Development of monoclonal antibodies to integrin receptors.

Integrins are heterodimeric cell surface receptors composed of an alpha and a beta subunit. They are involved in homotopic and heterotopic cell adhesion and also function as receptors for extracellular matrix molecules such as collagen, fibronectin and laminin. The family to which an integrin belongs is defined by the presence of a particular beta subunit paired with a unique alpha subunit. In this chapter we describe methods to produce monoclonal antibodies to the family of integrin subunits characterized by beta1 and provide detailed instructions for the development of a monoclonal antibody to the alpha6 integrin receptor expressed by human prostate carcinoma cells (PC3 cells). Data are presented that correlate the functional capabilities of an antibody with its biochemical characterization.

The keratinocyte-derived cytokine IL-7 increases adhesion of the epidermal T cell subset to the skin basement membrane protein laminin-5.

Human epidermis contains a subset of epidermal T cells that can mount an immune response by migrating through the skin and into the peripheral lymphnodes to proliferate before re-entering the epidermis. The cytokine IL-7 is shown to be localized to the basement membrane of normal human skin. Furthermore, culturing in the presence of IL-7 causes increased adhesion of epidermal T cells but not peripheral blood T cells to the major epidermal basement membrane protein, laminin-5. The mechanism for increased T cell adhesion to laminin-5 is due, at least in part, to an increase in the cell surface expression of the integrin alpha3beta1. Epidermal T cells cultured in IL-7 that are strongly adherent to laminin-5 are shown by flow cytometry to consist of a variety of subsets; therefore, the increase in cell adhesion is not due to an outgrowth of one T cell subset during culturing. We hypothesize that in vivo, exposure to IL-7 is required for epidermal T cell adhesion to laminin-5.

Adhesion of cultured human kidney mesangial cells to native entactin: role of integrin receptors.

Entactin is an extracellular matrix glycoprotein which binds to laminin and is found in most renal basement membranes and in the glomerular mesangial matrix. In the present study, we have characterized specific integrin receptors on cultured human mesangial cells (CHMC) responsible for adhesion to native entactin. The integrin receptors alpha 2 beta 1, alpha 3 beta 1, alpha 5 beta 1, alpha v beta 3, alpha v beta 5, and alpha 6 complexed with either beta 1 or beta 4 could be immune precipitated from detergent extracts of metabolically labeled CHMC. Adhesion assays with inhibitory anti integrin monoclonal antibodies (mab) demonstrated that CHMC use both alpha v beta 3 and a beta 1-containing integrin to bind surfaces coated with native entactin. Optimal binding of CHMC to native entactin required the participation of cations. Using wild type and mutant recombinant entactin fragments, the binding site for the alpha v beta 3 receptor was localized to the RGD sequence on the rod or E domain of entactin. CHMC adhesion to mutant full length recombinant entactin ligands lacking the E domain RGD sequence confirmed the presence of ligand binding site(s) for beta 1 integrin receptor(s). Differences in CHMC binding characteristics to recombinant and full length entactin compared to native bovine basement membrane entactin were observed. This suggests that tertiary molecular structure may contribute to entactin ligand binding properties. Primary amino acid residue sequences and tertiary structure of entactin may play roles in forming functional cell attachment sites in native basement membrane entactin.

Interactions of type IV collagen and its domains with human mesangial cells.

Type IV collagen (COL-IV) interacts with a variety of cell types. We present evidence that human mesangial cells (HMC) bind directly to COL-IV, its major triple helical domain, and the main non-collagenous, NC1 domain. A synthetic peptide, HEP-III, and its triple helical counterpart (THP-III), previously reported to be a heparin-binding domain, also promoted approximately 15% adhesion of HMC. HMC bound to solid-phase-immobilized, intact COL-IV (approximately 75%), isolated NC1 domain (approximately 15%), and a pepsin-derived triple helical fragment,which lacks Hep-III (approximately 65%). We further examined inhibition of HMC adhesion to COL-IV and its domains by using anti-integrin antibodies. Blocking monoclonal antibodies against the alpha2 integrin resulted in 70% inhibition of adhesion to COL-IV and 80% inhibition to HEP-III. Moderate inhibition was observed on the NC1 and triple helical fragments. Anti-alpha1 antibodies inhibited the binding of HMC to COL-IV, the NC1, and triple helical domains, but not to peptide HEP-III. Anti-beta1 antibodies inhibited almost completely (>95%) the adhesion to COL-IV, the NC1, and triple helical fragments; inhibition on HEP-III was approximately 30%. Affinity chromatography studies with solid-phase HEP-III and mesangial cell lysate also demonstrated the presence of integrin alpha2 beta1 along with alpha3 beta1. We conclude that alpha2 beta1 and alpha1 beta1 integrins mediate HMC adhesion to COL-IV. Peptide HEP-III is a major, specific site for alpha2 integrin-mediated binding of mesangial cells to COL-IV. Both the alpha1 beta1 and alpha2 beta1 integrins interact with the NC1 and triple helical fragments of COL-IV. Therefore, we demonstrate that several sites for integrin-mediated interactions exist on several collagenous and non-collagenous domains of COL-IV.

Abnormal expression of epiligrin and alpha 6 beta 4 integrin in basal cell carcinoma.

BACKGROUND: Basal cell carcinoma is characterized by local invasion, and only rarely metastasizes. The role of the containing basement membrane (BM) in this tumor is unclear. Several BM components have been shown to be absent or significantly reduced in BM surrounding infiltrating tumor. OBJECTIVE: The purpose of this study is to examine the expression of epiligrin, a BM-associated glycoprotein, and the integrin chains alpha 3, alpha 6, beta 1, and beta 4 in the basement membranes surrounding basal cell carcinoma. METHODS: Samples were obtained from 20 patients with basal cell carcinomas and subjected to a standard avidin biotin complex/alkaline phosphatase immunohistochemical technique using a panel of antibodies. RESULTS: There was a consistent abnormality of expression of epiligrin, alpha 6, and beta 4. CONCLUSION: We propose that reduced expression of epiligrin is involved in the pathogenesis of the local invasion by tumor and that an altered integrin ratio in basal cell carcinoma enhances tumor spread.

Characterization of monoclonal antibodies that recognize canine CD34.

Using a polyclonal antiserum against canine CD34, we previously found that CD34 is expressed on canine bone marrow progenitor cells in a manner analogous to that found in humans. To further characterize CD34+ cells and to facilitate preclinical canine stem cell transplant studies, monoclonal antibodies (MoAbs) were raised to CD34. A panel of 10 MoAbs was generated that reacted with recombinant CD34 and with CD34+ cell lines and failed to react with CD34- cell lines. Binding properties of five purified MoAbs were determined by BIAcore analysis and flow cytometric staining, and several MoAbs showed high affinity for CD34. Two antibodies, 1H6 and 2E9, were further characterized, and in flow cytometry studies typically 1% to 3% of stained bone marrow cells were CD34+. Purified CD34+ bone marrow cells were 1.8- to 55-fold enriched for colony-forming unit-granulocyte-macrophage and for long-term culture initiating cells as compared with bone marrow mononuclear cells, whereas CD34- cells were depleted of progenitors. Three autologous transplants were performed with CD34+ cell fractions enriched by immunomagnetic separation. After marrow ablative total body irradiation (920 cGy), prompt hematopoietic recovery was seen with transplanted cell doses of

Circulating activated endothelial cells in sickle cell anemia.

BACKGROUND: The vascular wall participates in the pathogenesis of sickle cell disease. To determine whether the endothelium is activated in this disease, we studied the number, origin, and surface phenotype of circulating endothelial cells in patients with sickle cell anemia. METHODS: We used immunohistochemical examination of buffy-coat smears to enumerate circulating endothelial cells, and we evaluated the surface phenotype by applying preparations of circulating endothelial cells. An immunofluorescence microscopy panel of antibodies was used, including a specific anti-endothelial-cell antibody, P1H12. RESULTS: Mean (+/-SD) numbers of circulating endothelial cells in normal blood donors, patients with sickle cell trait, and patients with hemolytic anemias not due to hemoglobin S were 2.6+/-1.6, 3.0+/-2.6, and 2.0+/-0.8 per milliliter of whole blood, respectively. Patients with sickle cell anemia who presented with acute painful episodes had 22.8+/-18.2 circulating endothelial cells per milliliter of blood (P<0.001 for the comparison with normal donors), and patients with no such events within one month before or after blood sampling had 13.2+/-11.8 circulating endothelial cells per milliliter of blood (P=0.002 for the comparison with normal donors and P=0.019 for the comparison with patients with acute events). Serial observations of three patients showed a tendency toward higher levels of circulating endothelial cells at the onset of acute painful crises. The average viability of circulating endothelial cells was 66+/-30 percent. In patients with sickle cell anemia, regardless of clinical status, the circulating endothelial cells were predominantly microvascular in origin (CD36-positive), and most of the cells expressed four markers of endothelial-cell activation: intercellular adhesion molecule 1, vascular-cell adhesion molecule 1, E-selectin, and P-selectin. CONCLUSIONS: Our studies suggest that the vascular endothelium is activated in patients with sickle cell anemia, regardless of the patients' clinical status. Adhesion proteins on activated endothelial cells may have a role in the vascular pathology of sickle cell disease.

Mapping epitopes to distinct regions of the extracellular domain of endoglin using bacterially expressed recombinant fragments.

Endoglin (CD105) is a homodimeric cell surface component of the TGF-beta 1 receptor complex, which is expressed at high levels on vascular endothelium and at lower levels on activated monocytes. It is also the target gene for the dominantly inherited vascular disorder hereditary hemorrhagic telangiectasia type 1. To date, each family has a distinct endoglin mutation, most of which generate premature stop codons. The purpose of the current study was to identify monoclonal antibodies capable of binding to normal and mutated forms of the protein. We generated stable transfectants of full-length human endoglin in murine fibroblasts and engineered and expressed in bacteria several fragments of the extracellular domain. Relatively pure polypeptides were recovered with good yield from inclusion bodies and were tested by ELISA and Western blot; 11 monoclonal antibodies were shown to react specifically with the endoglin transfectants. Ten of these monoclonal antibodies reacted with the bacterial fragments, and their epitopes were assigned to 3 distinct regions of endoglin. Monoclonal antibodies P3D1, TEC4 and GRE reacted with the N-terminal region of 204 amino acids encoded by exons 1 to 5. Monoclonal antibodies P4A4, 44G4, E-9, MAEND3 and PN-E2 all bound to a region of 54 amino acids encoded mostly by exon 7. Monoclonal antibodies CLE4 and RMAC8 reacted with the C-terminal region of the extracellular domain, coded for by exons 8 to 12. Knowing the localization of these epitopes will facilitate the structural and functional analysis of normal and mutated forms of endoglin.

Monoclonal antibody crosslinking of the alpha 4 or beta 1 integrin inhibits committed clonogenic hematopoietic progenitor proliferation.

Adhesion receptors can serve as primary signal transduction molecules that convey information into cells that can affect cell proliferation and differentiation. Since hematopoietic progenitors adhere to marrow stroma and fibronectin via the alpha 4 beta 1 integrin and CD44, we examined the role of these receptors in the transfer of proliferation-regulatory signals to progenitors. Actively proliferating colony-forming cells (CFCs) present in cultured CD34+ cells were incubated with mouse monoclonal antibodies against the alpha 4, beta 1, or CD44 receptors and crosslinking was performed with a secondary goat-anti-mouse antibody. The effect on CFC proliferation was examined with a 3H thymidine suicide assay. Compared with controls (39 to 51% kill), crosslinking the alpha 4 or beta 1 integrins significantly reduced CFC proliferation (12 to 26% kill, p = 0.01), indicating that proliferation-inhibitory signals are transmitted through the VLA-4 integrin. Cytochalasin D, a compound that prevents actin polymerization, prevented not only alpha 4 receptor capping, but also the inhibition of CFC proliferation observed following alpha 4 crosslinking. However, crosslinking of the CD44 receptor with the antibodies Hermes-3 and 50B4, which inhibit adhesion of CFC to fibronectin, failed to cap the CD44 receptor in the majority of CD34+ cells. Furthermore, crosslinking of the CD44 receptor with these antibodies also failed to inhibit proliferation of CFCs. These studies demonstrate that adhesion receptor crosslinking of the alpha 4 beta 1 integrin, together with subsequent changes in F-actin polymerization, negatively regulates hematopoietic progenitor proliferation in a manner independent of the shape change associated with adhesion.

Distribution of integrin subunits in human diabetic kidneys.

Integrins are cell-surface protein receptors that participate in cell adhesion to multiple extracellular matrix ligands, and consist of alpha and beta chain heterodimers. This study examined altered integrin distribution in diabetic nephropathy by investigating 12 human diabetic kidney biopsies, which were compared with normal human kidney. Diabetic nephropathy is characterized by mesangial expansion and progressive thickening of the glomerular basement membrane. Based on morphometric studies of mesangial expansion, diabetic nephropathy was determined to be moderate or severe. Three different patterns (P) of altered intensity of integrin staining were observed. In the mesangial integrin P, the intensity of integrin subunit staining of mesangial cells (alpha 1, alpha 2, alpha 3, beta 1, alpha V, alpha V beta 5) was increased in moderate diabetic nephropathy and further increased in severe diabetic nephropathy. In the epithelial integrin P, integrin subunits localized to epithelial cells (alpha V, beta 3, alpha V beta 3, alpha V beta 5) were increased to the same extent in moderate and severe diabetic nephropathy. In the endothelial integrin P, integrin subunits localized to endothelial cells (alpha 3, alpha 5, alpha 6, beta 1) were increased in moderate diabetic nephropathy but returned to normal kidney staining intensity in severe diabetic nephropathy. From these observations, it was concluded that there is significant alteration in the expression of integrin subunits in diabetic nephropathy that is related to the severity of diabetic mesangial expansion. Additionally, the spectrum of integrin subunit alteration appears to be unique to individual glomerular cell types. Given the role of integrins in cell-surface interactions with extracellular matrix components, abnormalities in the expression of these molecules may be important in the pathogenesis of diabetic nephropathy.

Expression of alpha 6 and beta 4 integrins in serous ovarian carcinoma correlates with expression of the basement membrane protein laminin.

The surface of a normal ovary is covered by a monolayer of epithelial cells that rest on a basement membrane. The glycoprotein laminin is the major noncollagenous protein present in the basement membrane. The integrins alpha 1 beta 1, alpha 2 beta 1, alpha 3 beta 1, alpha 6 beta 1, and alpha 6 beta 4 serve as cell surface receptors for laminin. During the progression of serous ovarian carcinoma, tumor cells are frequently exfoliated from the surface of the ovary, thereby losing contact with the basement membrane. This study was designed to determine whether alterations in integrin expression may be associated with the malignant phenotype of the primary ovarian tumor and exfoliated ovarian carcinoma cells in the ascites fluid. By immunohistochemical staining, the entire surface of epithelial cells of normal ovaries stained positively for beta 1, alpha 2, and alpha 3 integrins, whereas only the basal surface of the epithelial cells, where they are in contact with laminin, stained positively for alpha 6 and beta 4. The entire surface of epithelial cells of solid tumors from patients with serous ovarian carcinoma stained positively for beta 1, alpha 2, and alpha 3 integrins. In most cases, no intact basement membrane surrounded the tumor nests, and staining for alpha 6 and beta 4 was irregular. When present, the basement membrane stained positively for laminin, and the basal surface of the epithelial cells stained positively for alpha 6 and beta 4. Ovarian carcinoma ascites cells exhibited a distinct phenotype, with a significant decrease in expression of the alpha 6 and beta 4 integrin subunits. As alpha 6 and beta 4 integrin subunits are present at the basal surface of many epithelial cells and serve as receptors for laminin, it is possible that ovarian carcinoma epithelial cells may be released from the basement membrane of the ovary due to their deficit of alpha 6 and beta 4 integrin subunits.

Human SY5Y neuroblastoma cell interactions with laminin isoforms: neurite outgrowth on laminin-5 is mediated by integrin alpha 3 beta 1.

Laminin (Ln) isoforms may play important roles in neuronal development, particularly axon guidance, but neural receptors mediating interactions with Ln are not entirely understood. In this paper, we have compared the adhesive and process outgrowth activities of a human neuroblastoma cell line SY5Y on various laminin isoforms. Cell adhesion and process outgrowth were examined on murine Ln-1 (Englebreth-Holm-Swarm sarcoma laminin), human placental Ln-1 (human Ln-1[p]), human Ln-2 (merosin), human Ln-5 (kalinin/epiligrin/nicein), and human foreskin keratinocyte extracellular matrix extract (human HFK-ECM). Ln-5 was shown to evoke process outgrowth in amounts comparable to other Ln isoforms. Antibody perturbation experiments showed that adhesion and process outgrowth on murine Ln-1 was primarily mediated by the integrin alpha 1 beta 1, whereas adhesion and outgrowth on human Ln-5 and human HFK-ECM were mediated by alpha 3 beta 1. Adhesion to human Ln-1(p) and Ln-2 was not blocked by addition of anti-alpha 1 or anti-alpha 3 antibodies alone, but adhesion was partially perturbed when these antibodies were added in combination. Process outgrowth on human Ln-1(p) was blocked when either anti-alpha 3 or anti-beta 1 antibodies were added, indicating that alpha 3 beta 1 is the primary integrin heterodimer responsible for process extension on this substrate. These results demonstrate that Ln-5 and other Ln isoforms support comparable levels of adhesion and process outgrowth, but different integrin heterodimers, alone and in combination, are used by SY5Y cells to mediate responses.

REceptors in proximal tubular epithelial cells for tubulointerstitial nephritis antigen.

Tubulointerstitial nephritis antigen (TIN-ag) is a novel basement membrane macromolecule that is involved in human antitubular-basement-membrane-mediated tubulointerstitial nephritis. The presence of antibodies to TIN-ag may result in an alteration of proximal tubule epithelial cell interaction with surrounding matrix and contribute to the pathogenesis of immune-mediated tubulointerstitial disease. To study the adhesive interactions between TIN-ag and proximal tubule epithelial cells and the macromolecules that mediate these interactions, an immortalized proximal tubular epithelial cell line from normal adult human kidney (HK-2) was used. Plastic-coated TIN-ag was able to promote adhesion of HK-2 cells in a concentration-dependent manner. the strength of the adhesive interaction was comparable to that of type IV collagen or laminin. to explore which members of the integrin family of cell surface receptors were involved in this interaction, we performed fluorescence activated cell sorting (FACS) analysis and adhesion-inhibition studies using monoclonal antibodies against various integrins. Both approaches suggested that integrins alpha 3 beta 1 and alpha 5 beta 3 are crucial for the adhesion of proximal tubule epithelial cells on TIN-ag, and that they are probably using independent domains of TIN-ag for their action. These data will help us to understand the interactions between proximal tubule epithelial cells and the underlying basement membrane, and will provide tubule clues to the pathogenesis of kidney tubular diseases at the molecular level.

Glucose-induced alteration of integrin expression and function in cultured human mesangial cells.

Alteration in mesangial volume, due to an increase of the matrix surrounding mesangial cells, is a hallmark indicator of nephropathy in diabetes. Mesangial cells may also play a significant role in the development of nephropathy. Therefore, we examined the effect of glucose on the expression of integrins by cultured human mesangial cells and their ability to interact with collagen IV, a major component of the mesangial matrix. Human mesangial cells were grown in 5 and 25 mM glucose and their integrin profile was examined by immunoprecipitation and flow cytometry in each experimental condition. The results indicate that when mesangial cells were grown in 25 mM glucose, the expression of integrin subunit alpha 2, was increased, while the alpha 1 subunit was considerably decreased, as compared to cells grown in 5 mM glucose. Additionally, mesangial cells were tested for their ability to adhere to collagen IV in a solid-phase assay in the presence of neutralizing antibodies to integrin subunits. The results of these experiments indicate that both alpha 1 and alpha 2 complexed to beta 1 (alpha 2 beta 1 and alpha 1 beta 1) are major mesangial cell receptors for adhesion to collagen IV both in 5 and 25 mM glucose. The two receptors act in concert to mediate adhesion of mesangial cells to type IV collagen. When cell surface expression of the alpha 1 subunit in 25 mM glucose was reduced, the alpha 2 subunit was involved in adhesion to a greater extent than it was in 5 mM glucose. Immunoperoxidase histochemical studies localized both alpha 1 and alpha 2 integrin subunits in the mesangium of normal adult kidneys, suggesting that in vivo interaction with collagen IV could involve both of these receptors. These observations suggest that glucose-induced alterations in integrin expression may modify the ability of mesangial cells to interact with collagen IV.

Dermabrasive scar revision. Immunohistochemical and ultrastructural evaluation.

BACKGROUND: Dermabrasion of facial scars 4-8 weeks after injury frequently completely eliminates visible evidence of scar formation. However, efforts to define the cellular and structural mechanisms by which this phenomenon occurs have been limited in their success. OBJECTIVE: We investigated wound healing after dermabrasive scar revision. METHODS: The surgical scars of seven patients were abraded 6-8 weeks after injury. Comparative electron microscopic and immunohistochemical studies were performed on punch biopsy specimens taken before and after the dermabrasion. Ultrastructural changes in the basement membrane components and dermal structures were evaluated. Monoclonal antibody staining techniques were used to observe the presence, location, and temporal expression of tenascin, epiligrin, cadherins, and integrin subunits. RESULTS: We observed: 1) an increase in collagen bundle density and size with a tendency toward unidirectional orientation of fibers parallel to the epidermal surface, 2) an upregulation of tenascin expression throughout the papillary dermis, and 3) expression of alpha-6/beta-4 integrin subunit on the keratinocytes throughout the stratum spinosum. CONCLUSIONS: The mechanisms by which dermabrasive scar revision alters the events of primary cicatrix formation include modification of extracellular ligand expression, thereby influencing epithelial cell-cell interaction, and reorganization of connective tissue.

Selective assembly of laminin variants by human carcinoma cells.

BACKGROUND: The laminins are heterotrimeric basement membrane glycoproteins. Eight subunits that can be assembled into laminins have been characterized and are known as: A, B1, B2, S, M, K, B2t, B1k laminin chains. Although many neoplastic cells secrete laminins and some of them even assemble basement membranes, the pattern of production of various laminin subunits remains to be explored. EXPERIMENTAL DESIGN: The expression of laminin was examined in several human carcinoma cells using a panel of specific cDNA probes as well as polyclonal and chain specific monoclonal antibodies. For this purpose a human laminin S chain 2 kb cDNA was isolated and characterized and used together with existing probes for laminin chains. RESULTS: All carcinoma cell lines had a high level of expression of three light chains (B1, S and B2) mRNA. In contrast, the heavy chains of laminin, A and M, were expressed in negligible amounts as detected by Northern blotting and PCR. The only exception was the HU-1 lung adenocarcinoma cell line which expressed significant quantities of laminin M chain mRNA and lower levels of laminin A chain mRNA. The presence in the HU-1 cells of translated polypeptides was demonstrated by immunofluorescence staining. The cells contained both B1 and S chain laminin in the cell layer, but preferentially secreted the B1 chain into the culture supernatant as shown by Western blotting. The 300 to 400 kDa M chain immunoreactive band was found in laminin secreted into the culture medium of HU-1 cells. Immunoprecipitation of biosynthetically labeled proteins showed that the M chain was synthesized as a complex with B chains. Little or no A chain laminin was detected in the culture medium supernatant. HU-1 cells also synthesized the newly described laminin variant, epiligrin which was secreted into the medium. Thus, the HU-1 cells secreted two laminin variants: M-B1-B12 laminin and epiligrin into the culture medium. Immunostaining of HU-1 nude mice tumors showed that tumor basement membranes contained M, B1, and B2 laminin and epiligrin immunoreactivity but apparently no S chain. CONCLUSIONS: All human carcinoma cell lines produced laminin chains B1, B2 and S, but no or little A or M. The only exception was the lung carcinoma cell line HU-1. Human HU-1 carcinoma cells in culture synthesize several homologous laminin chains and regulate the process of assembly, secretion and deposition of laminin variants into tumor basement membranes. These data indicate that the tumor cells vary among themselves with regards to laminin production and that some of them, like HU-1 may produce essentially all laminin chains simultaneously.

Chlamydia trachomatis does not bind to alpha beta 1 integrins to colonize a human endometrial epithelial cell line cultured in vitro.

Chlamydia trachomatis is the leading cause of bacterially acquired sexually transmitted diseases in the United States and Europe. As an obligate intracellular pathogen, this bacterium must invade epithelial cells in order to survive and grow. Thus, multiple strategies probably exist for initial binding of chlamydiae to their target cells. Since a variety of bacteria have exploited integrins to colonize tissues, and a precedent existed for the involvement of extracellular matrix components in chlamydial attachment, this study first analyzed, by flow cytometry, integrins expressed by the human endometrial epithelial cell line HEC-1B. The genital cells were then exposed to monoclonal antibodies directed against those integrins and assayed for chlamydial attachment and inclusion development. Monoclonal antibodies bound to the alpha and/or beta 1 subunit of classic integrin receptors displayed by HEC-1B cells were not able to prevent colonization and infection of the epithelial cells by a genital isolate of C. trachomatis.

Activation of 72-kDa type IV collagenase/gelatinase by normal fibroblasts in collagen lattices is mediated by integrin receptors but is not related to lattice contraction.

The matrix metalloproteinase 72-kDa type IV collagenase (also known as gelatinase A) is thought to be involved in both normal connective tissue remodeling and invasive pathological processes. Like other matrix metalloproteinases, 72-kDa type IV collagenase is secreted by fibroblast monolayers as an inactive proenzyme, but is unique among this enzyme family in that it is not activated by serine proteinases such as plasmin. However, when fibroblasts are cultured in a collagen lattice, a situation thought to better approximate in vivo conditions, we have invariably found much of the secreted 72-kDa type IV collagenase in its enzymatically active 62-kDa form. Although collagen lattice contraction appeared to be required for the activation of 72-kDa type IV collagenase, we have found that the process of contraction can be dissociated from proenzyme activation. Both cytochalasin D and alpha-methylmannoside completely blocked lattice contraction, but not proenzyme activation. Furthermore, the monoclonal antibody M-13, which is directed against the beta 1 integrin chain, blocked collagen lattice contraction but not 72-kDa type IV procollagenase activation. At concentrations significantly higher than required to block lattice contraction or cell adhesion to collagen, M-13 was able to inhibit proenzyme activation. A second monoclonal antibody to the beta 1 integrin, P5D2, had little effect on collagen lattice contraction at low concentrations, but could significantly inhibit the activation of 72-kDa type IV procollagenase. Antibodies to the integrin alpha 2 chain also inhibited proenzyme activation. These data show that the activation of 72-kDa type IV collagenase proenzyme, like collagen lattice contraction, is mediated by beta 1 integrin receptors, possibly alpha 2 beta 1. Although both anti-beta 1 antibodies used are directed to the same site on the integrin chain, the fact that each antibody preferentially blocks a different event, either lattice contraction or activation of 72-kDa type IV collagenase, suggests the existence of branch points in the receptor-mediated signal transduction pathway.

Interferon-alpha restores normal adhesion of chronic myelogenous leukemia hematopoietic progenitors to bone marrow stroma by correcting impaired beta 1 integrin receptor function.

Treatment of chronic myelogenous leukemia (CML) with interferon-alpha frequently results in normalization of peripheral blood counts and, in up to 20% of patients, reestablishment of normal hematopoiesis. We hypothesize that interferon-alpha may restore normal adhesive interactions between CML progenitors and the bone marrow microenvironment and restore normal growth regulatory effects resulting from these progenitor-stroma interactions. We demonstrate that treatment with interferon-alpha induces a significant, dose-dependent increase in the adhesion of primitive long-term culture initiating cells and committed colony-forming cells (CFC) from CML bone marrow to normal stroma. Adhesion of CFC seen after interferon-alpha treatment could be inhibited by blocking antibodies directed at the alpha 4, alpha 5, and beta 1 integrins and vascular cell adhesion molecule, but not CD44 or intracellular adhesion molecule, suggesting that interferon-alpha induces normalization of progenitor-stroma interactions in CML. Because FACS analysis showed that the level of alpha 4, alpha 5, and beta 1 integrin expression after interferon-alpha treatment is unchanged, this suggests that interferon-alpha may restore normal beta 1 integrin function. Normalization of interactions between CML progenitors and the bone marrow microenvironment may then result in the restoration of normal regulation of CML progenitor proliferation, and explain, at least in part, the therapeutic efficacy of interferon-alpha in CML.

The decreased adhesion of Y79 retinoblastoma cells to extracellular matrix proteins is due to a deficit of integrin receptors.

PURPOSE: This study was designed to determine whether the Y79 retinoblastoma cell line, a prototype for retinoblastoma cells, exhibits differential adhesive properties toward extracellular matrix (ECM)/basement membrane (BM) proteins compared to normal human retinal (NHR) cells. A second goal was to determine whether differences in adhesion are related to differences in the expression of integrin subunits. METHODS: Y79 cells and NHR cells were tested for their ability to adhere and spread in microtiter wells adsorbed with the ECM/BM proteins laminin, fibronectin, and type IV collagen, as well as fragments of these proteins. The presence of cell surface integrins was determined by flow cytometry, using monoclonal antibodies (mAbs) against integrin subunits. For inhibition assays, cells were preincubated with mAbs against integrin subunits before the adhesion assays. RESULTS: NHR cells adhered to and spread on laminin, fibronectin, and type IV collagen, whereas Y79 cells only adhered moderately to fibronectin. NHR cells expressed high levels of beta 1, alpha 1, alpha 2, alpha 3, alpha 4, alpha 5, alpha 6, and alpha v integrin subunits, and they used these integrin subunits to adhere to all three ECM proteins. In contrast, Y79 cells expressed high levels of only the alpha 4 and beta 1 integrin subunits, used to adhere to fibronectin. CONCLUSIONS: Y79 cells have decreased adhesive capabilities toward ECM/BM proteins, compared to NHR cells. These differences can be attributed, in part, to their significantly lower levels of alpha 1, alpha 2, alpha 3, and alpha 5 integrin subunits, which serve as receptors for type IV collagen, laminin, and fibronectin.