Expression sequences and distribution of two primary cell adhesion molecules during embryonic development of Xenopus laevis

Studies of chicken embryos have demonstrated that cell adhesion molecules are important in embryonic induction and are expressed in defined sequences during embryogenesis and histogenesis. To extend these observations and to provide comparable evidence for heterochronic changes in such sequences during evolution, the local distributions of the neural cell adhesion molecule (N-CAM) and of the liver cell adhesion molecule (L-CAM) were examined in Xenopus laevis embryos by immunohistochemical and biochemical techniques. Because of the technical difficulties presented by the existence of multiple polypeptide forms of CAMs and by autofluorescence of yolk-containing cells, special care was taken in choosing and characterizing antibodies, fluorophores, and embedding procedures. Both N-CAM and L-CAM were found at low levels in pregastrulation embryos. During gastrulation, N-CAM levels increased in the presumptive neural epithelium and decreased in the endoderm, but L-CAM continued to be expressed in all cells including endodermal cells. During neurulation, the level of N-CAM expression in the neural ectoderm increased considerably, while remaining constant in non-neural ectoderm and diminishing in the somites; in the notochord, N-CAM was expressed transiently. Prevalence modulation was also seen at all sites of secondary induction: both CAMs increased in the sensory layer of the ectoderm during condensation of the placodes. During organogenesis, the expression of L-CAM gradually diminished in the nervous system while N-CAM expression remained high. In all other organs examined, the amount of one or the other CAM decreased, so that by stage 50 these two molecules were expressed in non-overlapping territories. Embryonic and adult tissues were compared to search for concordance of CAM expression at later stages. With few exceptions, the tissue distributions of N-CAM and L-CAM were similar in the frog and in the chicken from early times of development. In contrast to previous observations in the chicken and in the mouse, N-CAM expression was found to be high in the adult liver of Xenopus, whereas L-CAM expression was low. In the adult brain, N-CAM was expressed as three components of apparent molecular mass 180, 140, and 120 kD, respectively; in earlier stages of development only the 140-kD component could be detected. In the liver, a single N-CAM band appears at 160 kD, raising the possibility that this band represents an unusual N-CAM polypeptide. L-CAM appeared at all stages as a 124-kD molecule.(ABSTRACT TRUNCATED AT 400 WORDS)     

Significance of cell adhesion molecules, CD56/NCAM in particular, in human tumor growth and spreading

Cell adhesion molecules (CAM) represent a large group of cell surface protein moieties with distinctive biological functions. In physiological terms they ascertain cell to cell contact such as cell cohesion of epithelia, condition cell migration and transmigration via biological membranes such as blood vessel walls, provide means for homing cells in a new microenvironment etc. These features of CAM are exploited by tumor cells to grow and spread in a tumor bearing host. CD56/N-CAM antigen is 140 kD isoform of neural cell adhesion molecule. N-CAM belongs to the large Ig superfamily of CAMs. CD56 can be traced at various sites, including nervous tissue, neuro-muscular junctions, neuroendocrine and endocrine organs. It is well known as a differentiation antigen of natural killer (NK) cells. Its role and function are far from clear, but its adhesion properties are evident in cell-cell (homophilic) interactions. CD56 has been, however, demonstrated the cells various human malignancies. Tumors of the nervous system such as neuroblastoma, are well known to express this marker. Malignant lymphomas of T-NK cell origin bear CD56, as well as multiple myeloma, melanoma and some cancers of epithelial origin. These data suggest that CD56/N-CAM antigen is, in some unknown manner involved in tumor biology.     

Immunoelectron microscopic localization of the neural cell adhesion molecules L1 and N-CAM during postnatal development of the mouse cerebellum

The cellular and subcellular localization of the neural cell adhesion molecules L1 and N-CAM was studied by pre- and postembedding immunoelectron microscopic labeling procedures in the developing mouse cerebellar cortex. The salient features of the study are: L1 displays a previously unrecognized restricted expression by particular neuronal cell types (i.e., it is expressed by granule cells but not by stellate and basket cells) and by particular subcellular compartments (i.e., it is expressed on axons but not on dendrites or cell bodies of Purkinje cells). L1 is always expressed on fasciculating axons and on postmitotic, premigratory, and migrating granule cells at sites of neuron-neuron contact, but never at contact sites between neuron and glia, thus strengthening the view that L1 is not involved in granule cell migration as a neuron-glia adhesion molecule. While N-CAM antibodies reacting with the three major components of N-CAM (180, 140, and 120 kD) show a rather uniform labeling of all cell types, antibodies to the 180-kD component (N-CAM180) stain only the postmigratory granule cell bodies supporting the notion that N-CAM180, the N-CAM component with the longest cytoplasmic domain, is not expressed before stable cell contacts are formed. Furthermore, N-CAM180 is only transiently expressed on Purkinje cell dendrites. N-CAM is present in synapses on both pre- and post-synaptic membranes. L1 is expressed only preterminally and not in the subsynaptic membranes. These observations indicate an exquisite degree of fine tuning in adhesion molecule expression during neural development and suggest a rich combinatorial repertoire in the specification of cell surface contacts.     

Expression of cell-adhesion molecules in embryonic induction. II. Morphogenesis of adult feathers

The developmental appearance of cell-adhesion molecules (CAMs) was mapped during the morphogenesis of the adult chicken feather. Neural CAM (N-CAM), liver CAM (L-CAM), and neuron-glia CAM (Ng-CAM), as well as substrate molecules (laminin and fibronectin), were compared in newborn chicken skin by immunohistochemical means. N-CAM was found to be enriched in the dermal papilla, which was closely apposed to L-CAM-positive papillar ectoderm. The two CAMs were then co-expressed in cells of the collar epithelium. Subsequently generated barb epithelia expressed only L-CAM, but N-CAM reappeared periodically on cells between developing barbs and barbules. N-CAM first appeared on a single L-CAM-positive basilar cell located in each valley flanked by two adjacent barb ridges. Subsequently, the expression of N-CAM extended one cell after another to include the whole basilar layer. N-CAM also appeared in the L-CAM-positive axial-plate epithelia, beginning in a single cell located at the ridge base. The two collectives of N-CAM-positive epithelia constituting the marginal and axial plates then disintegrated, leaving interdigitating spaces between keratinized structures that had previously expressed L-CAM. The morphological transformation from an epithelial cylinder to a three-level branched feather pattern is thus achieved by coupling alternating CAM expression in linked cell collectives with specific differentiation events, such as keratinization. During all of these morphogenetic processes, laminin and fibronectin formed a continuous basement membrane separating pulp from feather epithelia, and were excluded from the sites involved in periodic appearances of N-CAM. The same staining pattern described for developing chickens persisted in the feather follicles of adult chicken tissue that have gone through several cycles of molting. Cyclic expression of the two different CAMs underlies each of the different morphological events that are generated epigenetically during feather morphogenesis.     

Patterns of N-CAM expression during myogenesis in Xenopus laevis

The neural cell adhesion molecule (N-CAM) is seen in the membrane of nerves and muscles from several vertebrate species. Using indirect immunofluorescence, we have examined the expression of this protein during embryonic and postembryonic myogenesis in the African clawed frog, Xenopus laevis. While good staining for N-CAM was seen in neuronal tissues at all stages examined, no staining of embryonic muscle was observed, including both mononucleated and polynucleated myoblasts. In contrast, limb muscles formed at metamorphosis showed strong expression of N-CAM. The developing limb muscles eventually lose their N-CAM, but will reexpress it dramatically when denervated. These observations suggest that myogenesis programs executed at different stages of development can display distinct patterns of N-CAM expression.     

Expression of neural cell adhesion molecule (N-CAM) in perisinusoidal stellate cells of the human liver

Neural cell adhesion molecule (N-CAM) is distributed in most nerve cells and some non-neural tissues. The present immunohistochemical study has revealed, for the first time, the expression of N-CAM in perisinusoidal stellate cells of the human liver. Liver specimens were stained with monoclonal antibody against human Leu19 (N-CAM) by a streptoavidin-biotin-peroxidase-complex method. Light- and electron-microscopic analyses have shown that N-CAM-positive nerve fibers are distributed in the periportal and intermediate zones of the liver lobule. Perisinusoidal stellate cells in these zones are also positive for N-CAM. N-CAM is expressed on the surface of the cell, including cytoplasmic projections. Close contact of N-CAM-positive nerve endings with N-CAM-positive stellate cells has been observed. On the other hand, stellate cells in the centrilobular zone exhibit weak or no reaction for N-CAM. Perivascular smooth muscle cells and fibroblasts in the portal area and myofibroblasts around the central veins are negative for N-CAM. The present results indicate that the perisinusoidal stellate cells in the periportal and intermediate zones of the liver lobule characteristically express N-CAM, unlike other related mesenchymal cells, and suggest that the intralobular heterogeneity of N-CAM expression by stellate cells is related to the different maturational stages of these cells.     

Functions and expression of liver N-CAM

In conclusion we have shown that: (a) liver N-CAM is a functionally active adhesion molecule, (b) its expression during the morphogenetic processes occurring during Xenopus laevis metamorphosis correlates with morphological changes in the cell-to-cell interaction; (c) Thyroxine is not directely involved in the activation of the expression of liver N-CAM.     

Expression of cell-adhesion molecules in embryonic induction. I. Morphogenesis of nestling feathers

The potential relationship of cell adhesion to embryonic induction during feather formation was examined by immunohistochemical analysis of the spatiotemporal distribution of three cell-adhesion molecules (CAMs), neural CAM (N-CAM), liver CAM (L-CAM), and neuron-glia CAM (Ng-CAM), and of substrate molecules (laminin and fibronectin) in embryonic chicken skin. The N-CAM found at sites of embryonic induction in the feather was found to be similar to brain N-CAM as judged by immuno-cross-reactivity, migratory position in PAGE, and the presence of embryonic to adult conversion. In contrast to the N-CAM found in the brain, however, only one polypeptide of Mr 140,000 was seen. N-CAM-positive dermal condensations were distributed periodically under L-CAM-positive feather placodes at those sites where basement membranes are known to be disrupted. After initiation of induction, L-CAM-positive placode cells became transiently N-CAM-positive. N-CAM was asymmetrically concentrated in the dorsal region of the feather bud, while fibronectin was concentrated in the ventral region. During feather follicle formation, N-CAM was expressed in the dermal papilla and was closely apposed to the L-CAM-positive papillar ectoderm, while the dermal papilla showed no evidence of laminin or fibronectin. The collar epithelium was both N-CAM- and L-CAM-positive. During the formation of the feather filament, N-CAM appeared periodically and asymmetrically on basilar cells located in the valleys between adjacent barb ridges. In contrast to the two primary CAMs, Ng-CAM was found only on nerves supplying the feather and the skin. These studies indicate that at each site of induction during feather morphogenesis, a general pattern is repeated in which an epithelial structure linked by L-CAM is confronted with periodically propagating condensations of cells linked by N-CAM.     

Expression of cell adhesion molecule E-cadherin in Xenopus embryos begins at gastrulation and predominates in the ectoderm

The expression of the Ca2+-dependent epithelial cell adhesion molecule E-cadherin (also known as uvomorulin and L-CAM) in the early stages of embryonic development of Xenopus laevis was examined. E-Cadherin was identified in the Xenopus A6 epithelial cell line by antibody cross-reactivity and several biochemical characteristics. Four independent mAbs were generated against purified Xenopus E-cadherin. All four mAbs recognized the same polypeptides in A6 cells, adult epithelial tissues, and embryos. These mAbs inhibited the formation of cell contacts between A6 cells and stained the basolateral plasma membranes of A6 cells, hepatocytes, and alveolar epithelial cells. The time of E-cadherin expression in early Xenopus embryos was determined by immunoblotting. Unlike its expression in early mouse embryos, E-cadherin was not present in the eggs or early blastula of Xenopus laevis. These findings indicate that a different Ca2+-dependent cell adhesion molecule, perhaps another member of the cadherin gene family, is responsible for the Ca2+-dependent adhesion between cleavage stage Xenopus blastomeres. Detectable accumulation of E-cadherin started just before gastrulation at stage 9 1/2 and increased rapidly up to the end of gastrulation at stage 15. In stage 15 embryos, specific immunofluorescence staining of E-cadherin was discernible only in ectoderm, but not in mesoderm and endoderm. The ectoderm at this stage consists of two cell layers. The outer cell layer of ectoderm was stained intensely, and staining was localized to the basolateral plasma membrane of these cells. Lower levels of staining were observed in the inner cell layer of ectoderm. The coincidence of E-cadherin expression with the process of gastrulation and its restriction to the ectoderm indicate that it may play a role in the morphogenetic movements of gastrulation and resulting segregation of embryonic germ layers.     

Differential contributions of Ng-CAM and N-CAM to cell adhesion in different neural regions

Individual neurons can express both the neural cell adhesion molecule (N-CAM) and the neuron-glia cell adhesion molecule (Ng-CAM) at their cell surfaces. To determine how the functions of the two molecules may be differentially controlled, we have used specific antibodies to each cell adhesion molecule (CAM) to perturb its function, first in brain membrane vesicle aggregation and then in tissue culture assays testing the fasciculation of neurite outgrowths from cultured dorsal root ganglia, the migration of granule cells in cerebellar explants, and the formation of histological layers in the developing retina. Our strategy was initially to delineate further the binding mechanisms for each CAM. Antibodies to Ng-CAM and N-CAM each inhibited brain membrane vesicle aggregation but the binding mechanisms of the two CAMs differed. As expected from the known homophilic binding mechanism of N-CAM, anti-N- CAM-coated vesicles did not co-aggregate with uncoated vesicles. Anti- Ng-CAM-coated vesicles readily co-aggregated with uncoated vesicles in accord with a postulated heterophilic binding mechanism. It was also shown that N-CAM was not a ligand for Ng-CAM. In contrast to assays with brain membrane vesicles, cellular systems can reveal functional differences for each CAM reflecting its relative amount (prevalence modulation) and location (polarity modulation). Consistent with this, each of the three cellular processes examined in vitro was preferentially inhibited only by anti-N-CAM or by anti-Ng-CAM antibodies. Both neurite fasciculation and the migration of cerebellar granule cells were preferentially inhibited by anti-Ng-CAM antibodies. Anti-N-CAM antibodies inhibited the formation of histological layers in the retina. The data on perturbation by antibodies were correlated with the relative levels of expression of Ng-CAM and N-CAM in each of these different neural regions. Quantitative immunoblotting experiments indicated that the relative Ng-CAM/N-CAM ratios in comparable extracts of brain, dorsal root ganglia, and retina were respectively 0.32, 0.81, and 0.04. During culture of dorsal root ganglia in the presence of nerve growth factor, the Ng-CAM/N-CAM ratio rose to 4.95 in neurite outgrowths and 1.99 in the ganglion proper, reflecting both polarity and prevalence modulation. These results suggest that the relative ability of anti-Ng-CAM and anti-N-CAM antibodies to inhibit cell-cell interactions in different neural tissues is strongly correlated with the local Ng-CAM/N-CAM ratio.(ABSTRACT TRUNCATED AT 400 WORDS)     

Selective expression of the 180-kD component of the neural cell adhesion molecule N-CAM during development

The rodent neural cell adhesion molecule (N-CAM) consists of three glycoprotein chains of 180, 140, and 120 kD in their adult forms. Although the proportions of the three components are known to change during development and differ between brain regions, their individual distribution and function are unknown. Here we report studies carried out with a monoclonal antibody that specifically recognizes the 180-kD component of mouse N-CAM (N-CAM180) in its highly sialylated embryonic and less glycosylated adult forms. In primary cerebellar cell cultures, N-CAM180 antibody reacts intracellularly with all types of neural cells including astrocytes, oligodendrocytes, and neurons. During cerebellar, telencephalic, and retinal development N-CAM180 is detectable by indirect immunohistology in differentiated neural cells, but, in contrast to total N-CAM, not in their proliferating precursors in the ventricular zone and primordial and early postnatal external granular layer. In monolayer cultures of C1300 neuroblastoma cells, N-CAM180 appears by immunofluorescence more concentrated at contact points between adjacent cells, while N-CAM comprising the 180- and 140-kD component shows a more uniform distribution at the plasma membrane. Treatment of neuroblastoma cells with dimethylsulfoxide, which promotes differentiation, induces a shift toward the predominant expression of N- CAM180. These observations support the notion that N-CAM180 is expressed selectively in more differentiated neural cells and suggest a differential role of N-CAM180 in the stabilization of cell contacts.     

Overexpression of Fyn tyrosine kinase causes abnormal development of primary sensory neurons in Xenopus laevis embryos

The expression and function of the Src family protein tyrosine kinase Fyn in Xenopus laevis embryos have been examined. In situ hybridization analysis demonstrated nervous system-specific expression of Fyn mRNA in tail-bud embryos. However, a class of primary sensory neurons; that is, Rohon-Beard (RB) neurons, which is positive for immunoglobulin superfamily cell adhesion molecules (CAM), neural cell adhesion molecule (N-CAM) and contactin, is devoid of Fyn expression. Injection of Fyn mRNA into one of the blastomeres at the 2-cell stage led to overexpression of Fyn in the injected half of the tail-bud embryos. Immunolabeling of the embryos with anti-HNK-1 antibody revealed that the peripheral axons of RB neurons were partially misguided and bound to each other to form abnormal subcutaneous fascicles. Similar abnormality was induced by injection of the Fyn overexpression vector. The incidence of abnormality appeared dose-dependent, being 68-92% of the injected embryos at 50-400 pg of mRNA. Co-injection of the contactin antisense vector depleted contactin mRNA accumulation without affecting Fyn overexpression and reduced the incidence of the abnormal RB-cell phenotype. However, the N-CAM antisense was ineffective in reducing this abnormality. These results suggest that Fyn can modify signals regulating axonal guidance or fasciculation in the developing X. laevis nervous system and that contactin may affect this action of Fyn.     

Expression of the cell adhesion molecules, L-CAM and N-CAM during avian scale development

To examine the involvement of cell adhesion molecules in the inductive epithelial-mesenchymal interactions during avian scale development, a study of the spatiotemporal distribution of L-CAM and N-CAM was undertaken. During scutate scale development, L-CAM and N-CAM are expressed together in cells of the transient embryonic layers destined to be lost at hatching. The ongoing linkage of the cells of these layers by both CAMs sets them apart, early in development, as unique cell populations. L-CAM and N-CAM were also expressed simultaneously at the basal surface of the early germinative cells where signal transduction is presumed to occur. In spite of the differences in cell shape, adhesion, density and proliferative state between populations of epidermal placode and interplacode cells, the expression of L-CAM and N-CAM appeared to be uniform and nondiscriminating for these discrete cell lineages. The same pattern of L-CAM and N-CAM expression was observed during morphogenesis of reticulate scales that develop without placode formation. While L-CAM and N-CAM are present during the early stages of scale development and most likely function in cell adhesion, the data do not support a role for these adhesion molecules in the formation of the morphogenetically critical placode and interplacode cell populations. In both scale types, L-CAM became predominantly epithelial, and N-CAM became predominantly dermal as histogenesis occurred. Initially, N-CAM was concentrated near the basal lamina where it may be involved in the reciprocal epidermal-dermal interactions required for morphogenesis. However, as development of the scales progressed, N-CAM disappeared from the tissues. L-CAM expression continued in the epidermis and was intense on all suprabasal cells undergoing differentiation into either an alpha-stratum or beta-stratum. However, L-CAM was more prevalent on the basal cells of alpha-keratinizing regions than on the basal cells of beta-keratinizing regions.     

Comparison of two cell-adhesion molecules, uvomorulin and cell-CAM 105

Two cell adhesion molecules, cell-CAM 105 and uvomorulin (UM), were compared by analysing their antigenic structures, their activity in cell aggregation assays and their expression in various tissues. Cell-CAM 105 is a membrane glycoprotein which mediates the intercellular adhesion of reaggregating rat hepatocytes, and UM was first described to be involved in the compaction of preimplantation mouse embryos and embryonal carcinoma cells. UM is not only expressed during embryonic development but also in various adult tissues including liver, epithelia of lung, gut, kidney and uterus. A similar distribution for UM was found in rat tissues on cell types where cell-CAM 105 is known to be present. Our studies show that (i) cell-CAM 105 and UM are distinct and different proteins; (ii) uvomorulin is involved in the compaction of rat preimplantation embryos but Fab anti-UM has no effect on reaggregating rat hepatocytes, where Fab anti-cell CAM is effective; (iii) distribution studies show that UM is expressed on a broader range of epithelial cells while cell-CAM 105 is more restricted to hepatocytes and simple epithelia. In cases where both cell adhesion molecules are expressed on the same cell types they can be localized to different parts of the cell surface.     

Clusters of neural cell adhesion molecule at sites of cell-cell contact

I have examined the distribution of neural cell adhesion molecule (N-CAM) in cultured C2 myogenic cells and other cell lines to determine if N-CAM accumulates at sites of cell-cell contact. C2 cells growing in log phase display large clusters of neural cell adhesion molecule where they contact each other. These clusters are remarkably stable, do not form at cell-substrate contacts, and appear not to be enriched in a number of other cytoskeletal, membrane, or extracellular proteins. Thus, N-CAM clusters form preferentially in response to cell-cell contact and are specifically enriched in N-CAM. As C2 cultures mature and differentiate, clusters persist at contacts between aligning myoblasts and between myotubes, consistent with a role in myogenesis. N-CAM is also enriched at cell-cell contacts in cultures of PC12, NRK, and CHO cells. These cells have significant amounts of N-CAM as detected on immunoblots. Clusters are not seen in L929 cells, which do not have detectable amounts of N-CAM. Coculture of these cells with C2 cells results in the clustering of N-CAM at heterologous contacts between C2 cells and NRK, CHO, or PC12 cells, but not between C2 cells and L929 cells. These results suggest that N-CAM specifically accumulates where N-CAM-bearing cells contact one another. Clustering of N-CAM may be an important step in strengthening intercellular adhesion.     

Expression of the adhesion molecules L1, N-CAM and J1/tenascin during development of the murine small intestine

We have previously studied the immunohistological localization of the three adhesion molecules L1, N-CAM and J1/tenascin in adult mouse small intestine and shown that L1 expression in epithelial crypt cells underlies the adhesion of these cells to one another [63]. To obtain further insight into the functional roles of L1, N-CAM and J1/tenascin in this organ we studied their expression starting at embryonic day 14 during embryonic and early postnatal morphogenesis and during epithelial cell migration in the adult. Expression of L1 was restricted to neural cells until approximately postnatal day 5, when L1 started to be detectable on crypt but not on villus cells, predominantly on the basolateral membrane infoldings. As in brain, L1-specific mRNA was approximately 6 kb in size. L1 from intestine appears to differ from the brain-derived equivalent in possessing a higher level of glycosylation. N-CAM was detectable from embryonic day 14 onward in neural and also in mesenchymal cells. Expression by smooth muscle cells decreased during development. In the villus core, N-CAM was strongly detectable at contact sites between smooth muscle cells forming the cellular scaffold of the villus. From embryonic day 14 onward, N-CAM appeared in both 180- and 140-kDa forms. J1/tenascin was present in both neural and mesenchymal cells from embryonic day 14 onward. Starting at embryonic day 17, J1/tenascin appeared concentrated at the boundary between mesenchyme and epithelium in an increasing gradient from the crypt base to the villus top. From embryonic day 14 onward J1/tenascin consisted of the 190- and 220-kDa components. J1/tenascin from intestine differed from brain-derived J1 in its carbohydrate composition. These observations show that the three adhesion molecules are expressed by distinct cell populations and may serve as cell-type-specific markers in pathologically altered intestinal tissue.     

Neural cell adhesion molecule expression is regulated by Schwann cell- neuron interactions in culture

To investigate the cellular and molecular signals underlying regulation of cell adhesion molecule expression, the influence of interactions between dorsal root ganglion neurons and Schwann cells on their expression of L1 and N-CAM was quantitated by immunogold electronmicroscopy. The numbers of antibody binding sites on cell surfaces of neurons and glia were compared between pure populations and co-cultures. After 3 d of co-culture, expression of L1 was reduced by 91% on Schwann cells and 36% on neurons, with expression in pure cultures being taken as 100%. N-CAM expression was unchanged on neurons and reduced by 43% on Schwann cells. Within 3 d after removal of neurons from Schwann cell-neuron co-cultures by immunocytolysis, expression of L1 and N-CAM on Schwann cell surfaces increased by 69 and 84%, respectively. Cell surface antigens recognized by an antibody to mouse liver membranes were unchanged in co-cultures. Furthermore, in co-cultures of neurons and sciatic nerve fibroblasts neither of the three antibodies detected any changes in expression of antigens when pure and co-cultures were compared. These observations suggest that adhesion molecules are not only involved in neuron-Schwann cell recognition and neurite outgrowth on Schwann cells (Seilheimer, B., and M. Schachner. 1988. J. Cell Biol. 107: 341-351), but that cell interactions, in turn, modulate the extent of adhesion molecule expression.