[0001] This application is a continuation-in-part application of U.S. Ser. No. 08/635,572, filed on Apr. 22, 1996, which is hereby incorporated by reference.
[0003] 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.
[0004] 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-α] (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).
[0005] 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).
[0006] 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).
[0007] 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 β
[0008] 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.
[0009] 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 β
[0010] 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 β
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[0028] 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 β
[0029] In one embodiment of the invention, the foreign body is a prosthetic device, a catheter, or a suture.
[0030] In another embodiment of the invention, the subject is a mammal such as a human.
[0031] In yet another embodiment of the invention, the leukocyte cells are polymorphonuclear leukocyte cells such as neutrophils, basophils, and eosinophils.
[0032] In another embodiment of the invention, the leukocyte cells are monocytes or macrophages.
[0033] In yet another embodiment of the invention, the agent is a peptide such as a peptide containing a β
[0034] In another embodiment of the invention, the agent is an antibody or a fragment thereof that specifically binds to the β
[0035] 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.
[0036] In one embodiment of the invention, the foreign body is a prosthetic device, a catheter, or a suture.
[0037] In another embodiment of the invention, the subject is a mammal such as a human.
[0038] In yet another embodiment of the invention, the fibrinolytic agent is plasminogen activator such as urokinase, streptokinase, or tissue plasminogen activator.
[0039] 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 β
[0040] In one embodiment of the invention, the subject is a mammal such as human.
[0041] In another embodiment of the invention, the leukocyte cells are polymorphonuclear leukocyte cells such as neutrophils, basophils, and eosinophils.
[0042] In yet another embodiment of the invention, the leukocyte cells are monocytes or macrophages.
[0043] In another embodiment of the invention, the leukocyte cells are lymphocyte cells such as NK cells and killer cells.
[0044] In yet another embodiment of the invention, the agent is a peptide such as a peptide containing a β
[0045] In another embodiment of the invention, the agent is an antibody or a fragment thereof that specifically binds to the β
[0046] 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 β
[0047] In one embodiment of the invention, the subject is a mammal such as human.
[0048] In another embodiment of the invention, the leukocyte cells are polymorphonuclear leukocyte cells such as neutrophils, basophils, and eosinophils.
[0049] In yet another embodiment of the invention, the leukocyte cells are monocytes or macrophages.
[0050] In yet another embodiment of the invention, the agent is a peptide such as a peptide containing a β
[0051] 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.
[0052] 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.
[0053] Human monocyte recombinant IL-8 (Ser-IL-8)
[0054] Becton-Dickinson cell culture inserts (pore sizes 3 or 8 μm: Franklin Lakes, N.J.) were overlaid with the following proteins:
[0055] Fibrin gels: 1 Unit of thrombin (a gift from Dr. John Fenton, Albany Medical College, Albany, N.Y.), in 5 μl of PBS was added first to each insert. 0.1 ml phosphate buffered saline supplemented with Ca
[0056] Collagen type IV and Matrigel matrices: 0.1 ml PBS containing 100 μg human placental collagen IV (Fluka Chemical Corp., Ronkonkoma, N.Y.), or 80 μg 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.
[0057] 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 μl of this mixture was placed into each insert containing thrombin and allowed to clot as described above. One unit of PPACK in 100 μl PBS then was added and the inserts were washed with 250 μl of PBS.
[0058] Fibrinogen or fibronectin: 0.1 ml of PBS containing 100 μg/ml fibrinogen or fibronectin (New York Blood Center, New York, N.Y.), was placed into each insert (pore size 3 μm). Inserts were incubated at 37° C. for 60 min and washed with 250 μl 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.
[0059] 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 >95% as determined by Wright-Giemsa staining (7). 10
[0060] PMNs were suspended in medium containing 10 μM 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× 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× 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 μm 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 3×3×3 Gaussian convolution filter. Each experiment was repeated at least twice using duplicate samples.
[0061] Fibrin coated Terasaki tissue culture plates were prepared as described (10). 5 μl of PBSG-HSA, containing PMNs (10
[0062] 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 μl of the various solutions were used per well. 20 μl of PMNs (10
[0063] 1.0 ml PBS containing 1 mg human fibrinogen, 1 mCi of Na
[0064] 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° 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
[0065] 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 (
[0066] The percent of PMNs that migrated through fibrin gels varied with the concentration of IL-8 or LTB4 placed in the bottom compartment (
[0067] 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 (
[0068] 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.
[0069] PMNs migrated through fibrin gels more rapidly in response to an optimal concentration of LTB4 than to an optimal concentration of IL-8 (
[0070] 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° 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; (
[0071] 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
[0072] 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 (
[0073] 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 (
[0074] 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° C. As expected, TNF, fMLP, LTB4 and IL-8 were equally effective in stimulating PMN adherence to fibrin (>200 chemoattractant-stimulated PMNs vs ˜10 unstimulated PMNs adhered per mm
[0075] Previous studies (10) showed that the exclusion of fluorescein-conjugated F(ab)
[0076] 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 (
[0077] 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 (
[0078] To determine the machanism by which fibrin blocks PMN migration in response to fMLP, the effects of anti-integrin antibodies were tested (
[0079] 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) (
[0080] 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 β
[0081] 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.
[0082] 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 (
[0083] The zones of close apposition formed between fMLP- or TNF-stimulated PMNs and fibrin gels are impermeant to molecules of >10 kDa thereby excluding virtually all plasma protease inhibitors such as alpha
[0084] That fMLP and TNF promote PMN migration through fibrinogen-coated filters (
[0085] 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 β
[0086] 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 (
[0087] 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.
[0088] 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.
[0089] 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 β
[0090] 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 >95% as determined by Wright-Giemsa staining.
[0091] 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.
[0092] Cultured monocytes were prepared by allowing 10
[0093] Cell culture inserts containing polyethylene terephthalate filters, 8-μm pore size (Becton-Dickinson), were overlaid with 0.1 ml of Matrigel (20-25 μg 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
[0094] 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 2-10×10
[0095] PMN: 250 μl of PBS [supplemented with 5.5 mM glucose and 0.1% human serum albumin (HSA) (PBSG-HSA)] containing 1×10
[0096] 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° 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 4 M guanidine-HCl/0.1 M Tris (pH 7.6), and fractionated on a 1.5×100 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 4 M guanidine-HCl; the tenascin containing fractions were then dialyzed extensively vs PBS.
[0097] Chick tenascin was radiolabeled using chloramine T (67), and mixed with unlabeled tenascin at a 1:100 protein ratio. 250 μl 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° C. in a humidified 95%air/5% CO
[0098] Monoclonal antibody P4C10 (anti-β chain of human beta-1 integrin), was from GIBCO-BRL. Fluorescein conjugated monoclonal antibody against the β chain of human beta-2 integrins (anti-β
[0099] Chondroitin sulfate proteoglycan monomers were purified and antibodies prepared against these proteoglycan monomers as described (37). The F(ab)′
[0100] 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 (
[0101] Addition of chick brain tenascin to the Matrigel significantly reduced monocyte and PMN migration in response to TNF, fMLP or LTB4 (
[0102] The effect of chick brain tenascin on TNF-stimulated migration of monocytes varied with the amount of tenascin added (
[0103] 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 (
[0104] Monocytes or PMN were added to the upper compartment of inserts coated with Matrigel alone or with Matrigel and tenascin. F(ab)′
[0105] Because of their affinity for tenascin, proteoglycans may contaminate some tenascin preparations (46). Therefore, the effects of F(ab)′
[0106] Endothelial cell attachment and spreading on human tenascin has been shown to be partially mediated by β
[0107] LTB4 was added to the lower compartment and the inserts were incubated at 37° C. for 24 h for monocytes or 4 h for PMN. A monoclonal antibody directed against β
[0108] To confirm that the effect of P4C10 was due to its interaction with β
[0109] 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.
[0110] 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.
[0111] 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.
[0112] 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).
[0113] 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.
[0114] Anti-β
[0115] 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.
[0116] β
[0117] The results of Example 1 show that β
[0118] 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.
[0119] The role of β
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[0121] 2. Miller, M. D., and Krangel, M. S. (1992). Biology and biochemistry of the chemokines: a family of chemotactic and inflammatory cytokines.
[0122] 3. Snyderman, R., and Uhing, R. J. (1992). Chemoattractant stimulus-response coupling.
[0123] 4. Kishimoto, T. K., and Anderson, D. C. (1992). The role of integrins in inflammation.
[0124] 5. Lasky, L. A., and Rosen S. D. (1992). The selectins. Carbohydrate binding adhesion molecules of the immune system.
[0125] 6. Downey, G. P. (1994). Mechanisms of leukocyte motility and chemotaxis.
[0126] 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 Aα of fibrinogen.
[0127] 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.
[0128] 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.
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