 
of polymorphonuclear and mononuclear phagocytes across reconstituted 
basement membrane (Matrigel)-coated filters in a .beta..sub.1 
integrin-dependent process. 
EXPERIMENTAL PROCEDURES 
Cells 
Polymorphonuclear leukocytes (PMN), were prepared as described (7) from 
heparinized human blood by sedimentation on Ficoll-Hypaque gradients. 
Contaminating red blood cells were removed by hypotonic lysis. The purity 
of PMN isolated by this method was &gt;95% as determined by Wright-Giemsa 
staining. 
Mononuclear cells were isolated by centrifugation of heparinized human 
blood on Ficoll-Hypaque gradients as described (65,66). The mononuclear 
cell fraction was resuspended in RMPI 1640 medium supplemented with 10% 
pooled human serum or autologous serum and used immediately for monocyte 
migration studies. For some experiments, monocytes were obtained by 
centrifugation of whole blood or of white blood cells concentrated from a 
unit of blood (leukopak), on Nycodenz gradients as described (39). More 
than 90% of the nucleated cells obtained by this Nycodenz method were 
monocytes, as assessed by their ability to phagocytose IgG-coated red 
blood cells. 
Cultured monocytes were prepared by allowing 10.sup.7 total mononuclear 
cells, suspended in 10 ml of RMPI 1640 medium supplemented with 10% pooled 
human serum (1640+HS), or 10% autologous serum (1640+AS), to adhere to 
Falcon T-150 tissue culture Petri dishes for 2 h at 37.degree. C. 
Non-adherent cells were removed by washing, leaving an adherent cell 
population consisting of greater than 98% monocytes as measured by their 
capacity to phagocytose IgG coated sheep red blood cells. For migration 
studies, monocytes were maintained in culture for 24 h in RPMI 1640+HS or 
AS, and detached from the dishes by gentle pipetting of 10 ml of ice cold 
phosphate buffered saline (without Ca.sup.2+ or Mg.sup.2+) containing 1.0 
mM EDTA. The cells recovered were resuspended in RPMI-1640+HS as described 
(65). 
Protein-coated filters 
Cell culture inserts containing polyethylene terephthalate filters, 8-.mu.m 
pore size (Becton-Dickinson), were overlaid with 0.1 ml of Matrigel (20-25 
.mu.g protein/filter) (Collaborative Research, Bedford, Mass.), and 
incubated at room temperature until they dried. These Matrigel-coated 
filters were washed with phosphate buffered saline containing 1.0 mM 
Mg.sup.2+ and 1.0 mM Ca.sup.2+ (PBS). 0.1 ml of a PBS solution (pH 7.2) 
containing the indicated amount of purified chick brain tenascin was added 
to some of the inserts. The filters were incubated at room temperature 
until they dried. These protein-coated filters were washed again with PBS 
and used within 12 h for cell migration studies. To prepare collagen 
I-coated inserts, filters were coated with rat tail collagen I (ICN 
Biochemicals, Costa, Mesa, Calif.) (400 .mu.g/ml in PBS), by adding 0.1 ml 
of this solution to an insert and incubating the inserts at room 
temperature for 24 h. Some of these collagen I-coated filters were then 
incubated with tenascin as described above. 
Cell migration 
Monocytes: Cell culture inserts were placed in 16 mm wells containing 0.5 
mls of RPMI-1640+HS or AS in the presence or absence of a chemoattractant. 
0.5 ml of RPMI-1640+HS or AS serum containing between 2-10.times.10.sup.5 
mononuclear phagocytes was added to the upper chamber of the inserts and 
the inserts were placed in a humidified CO.sub.2 incubator at 37.degree. 
C. After 24 h the medium in the lower chamber was recovered and its cell 
content assayed using either a Coulter counter or a hemocytometer. No 
significant increase was observed in the number of monocytes that migrated 
into the lower compartment at times greater than 24 h. Cell counts 
reported are the average number of cells recovered from the medium in the 
lower compartment of chemotaxis chambers. Over 90% of the cells in the 
lower chamber were identified as monocytes based on their morphology, 
their capacity to phagocytose IgG coated sheep red cells, and their 
staining with fluorescein-conjugated anti .alpha..sub.m .beta..sub.2 
monoclonal antibody (Oncogene Sciences, Uniondale, N.Y.). Unless otherwise 
indicated, all values are the average of duplicate samples run in parallel 
in a representative experiment. Each experiment was repeated at least 
three times with similar results. 
PMN: 250 .mu.l of PBS supplemented with 5.5 mM glucose and 0.1% human 
serum albumin (HSA) (PBSG-HSA)! containing 1.times.10.sup.6 PMNs was 
placed in the upper compartment of each insert. 250 .mu.l of PBSG-HSA with 
or without chemoattractant, as indicated, was added to the bottom 
compartment. The inserts were incubated for about 4 h at 37.degree. C. in 
a humidified atmosphere containing 95% air/5% CO.sub.2. No further 
increase in the number of PMNs was observed in the lower compartment 
beyond 4 h. The medium in the lower compartment was collected and its 
content of PMN determined using a Coulter Counter, as described in Example 
1. 
Tenascin 
To isolate tenascin, 14-day embryonic chick brains were homogenized in the 
presence of protease inhibitors and the extracts were clarified as 
previously described (61). Dry CsCl was added to a final concentration of 
0.5 g/ml and the extract was centrifuged (18 h, 45,000 rpm, 20.degree. C.) 
in a Beckman VAC 50 rotor. The resulting density gradients were 
fractionated into five 8-ml fractions. The third and fourth fractions a 
rich source of relatively pure tenascin, were pooled, dialyzed versus 10 
mM Tris pH 8.0, then incubated with chondroitin ABC lyase (Seikagaka 
America), in the presence of protease inhibitors (61), to degrade 
contaminating proteoglycans. The sample then was lyophilized, resuspended 
in 4M guanidine-HCl/0.1M Tris (pH 7.6), and fractionated on a 
1.5.times.100 cm column containing Sephacryl S-500 (Pharmacia, Piscataway, 
N.J.), equilibrated in the same buffer. Tenascin-rich fractions were 
pooled, dialyzed, lyophilized, resuspended in a small volume of guanidine 
buffer, and finally dialyzed extensively vs PBS. This procedure yielded 
large amounts of purified tenascin which migrated as characteristic 220, 
200, and 190 kD polypeptides when analyzed by SDS-PAGE under reducing 
conditions (44). Human tenascin was obtained from GIBCO-BRL, (Grand 
Island, N.Y.). To remove the detergent in the preparation, the sample was 
run over a gel filtration column in the presence of 4M guanidine-HCl; the 
tenascin containing fractions were then dialyzed extensively vs PBS. 
Measurement of tenascin bound to matrices 
Chick tenascin was radiolabeled using chloramine T (67), and mixed with 
unlabeled tenascin at a 1:100 protein ratio. 250 .mu.l of PBS containing 
varying amounts of this mixture was added to inserts coated with Matrigel 
or collagen I. The inserts were incubated at room temperature for 4 h at 
37.degree. C. in a humidified 95% air/5% CO.sub.2 atmosphere and washed 
with PBS. The filters were cut from the inserts with a scalpel, and 
assayed for .sup.125 I in a Beckman gamma counter. 
Reagents 
Monoclonal antibody P4C10 (anti-.beta. chain of human beta-1 integrin), was 
from GIBCO-BRL. Fluorescein conjugated monoclonal antibody against the 
.beta. chain of human beta-2 integrins (anti-.beta..sub.2 -CP14F) was 
obtained from Oncogene Sciences (Uniondale, N.Y.). Fluorescein conjugated 
anti CD11b monoclonal antibody was from AMAC Inc. (Westbrook, Me.). 
Monoclonal anti-CD11c/CD18 (LeuM5) was from Organon-Toknika Inc.(Malvern, 
Pa.). F(ab)'.sub.2 fragments of anti-tenascin antibodies were prepared as 
described (46). Chondroitin sulfate proteoglycan monomers were purified 
and antibodies prepared against these proteoglycan monomers as described 
(37). The F(ab)'.sub.2 fragments used in the present study were prepared 
from total anti-proteoglycan IgG. These antibodies were not further 
purified by affinity chromatography and therefore recognize both 400 kD 
and 250 kD proteoglycan core proteins (67). 
Results 
Tenascin blocks chemotaxis of monocytes and neutrophils, through 
matrigel-coated filters 
About 20% of freshly isolated human monocytes, 15% of cultured monocytes 
and 10% of PMNs migrated through Matrigel-coated culture inserts in 
response to fMLP, LTB4, or TNF (FIGS. 11A, 11B, and 12). Monocytes began 
to appear in the lower compartment at 12 h, and reached maximum numbers by 
16-24 h. PMNs began to appear in the lower compartment by 2 h, and reached 
a maximum by 4 h. Fewer than 2% of added monocytes or PMN, migrated into 
the lower compartment in the absence of a chemoattractant (FIGS. 11A, 11B, 
and 12). 
Addition of chick brain tenascin to the Matrigel significantly reduced 
monocyte and PMN migration in response to TNF, fMLP or LTB4 (FIGS. 11A, 
11B and 12). The presence of tenascin had no significant effect on the 
limited number of monocytes that migrated in the absence of 
chemoattractant (FIGS. 11A and 11B). In contrast, tenascin further reduced 
the small number of PMN that migrated across Matrigel in the absence of 
chemoattractant (FIG. 12). The extent to which tenascin inhibited 
chemoattractant-stimulated monocyte or PMN migration varied from 65-80% 
depending on the chemoattractant used (FIGS. 11A, 11B, and 12). The 
ability of tenascin obtained from cultured human glioma cells to 
effectively inhibit leukocyte migration was examined. Indeed, the addition 
of 5 .mu.g of human tenascin to the Matrigel-coated filters reduced the 
number of monocytes or PMNs migrating in response to a chemoattractant by 
at least 50% (FIGS. 13A and 13B). 
The effect of chick brain tenascin on TNF-stimulated migration of monocytes 
varied with the amount of tenascin added (FIG. 14). Addition of about 0.75 
.mu.g tenascin caused half maximal inhibition of TNF-stimulated monocyte 
migration (FIG. 14). To confirm that tenascin bound to the Matrigel, 
Matrigel-coated inserts were incubated with varying amounts of 
concentrations of .sup.125 I-labeled chick tenascin for 4 h at 37.degree. 
C. The inserts then were washed with PBS and the bound radioactivity was 
determined. Near plateau binding of radiolabeled tenascin was obtained 
with the addition of 0.625-1.25 .mu.g of chick tenascin to the Matrigel 
coated filters (FIG. 15). This resulted in the adsorption of about 0.2 
.mu.g of tenascin per filter. Once bound, less than 1% of the .sup.125 
I-labeled tenascin eluted from the filters into either the upper or lower 
compartment of the chambers during a 4-6 h incubation at 37.degree. C. 
Thus, .about.99% of tenascin remained bound to Matrigel-coated filter. The 
capacity of tenascin to inhibit monocyte migration (FIG. 14) was roughly 
proportional to the amount of tenascin that bound to the filter (FIG. 15), 
indicating that tenascin bound to the Matrigel matrix, not soluble 
tenascin, blocked monocyte and PMN migration. 
Tenascin blocks migration of monocytes through collagen I gels 
The effects of tenascin on monocyte migration through another extracellular 
matrix, collagen I were examined. Twelve percent of TNF- and 18% of 
LTB4-stimulated monocytes migrated through cell culture inserts coated 
with collagen I (FIG. 16). Addition of chick brain tenascin to collagen I 
coated inserts reduced TNF- or LTB4-stimulated monocyte migration by at 
least 60% (FIG. 16). Binding studies with .sup.125 I-labeled chick 
tenascin revealed that similar amounts of tenascin bound to collagen 
I-coated filters as to Matrigel-coated filters. 
Effect of F(ab)'.sub.2 anti-tenascin on the migration of monocytes across 
filters coated with matrigel and tenascin 
Monocytes or PMN were added to the upper compartment of inserts coated with 
Matrigel alone or with Matrigel and tenascin. F(ab)'.sub.2 anti-tenascin 
(2 .mu.g/ml) was added to the medium in the upper compartment and LTB4 
(10.sup.-7 M) was added to the lower compartment. F(ab)'.sub.2 fragments 
of anti-tenascin antibody had no significant effect on PMN migration 
across filters coated with Matrigel alone (FIG. 17A), but reduced monocyte 
migration across this matrix by about 40% (FIG. 17B). However, 
F(ab)'.sub.2 fragments of anti-tenascin antibody restored PMN chemotaxis 
across filters coated with Matrigel and tenascin to about 75% of control 
values (FIG. 17B), and increased substantially monocyte migration across 
tenascin coated Matrigel (FIG. 17A). 
Because of their affinity for tenascin, proteoglycans may contaminate some 
tenascin preparations (46). Therefore, the effects of F(ab)'.sub.2 
fragments of polyclonal anti-proteoglycan on monocyte migration across 
filters coated with tenascin and Matrigel were examined. F(ab)'.sub.2 
anti-proteoglycan (2-5 .mu.g/ml) did not reverse tenascin's inhibitory 
effect on monocyte migration across filters coated with Matrigel and 
tenascin, and did not significantly affect monocyte chemotaxis across 
filters coated with Matrigel alone. These studies suggest that tenascin 
inhibits migration by interacting with monocytes and not by blocking some 
matrix component required for their migration. 
Antibodies that block .beta..sub.1 integrins reverse the inhibitory effects 
of tenascin on monocyte and pmn migration through filters coated with 
matrigel and tenascin 
Endothelial cell attachment and spreading on human tenascin has been shown 
to be partially mediated by .beta..sub.1 integrins (60). Similarly, Prieto 
et al. (53,54), showed that anti-.beta..sub.1 integrin antibodies block 
the adhesion of glioma and carcinoma cell lines to tenascin. Therefore, 
the effects of anti .beta..sub.1 antibodies on monocyte and PMN chemotaxis 
through filters coated with Matrigel alone or with Matrigel and tenascin 
were examined. Monocytes or PMNs were preincubated for 30 min at 4.degree. 
C. in medium containing 2 .mu.g/ml of the test antibody. The suspension 
then was added to the upper compartment of the inserts. 
LTB4 was added to the lower compartment and the inserts were incubated at 
37.degree. C. for 24 h for monocytes or 4 h for PMN. A monoclonal antibody 
directed against .beta..sub.1 integrins (P4C10) had no effect on monocyte 
chemotaxis through filters coated with Matrigel alone but reversed the 
inhibitory effect of tenascin on monocyte migration through 
Matrigel/tenascin coated filters by about 50% (FIGS. 17A and 17B). Control 
experiments showed that monoclonal antibody P4C10 also reversed tenascin's 
inhibitory effect on TNF-stimulated monocyte chemotaxis by about 60%. 
P4C10 reversed almost completely the inhibitory effect of tenascin on 
LTB4- (FIG. 17B) or TNF-stimulated chemotaxis of PMN across filters coated 
with Matrigel and tenascin. 
To confirm that the effect of P4C10 was due to its interaction with 
.beta..sub.1 integrins, the effects of other anti-integrin antibody, 
A.sub.II B.sub.II, on chemotaxis of monocytes through filters coated with 
Matrigel and tenascin were examined. A.sub.II B.sub.II (2 .mu.g/ml) 
blocked the inhibitory effect of tenascin on monocyte chemotaxis, allowing 
LTB4 stimulated monocytes to migrate through Matrigel/tenascin coated 
filters. In contrast, an antibody (LeuM5) directed against .alpha..sub.x 
(p150/95), a member of the .beta..sub.2 integrin family found on both PMNs 
and monocytes, did not reverse tenascin's inhibitory effect on 
LTB4-stimulated chemotaxis of PMN or monocytes through 
Matrigel/tenascin-coated filters (FIGS. 17A and 17B). As expected, 
monoclonal antibody IB4, directed against leukocyte .beta..sub.2 
integrins, inhibited chemotaxis of monocytes and PMNs across filters 
coated with Matrigel alone or with both Matrigel and tenascin. Thus, 
antibodies directed against members of the .beta..sub.2 integrin family 
did not reverse tenascin's inhibitory effect on monocyte and PMN 
chemotaxis. These results indicate that blocking the interaction of 
monocytes or PMNs with tenascin, either by masking the tenascin on the 
matrix with F(ab)'.sub.2 anti-tenascin or by blocking .beta..sub.1 
integrins on the cells, reversed the inhibitory effect of tenascin on 
monocyte and PMN chemotaxis. 
Discussion 
PMNs migrate through matrices formed by, and containing, proteins that are 
"normal" constituents of basement membranes and of the ground substance of 
interstitial spaces (e.g., collagens I and IV, laminin), in response to 
all chemoattractants tested (fMLP, TNF, C5a, IL-8, LTB4) (68,69). In 
contrast, whether PMN migrate through matrices composed of, or containing, 
fibrin depends upon the specific chemoattractant with which they have been 
stimulated. For example, fMLP, TNF and C5a stimulate PMNs to adhere 
tightly to fibrin gels, but not to migrate into or through them. In 
contrast, IL-8 and LTB4 stimulate PMNs to migrate efficiently through 
these gels. 
The capacity of specific chemoattractants to signal cessation of migration 
when PMNs contact fibrin suggested that this might be a mechanism by which 
these cells are excluded from some tissue compartments, and concentrated 
in others. One example, however, hardly establishes a general principle. 
Therefore, other matrix proteins that block PMN and monocyte chemotaxis 
were sought. 
Tenascin inhibits PMN and monocyte chemotaxis through collagen I or 
Matrigel Matrices 
Addition of tenascin to three-dimensional matrices formed by collagen I or 
Matrigel signals cessation of movement of PMNs and monocytes in response 
to all three chemoattractants tested (fMLP, LTB4, TNF) (FIGS. 11-17). The 
capacity of tenascin to block chemotaxis of PMNs stimulated by LTB4 is of 
special note since LTB4 promotes PMN migration through fibrin gels (25). 
This finding supports our contention that leukocyte migration through 
extracellular matrix is regulated by both the proteins in the matrix and 
the specific chemoattractant. It demonstrates that a chemoattractant can 
have entirely different effects on a single class of leukocyte, depending 
upon the matrix proteins with which the leukocyte is in contact. 
One of tenascin's functions in adults is to inhibit PMN and monocyte entry 
into specific tissue compartments 
Tenascin is an unusual matrix protein. It is expressed widely in embryonic 
tissues where it regulates cell migration during organogenesis. Under 
physiological conditions in adults, tenascin is absent from most tissues, 
except lymphoid tissue (40,55), and brain (43). However, under 
pathological conditions, tenascin synthesis is stimulated. It is deposited 
in the extracellular matrix in areas of vascular injury (57), and tumor 
stroma (43,63), which are also areas of fibrin deposition (59,70-73). 
It is notable that tenascin and fibrin, matrix proteins deposited in and 
around diseased (e.g., malignant tumors), or injured tissues (e.g., 
atherosclerotic lesions), or areas in which T-cells concentrate 
(40,43,55,57,59,63,70-73), and chemically modified matrix proteins (e.g., 
non-enzymatically glycated collagen IV 74!), all signal phagocytic 
leukocytes to become sessile. Dvorak et al. (70), and Singh et al. (59) 
have presented evidence that tumor stroma protects the tumors from host 
immune effector cells. Viewed from this perspective, tenascin contributes 
to an immuno-inhibitory effect of tumor stroma. 
.beta..sub.1 integrins play no role in PMN or monocyte migration through 
Matrigel 
Anti-.beta..sub.1 integrins had no inhibitory effect on PMN or monocyte 
chemotaxis through Matrigel alone (FIGS. 17A and 17B), while antibodies 
directed against PMN and monocyte .beta..sub.2 integrins blocked PMN and 
monocyte chemotaxis under all circumstances tested (75), including through 
Matrigel (FIGS. 17A and 17B). These findings suggest that .beta..sub.1 
integrins play no role in PMN or monocyte chemotaxis through Matrigel. 
These studies did not examine whether antibodies directed against 
.beta..sub.2 integrins inhibited PMN or monocyte chemotaxis through 
Matrigel by blocking their adhesion to the Matrigel or by other 
mechanisms, such as stimulating an increase in their cAMP content (76). 
On the mechanism(s) by which tenascin blocks PMN and monocyte chemotaxis 
Chemoattractants that signal PMNs to remain sessile on fibrin gels, cause 
these cells to adhere more tightly and in greater numbers to fibrin than 
chemoattractants that promote PMN to migrate through fibrin gels. 
Similarly, chemoattractants stimulate monocytes to become sessile and 
adhere more tightly to glycated collagen IV than to native collagen IV 
(65,74). In contrast, no increase in the number of 
chemoattractant-stimulated PMN or monocytes that adhered to 
tenascin-impregnated Matrigel over Matrigel alone was observed. Thus, 
while fibrin and glycated matrices may inhibit chemotaxis by providing 
ligands to which chemoattractant-stimulated PMNs and monocytes bind very 
tightly, and in increased numbers, tenascin appears to exert its 
inhibitory effect by a different mechanism. 
.beta..sub.1 integrins have been reported to promote adhesion of normal and 
transformed cells to tenascin (53,54,60). F(ab)'.sub.2 anti-tenascin, and 
anti-.beta..sub.1 integrins reversed tenascin's inhibitory effect on both 
PMN and monocyte chemotaxis (FIGS. 17A and 17B). Since .beta..sub.1 
integrins appear to play no role in PMN or monocyte chemotaxis through 
Matrigel, the most straightforward explanation for the capacity of 
F(ab)'.sub.2 anti-tenascin and anti-.beta..sub.1 integrins to block 
tenascin's inhibitory effect on chemotaxis, is that the interaction of 
.beta..sub.1 integrins on PMN and monocytes with cognate ligands on 
tenascin signals these cells to stop migrating. 
The results of Example 1 show that .beta..sub.1 integrins regulate the 
migration of fMLP-stimulated PMNs through fibrin and that antibodies or 
peptides that block .beta..sub.1 integrins allow all PMNs to migrate 
through fibrin gel in response to chemoattractant that otherwise would 
cause PMNs to stop migrating when they encounter fibrin. Further work is 
needed to identify the cellular pathways via which .beta..sub.1 integrins 
signal PMNs and monocytes to stop moving, and to determine the 
physiological significance of this event. Cellular pathways and 
physiological significance notwithstanding, one practical consequence of 
these findings is that antibodies vs PMN and monocyte .beta..sub.1 
integrins may be therapeutically useful by facilitating entry of these 
cells into tissues and body compartments from which they otherwise are 
excluded. 
Tenascin has domains (53), which are homologous to regions in epidermal 
growth factor, fibronectin, and fibrinogen. Since fibrin and tenascin 
block monocyte and PMN migration, tenascin's fibrinogen-like terminal knob 
may play a critical role in signalling leukocytes to stop migrating. 
References 
1. Rot, A. (1991). The role of leukocyte chemotaxis in inflammation. In 
Biochemistry of Inflammation S. W. Evans and J. T. Whicher, eds). Kluwer 
Academic Publishers, Lancaster, 39-54. 
2. Miller, M. D., and Krangel, M. S. (1992). Biology and biochemistry of 
the chemokines: a family of chemotactic and inflammatory cytokines. Crit. 
Rev. Immunol. 12:17-46. 
3. Snyderman, R., and Uhing, R. J. (1992). Chemoattractant 
stimulus-response coupling. In Inflammation: basic principles and clinical 
correlates. J. I. Gallin, I. M. Goldstein, and R. Snyderman, eds. Raven 
Press, New York. 421-439. 
4. Kishimoto, T. K., and Anderson, D. C. (1992). The role of integrins in 
inflammation. In Inflammation: basic principles and clinical correlates. 
J. I. Gallin, I. M. Goldstein, and R. Snyderman, eds. Raven Press, New 
York. 353-406. 
5. Lasky, L. A., and Rosen S. D. (1992). The selectins. Carbohydrate 
binding adhesion molecules of the immune system. In Inflammation: basic 
principles and clinical correlates. J. I. Gallin, I. M. Goldstein, and R. 
Snyderman, eds. Raven Press, New York. 407-420. 
6. Downey, G. P. (1994). Mechanisms of leukocyte motility and chemotaxis. 
Current Opinion in Immunol. 6:113-124. 
7. Loike, J. D., Sodeik, B., Cao, L., Leucona, S., Weitz, J. I., Detmers, 
P. A., Wright, S. D., and Silverstein, S. C. (1991). CD11c/CD18 on 
Neutrophils recognizes a domain at the N terminus of the A.alpha. of 
fibrinogen. Proc. Natl. Acad. Sci, 88:1044-1048. 
8. Loike, J. D., Silverstein, R., Wright, S. D., Weitz, J. I., and 
Silverstein, S. C. (1992). The role of protected extracellular 
compartments in interactions between leukocytes, and platelets and 
fibrin/fibrinogen matrices. In Plasminogen activation in fibrinolysis, in 
tissue remodeling, and in development. P. Brakman, and C. Kluft. Editors. 
Ann. N.Y. Acad. Sci. 667:163-172. 
9. Wright, S. D., Weitz, J. I., Huang, A. J., Levin, J. M., Silverstein, S. 
C., and Loike, J. D. (1988). Complement receptor type three (CD11b/CD18) 
of human polymorphonuclear leukocytes recognizes fibrinogen. Proc. Natl. 
Acad. Sci. 85,7734-7738. 
10. Loike, J. D., Silverstein, R., Cao, L., Solomon, L., Weitz, J. I., 
Haber, E., Matsueda, G. R., Bernatowicz, M. S., and Silverstein, S. C. 
(1993). Activated platelets form protected zones of adhesion with 
fibrinogen and fibronectin-coated surfaces. J. Cell Biol. 121:945-955. 
11. Smith, W. B., Gamble, J. R., Clark-Lewis, I., and Vadas, M. A. (1993). 
Chemotactic desensitization of neutrophils demonstrates IL 8 dependent and 
IL-8 independent mechanisms of transmigration through cytokine activated 
endothelium. Immunology 78:491-497. 
12. Zigmond, S. H., and Hirsch, J. G. (1973). Leukocyte locomotion and 
chemotaxis. New methods for evaluation, and demonstration of a cell 
derived chemotactic factor. J. Exp. Med. 137, 387-410. 
13. Harvath L., and Leonard, E. J. (1982). Two neutrophil populations in 
human blood with different chemotactic activities: separation and 
chemoattractant binding. Infect. Immunol. 36:443-449. 
14. Furie M. B., Naperstek, B. L., and Silverstein, S. C. (1987). Migration 
of neutrophils across monolayers of cultured microvascular endothelial 
cells. An in vitro model of leucocyte extravasation. J. Cell. Sci. 
88:161-175. 
15. Weston, S. A. and Parish, C. R. (1990). New fluorescent dyes for 
lymphocyte migration studies. Analysis by flow cytometry and fluorescence 
microscopy. J. Immunol. Meth. 133:87-97. 
16. Howlett, A. R., Bissell, M. J. (1993). The influence of tissue 
microenvironment (stroma and extracellular matrix) on the development and 
function of mammary epithelium. Epithelila Cell Biol. 2:79-89. 
17. Klemke, R. L., Yebra, M., Bayna, E. M., and Cheresh, D. A. (1994). 
.alpha.v.beta.5 directed cell mobility but not adhesion on vitronectin. J. 
Cell Biol. 127:859-866. 
18. Milici, A. J., Furie, M. B., and Carly, W. W. (1985). The formation of 
fenestration and channels by capillary endothelium in vitro. Proc. Natl. 
Acad. Sci. 82:6181-6185. 
19. Calof A. L., and Lander, A. D. (1991). Relationship between neuronal 
migration and cell-substratum adhesion: laminin and merosin promote 
olfactory neuronal migration but are anti-adhesive. J. Cell Biol. 
115:779-794. 
20. Bronner-Fraser, M. (1994). Neural crest cell formation and migration in 
the developing embryo. FASEB J. 8:699-706. 
21. Detmers, P. A., Lo, S. K., Olsen-Egbert, E., Walz, A., Baggiolini, M., 
and Cohn, Z. A. (1990). Neutrophil-activating protein 1/interleukin 8 
stimulates the binding activity of the leukocyte adhesion receptor 
CD11b/CD18 on human neutrophils. J. Exp. Med. 171, 1155-1162. 
22. Lundgren-Akerlund, E., Berger, E., and Arfors, K. E. (1992). Effect of 
divalent cations on adhesion of PMNs to matrix molecules in vitro. J. 
Leuk. Biol. 51:603-608. 
23. Thompson, H. L., and Matsushima, K. (1992). Human polymorphonuclear 
leucocytes stimulated by TNF-.alpha. show increased adherence to 
extracellular matrix proteins which is mediated via the CD11b/18 complex. 
Clin. Exp. Immunol. 90:280-285. 
24. Pommier C. G., Inada, S., Fries, L. F. Takahashi, T., Frank, M. M., and 
Brown, E. J. (1983). Plasma fibronectin enhances phagocytosis of opsonized 
particles by human peripheral blood monocytes. J. Exp. Med. 157:1844-1854. 
25. Wright, S. D., Licht, M. R., Craigmyle, L. S., and Silverstein, S. C. 
(1985). Communication between receptors for different ligands on a single 
cell: ligation of fibronectin receptors induces a reversible alteration in 
the function of complement receptors on cultured human monocytes. J. Cell 
Biol. 99:336-339. 
26. Monboisse, J. C., Bellon, G., Randoux, A., Duffer, J., and Borel, J. P. 
(1990). Biochem. J. 270:459-462. 
27. Monboisse, J-C., Garnotel, R., Bellon, G., Ohno, N., Perreau, C., Borel 
J. P., and Kefalides. (1994). The .alpha.3 chain of type IV collagen 
prevents activation of human polymorphonuclear leukocytes. J. Biol. Chem. 
269:25475-25482. 
28. Nicosia, R. F. and Tuszynski, G. P. (1994). Matrix-bound thrombospondin 
promotes angiogenesis in vitro. J. Cell Biol. 124:183-193. 
29. Ohno, K., and Maier., P. (1994). Cultured rat hepatocytes adapt their 
cellular glycolytic activity and adenylate energy status to tissue oxygen 
tension: influences of extracellular matrix components, insulin and 
glucagon. J. Cell. Physiol. 160:358-366. 
30. Fuortes, M., Jen, W-W., and Nathan, C. (1993). Adhesion-dependent 
Protein Tyrosine Phosphorylation in Neutrophils Treated with Tumor 
Necrosis Factor J. Cell Biology 120:777-784. 
31. DiMilla, P. A., Stone, J. A., Quinn, J. A., Albelda, S. M., and 
Lauffenburger, D. A. (1993). Maximal migration of human smooth muscle 
cells on fibronectin and type IV collagen occurs at an intermediate 
attachment strength. J. Cell Biol. 122:729-737. 
32. Goodman, S. L., Risse, G., and von der Mark, K. (1989). The E8 
subfragment of laminin promotes locomotion of myoblasts over extracellular 
matrix. J. Cell Biol. 109:799-809. 
33. Weitz, J. I., Huang, A. J., Landman, S. L., Nicholson, S. C., and 
Silverstein, S. C. (1987). Elastase-mediated fibrinogenolysis by 
chemoattractant-stimulated neutrophils occurs in the presence of 
physiological concentrations of antiproteinases. J. Exp. Med. 
166:1836-1850. 
34. Lanir, N., Ciano, P. S., Van De Water, L., McDonagh, J., Dvorak, A. N., 
and Dvorak, H. P. (1988). Macrophage migration in fibrin gel matrices. II. 
Effects of clotting factor XIII, fibronectin, and glycosaminoglycan 
content on cell migration. J. Immunol. 140:2340-2349. 
35. Campbell, E. J., Senior, R. M., McDonald, J. A., and Cox, D. L. (1982). 
Proteolysis by neutrophils. Relative importance of cell-substract contact 
and oxidative inactivation of proteinase inhibitors in vitro. J. Clin. 
Invest. 70:845-852. 
36. Huhtala, P., Humpries, M., McCarthy, J., Werb Z., and Damsky, C. 
(1994). The RGD and CS-1 containing cell binding regions of fibronectin 
signal opposing effects on metalloproteinase expression via 
.alpha.5.beta.1. Mol. Biol. Cell 5:64a. 
37. Balsamo J, Ernst H, Zanin M K B, Hoffman S, Lilien J. (1995). The 
interaction of the retina cell surface 
N-acetylgalactosaminylphosphotransferase with an endogenous proteoglycan 
ligand results in inhibition of cadherin-mediated adhesion, J. Cell Biol, 
129:1391-1401. 
38. Bourdon, M. A., Ruoslahti, E. (1989). Tenascin mediates cell attachment 
through an RGD-dependent receptor. J. Cell Biology 108:1149-1155. 
39. Boyum, A., Lovhaug, D., Tresland, L., Nordlie, E. M. (1991). Separation 
of leucocytes: improved cell purity by fine adjustments of gradient medium 
density and osmolality. Scand. J. Immunol. 34:697-712. 
40. Chilosi, M., Lestani, M., Benedetti, A., Montagna, L., Pedron, S., 
Scarpa, A., Menestrina, F., Hirohashi, S., Pizzolo, G., and Semenzato, G. 
(1993). Constitutive expression of tenascin in T-dependent zones of human 
lymphoid tissues. Am. J. of Path. 143:1348-55. 
41. Chiquet-Ehrismann, R. (1993). Tenascin and other adhesion-modulating 
proteins in cancer.Review!. Sem. in Cancer Biol. 4:301-10. 
42. Chuong, C. M., and Chen, H. M. (1991). Enhanced expression of neural 
cell adhesion molecules and tenascin (tenascin) during wound healing. Am. 
J. Pathol. 138:427-440. 
43. Erickson, H. P. and Bourdon, M. A. (1989). Tenascin: an extracellular 
matrix protein prominent in specialized embryonic tissues and tumors. Ann. 
Rev. Cell. Biol. 5:71-92. 
44. Grumet, M., Hoffman, S. Crossin, K. L., and Edelman, G. M. (1985). 
Tenascin, an extracellular matrix protein of neural and non-neural tissues 
that mediates glial-neuron interaction. Proc. Natl. Aca. Sci. 
82:8075-9079. 
45. Herlyn, M., Graeven, U., Speicher, D., Sela, B. A., Bennicelli, J. L., 
Kath, R., Guerry, D. (1991). Characterization of tenascin secreted by 
human melanoma cells. Cancer Res. 51:4853-8. 
46. Hoffman, S., Crossin, K. L., and Edelman, G. M. (1988). Molecular 
forms, binding functions, and developmental expression patterns of 
cytotactin and cytotactin-binding proteoglycans, an interactive pair of 
extracellular matrix molecules. J. Cell Biol. 106:519-532. 
47. Hoffman, S. Dutton, S. L., Ernst, H., Boackle, M. K., Everman, D., 
Tourkin, A., and Loike, J. D. (1994). Functional characterization of 
anti-adhesion molecules. Perspectives in Development Neurbiology 
2:101-110. 
48. Joshi, P., Chung, C. Y., Aukhil, I., and Erickson, H. P. (1993). 
Endothelial cells adhere to the RGD domain and the fibrinogen-like 
terminal knob of tenascin. J. Cell Sci. 106:389-400. 
49. Juhasz, I., Murphy, G. F., Yan, H. C., Herlyn, M., Albelda, S. M. 
(1993). Regulation of extracellular matrix proteins and integrin cell 
substratum adhesion receptors on epithelium during cutaneous human wound 
healing in vivo. Am. J. of Path. 143:1458-69. 
50. Mackie., E. J., Halfter, W., and Liverani, D. (1988). J. Cell Biol. 
107:2757-2767. 
51. Mackie, E. J., Chiquet-Ehrismann, R., Pearson, C. A., Inaguma, Y. M., 
Taya, K., Kawarada, Y., and Sakakura, T. (1987). Proc. Natl. Acad. Sci., 
84:4621-4625. 
52. Pesheva, P., Probstmeier, R., Skubitz, A. P., McCarthy, J. B., Furcht, 
L. T. and Schachner, M. (1994). Tenascin-R J1 160/180 inhibits 
fibronectin-mediated cell adhesion--functional relatedness to tenascin-C. 
J. Cell Sci. 107:2323-33. 
53. Prieto, A. L., Andersson-Fisone, C. and Crossin, K. L. (1992). 
Characterization of multiple adhesive and counteradhesive domains in the 
extracellular matrix protein cytotactin. J. Cell Bio. 119:663-78. 
54. Prieto, A. L., Edelman, G. M., and Crossin, K. L. (1993). Multiple 
integrins mediate cell attachment to cytotactin/tenascin. Proc. Natl. 
Acad. Sci. USA 90:10154-8. 
55. Ruegg, C. R., Chiquet-Ehrismann, R., and Alkan, S. S. (1989). Tenascin, 
an extracellular matrix protein, exerts immunomodulatroy activities. Proc. 
Natl. Acad. Sci. 86:7637-7441. 
56. Sakai, T., Kawakatsu, H., Hirota, N., Yokoyama, T., Sakakura, T., and 
Saito, M. (1993). Specific expression of tenascin in human colonic 
neoplasms. British Journal of Cancer 67:1058-64. 
57. Sharifi, B. G., D. W. Lafleur, S. M. Schwartz, J. S. Forrester, J. A., 
Fagin. (1995). Expression of tenascin isoforms are selectively 
up-regulated following aortic balloon injury. The FASEB Journal, 9: a611. 
58. Shoji, T., Kamiya, T., Tsubura, A., Hatano, T., Sakakura, T., Yamamoto, 
M., Morii, S. (1992). Immunohistochemical staining patterns of tenascin in 
invasive breast carcinomas. Virchows. Arch. A. Pathol. Anat. Histopathol. 
421:53-6. 
59. Singh, S., Ross, S. R., Acena, M., Rowly, D. A., and Schreiber, H. 
(1992). Stroma is critical for preventing or permitting immunological 
destruction of antigenic cancer cells. J. Exp. Med. 175:139-146. 
60. Sriramarao, P., Mendler, M., and Bourdon, M. A. (1993). Endothelial 
cell attachment and spreading on human tenascin is mediated by alpha 2 
beta 1 and alpha v beta 3 integrins. J. Cell Sci. 105:1001-12. 
61. Tourkin A., Anderson T., LeRoy EC, Hoffman S. (1993). Eosinophil 
adhesion and maturation is modulated by laminin. Cell Adhesion and 
Communication, 1:161-176. 
62. Wehrle-Haller, B. and Chiquet, M. (1993). Dual function of tenascin: 
simultaneous promotion of neurite growth and inhibition of glial 
migration. J. Cell Sci. 106:597-610. 
63. Hiraiwa, N., Kida, H., Sakakura, T., and Kusakabe, M. (1993). Induction 
of tenascin in cancer cells by interactions with embryonic 
mesenchyme-mediated by a diffusible factor. J. Cell. Sci. 104:289. 
64. Koukouus, G. K., Gould, V. E., Bhattacharyya, A., Gould, J. E., 
Howeedy, A. A., and Virtanen, I. (1991). Tenascin in normal, reactive, 
hyperplastic and neoplastic tissue: biological and pathological 
implications. Hum. Pathol. 22:636. 
65. El Khoury, J., Loike, J., Cao, L., Thomas, C. and Silverstein, S. C. 
(1994). Macrophages adhere to glucose-modified basement membrane collagen 
via their scavenger receptors. J. Biol. Chem. 269:10197-10200. 
66. Loike, J. D., Somes, M., and Silverstein, S. C. (1986). Creatine 
uptake, metabolism, and efflux in human monocytes and macrophages. Am. J. 
Physiol. 251:C128. 
67. Friedlander, D. R., Hoffman, S., and Edelman, G. M. (1988). Functional 
mapping of cytotactin: proteolytic fragments active in cell-substrate 
adhesion. J. Cell Biol. 107:2329. 
68. Lundgren-Akerlund, E., Olofsson, A. M., Bergerand, E., and Arfors, K. 
E. (1993). CD11b/CD18-dependent polymorphonuclear leucocyte interaction 
with matrix proteins in adhesion and migration. Scan. J. Immunol. 37:569. 
69. Islam, L. N., McKay, I. C., and Wilkinson, P. C. (1985). The use of 
collagen or fibrin gels for the assay of human neutrophil chemotaxis. J. 
Immunol. Meth. 85:137. 
70. Dvorak, H. F. (1986). Tumors: Wounds that do not heal. Similarities 
between tumor stroma generation and wound healing. N. Engl. J. Med. 
315:1650-1659. 
71. Willhelm, O., Hafter, R., Coppenrath, E., Pflanz, M., Schmitt, M., 
Babic, R., Linke, R., Gossner, W., and Graeff, H. (1988). 
Fibrin-fibronectin compounds in Human ovarian tumor ascites and their 
possible relation to the tumor stroma. Can. Res. 48:3507. 
72. Strickland D. K., Kounnas, M. Z., and Argraves, W. S. (1995). LDL 
receptor-related protein: a multiligand receptor for lipoprotein and 
proteinase catabolism. Review! FASEB J. 9:890. 
73. Costantini, V., Zacharski, L. R., Memoli, V. A., Kisiel, W., Kudryk, B. 
J., Rousseau, S. M., and Stump. D. C. (1992). TI-Fibrinogen deposition and 
macrophage-associated fibrin formation in malignant and nonmalignant 
lymphoid tissue. J. Lab. & Clin. Med. 119:124. 
74. Schmidt A. M., Yan, S. D., Brett, J., Mora, R., Nowygrod, R., and 
Stern, D. (1993). Regulation of human mononuclear phagocyte migration by 
cell surface-binding proteins for advanced glycation end products. J. 
Clin. Invest. 91:2155. 
75. Gao, J. X., Wilkins, J., and Issekutz, A. C. (1995). Migration of human 
polymorphonuclear leukocytes through a synovial fibroblast barrier is 
mediated by both beta 2 (CD11/CD18) integrins and the beta 1 (CD29) 
integrins VLA-5 and VLA-6. Cell. Immunol. 163:178. 
76. Gresham, H. D., Graham, I. L., Anderson, D. C., and Brown, E. J. 
(1991). Leukocyte adhesion-deficient neutrophils fail to amplify 
phagocytic function in response to stimulation. Evidence for 
CD11b/CD18-dependent and -independent mechanisms of phagocytosis. J. Clin. 
Invest. 88:588. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 2 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
GlyArgGlyAspSerPro 
15 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
GlyArgGlyGluSerPro 
15 
__________________________________________________________________________