MDCK cysts: An in vitro model of epithelial cyst formation and growth

Summary MDCK cells dissociated from monolayer culture were either dispersed within medium-hydrated collagen gel or seeded atop a collagen substrate which was immediately overlaid with collagen gel. Individual cells exhibited clonal growth in three dimensions to form spherical cysts in which a simple epithelium surrounded a fluid-filled lumen. The cells of MDCK cysts were polarized with apical surface bordering the lumen. MDCK cysts increased in diameter with continued culture. Maximum cyst size was dependent on seeding density and was influenced by medium composition. MDCK cysts could be isolated from the collagen substrate by digestion with collagenase. Also, collagen gel could be dissected from the cyst wall to give unrestricted access to regions of the basolateral cell surface. This novel method of renal cell culture provides a study system to model the influence of the extracellular matrix on kidney epithelial cell structure and function. It also offers an in vitro model of general application to the study of epithelial cyst formation and growth.     

Bioadhesive hydrogel microenvironments to modulate epithelial morphogenesis

Epithelial cells polarize and differentiate into organotypic cell aggregates in response to cell-cell and cell-matrix interactions. For example, Madin-Darby Canine Kidney (MDCK) cells form spherical cell aggregates (cysts) with distinct apical and basolateral polarity when cultured three dimensionally (embedded) in type I collagen gels. To investigate the effects of individual extracellular factors on epithelial morphogenesis, we engineered fast degrading protease-responsive polyethylene glycol (PEG) hydrogels functionalized with controlled densities of various bioligands (RGD peptide, laminin-1 (LN)) to allow 3D culturing of MDCK cells, cyst expansion, and morphogenesis/polarization. Cysts formed after 15 days of culture in these hydrogels were analyzed with multiphoton fluorescence microscopy for markers of apical and basolateral membrane domains. Epithelial cysts formed in bioadhesive ligand-functionalized PEG gels exhibited a higher frequency of central lumen and interior apical pole formation as well as basolateral polarization compared to those of unmodified PEG hydrogels. These results demonstrate that incorporation of specific bioadhesive motifs into synthetic hydrogels provides 3D culture environments that support epithelial morphogenesis. These microenvironments provide a flexible and controlled system for systematic investigations into normal and pathologic morphogenic behaviours as well as synthetic environments for promoting tissue morphogenesis for regenerative medicine applications.     

Explant culture of human polycystic kidney

Autosomal dominant polycystic kidney disease is characterized by the formation of large fluid-filled epithelial cysts. To obtain renal cyst wall epithelium for in vitro study, we employed an explant culture technique using medium-hydrated collagen gel as the culture substrate. Pieces of excised cyst wall were submerged within collagen gel. Cells of the cyst lining migrated to form a polarized epithelium at the surface of the surrounding collagen gel. Regions of the outgrowth were isolated by microdissection and used as a source of cells for subculture. Cyst-derived epithelium retained the ultrastructural features of the renal cyst of origin. The cells were cuboidal and bore short apical microvilli. Cells often were joined by apical tight junctions. Intercellular channels were narrow and bordered by short microvillus projections at the basolateral membrane. Epithelial cells rested on a densely staining basal lamina. The explants commonly developed small solitary cysts within their wall that were filled with fluid. These mural cysts were lined by a simple epithelium morphologically similar to the cells that lined the explant. Explantation of human renal cyst wall to culture within collagen gel provides a reproducible method to isolate autosomal dominant polycystic kidney disease epithelium for subculture. This method offers an alternative to the use of proteolytic enzymes to establish morphologically stable cultures of cyst lining cells for use in experimental renal cystic disease.     

Effects of src kinase and TGFbeta1 on the differentiation and morphogenesis of MDCK cells grown in three-dimensional collagen and Matrigel environments.

This study attempted to analyse in detail the effect of src kinase on the growth and differentiation of MDCK cells in different extracellular matrix (ECM) environments. A method was developed to label the membrane proteins in situ and the distribution of cytoskeletal and junctional proteins was visualized in three-dimensional cell complexes, using optical sections generated by confocal microscopy. Independently of the ECM, non-transformed MDCK cells formed differentiated cell cysts with one or a few lumina, with the apical side facing the lumen; ZO-1 was expressed at the tight junctions close to the apical side and beta-catenin, E-cadherin and fodrin along the entire lateral walls. The phenotype of src kinase activated MDCK cells was strongly dependent on the ECM and varied from an irregular cluster in collagen I, to tubular structures in laminin or proteoglycans, and finally to a polarized cell cyst in Matrigel. In collagen I, E-cadherin and beta-catenin were seen partially along the lateral walls and partially in the cytoplasm of src-transformed MDCK cells; fodrin was released into the cytoplasm and ZO-1 was not visualized. When the src-transformed cells were cultivated in Matrigel, their junctional proteins were recruited to the cell membranes and ZO-1 reappeared at the apical face. Thus, the components of Matrigel could overcome the deleterious effect of src on the polarity of MDCK cells. TGFbeta1, together with its receptors and other soluble factors in Matrigel, were responsible for the induction of differentiation. The results show that tyrosine phosphorylation sensitizes the epithelial MDCK cells to ECM and TGFbeta1.     

Epithelial cell polarity affects susceptibility to Pseudomonas aeruginosa invasion and cytotoxicity.

Intact tissues are relatively resistant to Pseudomonas aeruginosa-induced disease, and injury predisposes tissue to infection. Intact epithelia contain polarized cells that have distinct apical and basolateral membranes with unique lipids and proteins. In this study, the role of cell polarity in epithelial cell susceptibility to P. aeruginosa virulence mechanisms was tested. Madin-Darby canine kidney (MDCK) cells, human corneal epithelial cells, and primary cultures of two different types of airway epithelial cells were grown on Transwell filters or in plastic tissue culture wells. P. aeruginosa invasion of cells was quantified by gentamicin survival assays with two isolates that invade epithelial cells (6294 and PAO1). Cytotoxic activity was assessed by trypan blue exclusion assays with two cytotoxic strains (6206 and PA103). Basolateral surfaces of cells were exposed by one of two methods: EGTA pretreatment of epithelial cells or growth of cells in low-calcium medium. Both methods of exposing basolateral membranes increased epithelial cell susceptibility to P. aeruginosa invasion and cytotoxicity. Migrating cells were also found to be more susceptible to P. aeruginosa invasion than confluent monolayers that had established membrane polarity. Monolayers of MDCK cells that had been selected for resistance to killing by concanavalin A were resistant to both cytotoxicity and invasion by P. aeruginosa because they were more efficiently polarized for their susceptibility to P. aeruginosa virulence factors than regular MDCK cells and not because they were defective in glycosylation. These results suggest that there are factors on the basolateral surfaces of epithelial cells that promote interaction with P. aeruginosa or that there are inhibitory factors on the apical cell surface. Thus, cell polarity of intact epithelia is likely to contribute to defense against P. aeruginosa infection.     

Patterned entry and egress by Epstein-Barr virus in polarized CR2-positive epithelial cells

In polarized epithelium direction of viral entry and release correlates with proclivity of a virus to establish local versus systemic infection. The Epstein-Barr virus (EBV), whose principal tissue reservoir is B lymphocytes, also has disease manifestations in epithelium, suggesting intertissue spread potentially influenced by epithelial cell polarity. We stably transfected the B lymphocyte EBV receptor (CR2/CD21) into Madin-Darby canine kidney (MDCK) epithelial cells used extensively to study effects of cell polarity on infection by both DNA and RNA viruses. CR2/CD21 was detected on both apical and basolateral surfaces of polarized MDCK cells, with predominant expression basolaterally. However, infectivity was up to four-fold greater apically, suggesting that endogenous cell surface molecules, sorted asymmetrically onto polarized plasma membranes, may be involved in EBV entry into MDCK cells. EBV gp350/220, a replicative cycle glycoprotein added to the virus envelope on egress through the cell membrane, was immunolocalized by confocal microscopy to basolateral cell surfaces only. Apical entry of EBV with subsequent basolateral release of newly replicated virus favors systemic infection by viral dissemination to underlying lymphocytic aggregations. Under conditions of long-term culture, latent EBV was not stably maintained in these cells, suggesting that the epithelial phase of acute EBV infection may be transient.     

Vectorial function of major histocompatibility complex class II in a human intestinal cell line.

This study explores the expression and the function of major histocompatibility complex class II in the intestinal epithelial cell line CaCo-2, which has been widely used as a model for the human gastrointestinal epithelium. Human leucocyte antigen (HLA)-DR expression on CaCo-2 cells is induceable by interferon-gamma (IFN-gamma), but responsiveness to IFN-gamma is dependent on cell differentiation and IFN-gamma availability at the basolateral cell surface. HLA-DR expression is concentrated in apical cytoplasmic vesicles and on the basolateral cell surface. Invariant chain is expressed in apical vesicles but is absent from the cell surface. Immunoprecipitation studies show a slow rate of dissociation of HLA-DR from Ii. Double labelling shows some overlap between HLA-DR expression and basolateral endosomal markers but no overlap with apical endosomal markers. Functional studies show processing and presentation of lysozyme endocytosed from the basolateral, but not apical surfaces. CaCo-2 cells may provide a useful model with which to dissect the antigen-processing pathways in polarized epithelial cells. The regulated access of antigens taken up from the gut lumen to the processing compartments may prevent overloading the immune system with antigens derived from normal gut contents.     

Sensitivity of polarized epithelial cells to the pore-forming toxin aerolysin

Aerolysin is one of the major virulence factors produced by Aeromonas hydrophila, a human pathogen that produces deep wound infection and gastroenteritis. The toxin interacts with target mammalian cells by binding to the glycan core of glycosylphosphatidyl inositol (GPI)-anchored proteins and subsequently forms a pore in the plasma membrane. Since epithelial cells of the intestine are the primary targets of aerolysin, we investigated its effect on three types of polarized epithelial cells: Caco-2 cells, derived from human intestine; MDCK cells, a well-characterized cell line in terms of protein targeting; and FRT cells, an unusual cell line in that it targets its GPI-anchored proteins to the basolateral plasma membrane in contrast to other epithelial cells, which target them almost exclusively to the apical surface. Surprisingly, we found that all three cell types were sensitive to the toxin from both the apical and the basolateral sides. Apical sensitivity was always higher, even for FRT cells. In contrast, FRT cells were more sensitive from the basolateral than from the apical side to the related toxin Clostridium septicum alpha-toxin, which also binds to GPI-anchored proteins but lacks the lectin binding domain found in aerolysin. These observations are consistent with the notion that a shuttling mechanism involving low-affinity interactions with surface sugars allows aerolysin to gradually move toward the membrane surface, where it can finally encounter the glycan cores of GPI-anchored proteins.     

Coordinate regulation of Salmonella enterica serovar Typhimurium invasion of epithelial cells by the Arp2/3 complex and Rho GTPases

Salmonella enterica serovar Typhimurium can infect epithelial cells via the basolateral surface after breaching the intestinal epithelium, yet little is known about this process. Here, we show that actin polymerization driven by the Arp2/3 complex is critical to both basolateral and apical bacterial invasion of polarized MDCK cells. While there is also a dependence upon toxin B-sensitive Rho GTPases, none of the four GTPases known to be activated by S. enterica serovar Typhimurium SopE are individually required for basolateral internalization. These results underscore that the specific factors required for Salmonella invasion differ between membrane domains of polarized epithelia.     

Ric‐8A,an activator protein of Gαi,controls mammalian epithelial cell polarity for tight junction assembly and cystogenesis

Correct cyst morphogenesis of epithelial cells requires apical–basal polarization, which is partly regulated by mitotic spindle orientation, a process dependent on the heterotrimeric G protein subunit Gαi and its binding protein LGN. Here, we show that in three‐dimensional culture of mammalian epithelial Madin–Darby canine kidney (MDCK) cells, the Gαi‐activating protein Ric‐8A is crucial for orientation of the mitotic spindle and formation of normal cysts that comprise a single layer of polarized cells with their apical surfaces lining an inner lumen. Consistent with the involvement of LGN, cystogenesis can be well organized by ADP‐ribosylated Gαi, retaining the ability to interact with LGN, but not by the interaction‐defective mutant protein Gαi2 (N150I). In monolayer culture of MDCK cells, functional tight junction (TJ) assembly, a process associated with epithelial cell polarization, is significantly delayed in Ric‐8A‐depleted cells as well as in Gαi‐depleted cells in a mitosis‐independent manner. Ric‐8A knockdown results in a delayed cortical delivery of Gαi and the apical membrane protein gp135, and an increased formation of intercellular lumens surrounded by membranes rich in Gαi3 and gp135. TJ development also involves LGN and its related protein AGS3. Thus, Ric‐8A regulates mammalian epithelial cell polarity for TJ assembly and cystogenesis probably in concert with Gαi and LGN/AGS3.     

Vectorial secretion of perivitelline membrane glycoprotein ZPC of Japanese quail (Coturnix japonica) in polarized Madin-Darby canine kidney cells

The avian perivitelline membrane (PL), which is an investment homologous to the mammalian zona pellucida, is found between the surface of the oocyte and the apical surface of ovarian granulosa cells. Our previous study demonstrated that ZPC, one of the components of PL, is synthesized in ovarian granulosa cells. However, how the secretion of ZPC is regulated in the cells has been insufficiently investigated. We studied the secretion of quail ZPC expressed in polarized Madin-Darby canine kidney (MDCK) cells in a dual-chamber apparatus. Western blot analyses of the conditioned medium demonstrated that the majority of the secreted ZPC were distributed in the apical compartment. When ZPC lacking N-linked oligosaccharides was transfected into the cells, the 31-kDa immunoreactive band was detected in both the apical and the basolateral medium. Interestingly, immunohistochemical observations of the follicular wall demonstrated that the predominant intracellular form of ZPC in the cells localized in the apical side of the perinuclear region apposed to the PL, but not the basolateral side, indicating the possibility that ZPC could be selectively transported toward the apical surface in vivo. Taken together, these results indicated that ZPC expressed in MDCK cells are selectively released to the apical compartment, and that the N-linked carbohydrates might possess information that causes the efficient transport of ZPC to the apical surface of the cells.     

Secretion of rubella virions and virus-like particles in cultured epithelial cells.

Rubella virus (RV) is an enveloped RNA virus that causes systemic infections in humans. More importantly, first trimester in utero infection leads to a collection of devastating birth defects known as congenital rubella syndrome. Epithelial cells are the first line of defense against viruses and consequently, the polarity of virus secretion is an important factor affecting viral spread. As a first step toward understanding how RV interacts with epithelial cells, we have examined the release of RV-like particles and virions from polarized cells in culture. RV structural proteins were targeted to the Golgi complex and virus particle formation occurred on intracellular membranes in three different polarized epithelial cells. Polarized cells could be infected from the apical and basal membranes, indicating that receptors are not confined to one surface. The secretion of virus-like particles and infectious virions varied according to cell type. In two of the three polarized cell lines examined, virus was released primarily from the apical surface, but significant quantities were also secreted from the basolateral membrane. Release of virus from the apical surface may facilitate virus spread from person to person, whereas basolateral secretion could be important for establishing a systemic infection and/or crossing the placenta prior to fetal infection.     

Ecotropic murine leukemia virus envelope protein affects interaction of cationic amino acid transporter 1 with clathrin adaptor protein complexes, leading to receptor downregulation

Mouse cationic amino acid transporter 1 (mCAT1) serves as the receptor for ecotropic murine leukemia virus (eMuLV). It has been shown that mCAT1 is expressed on the basolateral surface of polarized epithelial MDCK cells. However, little is known about the mechanisms involved in the intracellular trafficking of mCAT1. Using the green fluorescent protein-tagged mCAT1 expressed in MDCK cells, we report here that mCAT1 is physically associated with clathrin adaptor protein complex 1 (AP-1) implicated in protein trafficking from trans-Golgi network (TGN) to the basolateral surface. When the cells were infected with eMuLV, reduction of cell surface mCAT1, as well as a concomitant decrease in mCAT1-AP-1 association, was observed while association of mCAT1 with AP-3 involved in the TGN-to-lysosome trafficking was increased. Similar results were obtained when eMuLV envelope protein alone was expressed. The results may provide useful insights into the mechanism by which a simple retrovirus downregulates its receptor.     

Distinct transport characteristics of basolateral peptide transporters between MDCK and Caco-2 cells

The cellular polarity of [14C]glycylsarcosine (Gly-Sar) transport in Madin-Darby canine kidney (MDCK) cells was compared with that in the human intestinal cell line Caco-2. In MDCK cells, [14C]Gly-Sar accumulation was greater at the basolateral side than at the apical side, and there was little net transcellular transport. In Caco-2 cells, [14C]Gly-Sar accumulation was greater at the apical side and unidirectional transcellular transport occurred from the apical to basolateral side. Efflux of [14C]Gly-Sar from MDCK cells to either side was negligible, whereas that from Caco-2 cells was significantly faster to the basolateral side. The basolateral peptide transporter in MDCK cells possessed a similar substrate specificity, but a much higher substrate affinity, than that in Caco-2 cells. The basolateral peptide transporter in MDCK cells did not accumulate Gly-Sar during hypertonic stress. These findings indicate that the basolateral peptide transporter in MDCK cells is involved in the cellular uptake of small peptides, but not in the extrusion of small peptides to the extracellular space.     

Primary culture of rabbit gallbladder epithelial cells in collagen gel matrix

Primary culture of gallbladder epithelial cells obtained from normal rabbits was attempted in collagen gel matrix for up to 6 weeks. Fluid medium containing 0.02% ethylenediaminetetraacetic acid and 0.25% trypsin was poured into the gallbladder lumen. The pellets obtained by centrifugation of recovered fluid contained many isolated epithelial cells and a few small cell clumps. These pellets were dispersed and embedded inside collagen gel matrix and cultured in William's medium E supplemented with fetal calf serum and epidermal growth factor. The three-dimensional outgrowth from individual cells and small cell clumps consisted predominantly of spherical cystic masses 2-4 days later. These cysts contained mucin and were covered by a single layer of cuboidal or low columnar epithelial cells. Electron microscopy revealed the epithelial arrangement of cells lining the cyst walls, and these cells were similar to gallbladder epithelial cells in vivo. These epithelial cells showed active mucin secretion into the cystic cavities. Cytokeratin was diffusely present in the cytoplasm. This isolation and culture system provides a reproducible and consistent method for sustained growth of normal gallbladder epithelial cells from normal tissue in primary culture and seems valuable for investigating pathologic conditions of the gallbladder.     

Significance of basolateral domain of polarized MDCK cells for Sendai virus-induced cell fusion

Summary Fusion (fusion from within) of polarized MDCK monolayer cells grown on porous membranes was examined after infection with Sendai viruses. Wild-type virus, that buds at the apical membrane domain, did not induce cell fusion even when the F glycoprotein expressed at the apical domain was activated with trypsin. On the other hand, a protease activation mutant, F 1-R, with F protein in the activated form and that buds bipolarly at the apical and basolateral domains, caused syncytia formation in the absence of exogenous protease. Anti-Sendai virus antibodies added to the basolateral side, but not at the apical side, inhibited cell fusion induced by F 1-R. In addition, T-9, a mutant with bipolar budding phenotype of F 1-R but with an uncleavable F protein phenotype like wild-type virus, induced cell fusion exclusively when trypsin was added to the basolateral medium. By electron microscopy, cell-to-cell fusion was shown to occur at the lateral domain of the plasma membrane. These results indicate that in addition to proteolytic activation of the F protein, basolateral expression of Sendai virus envelope glycoproteins is required to induce cell fusion.     

Influenza A virus hemagglutinin containing basolateral localization signal does not alter the apical budding of a recombinant influenza A virus in polarized MDCK cells

Morphogenesis of influenza virus is a complex multistep process involving transport of all viral components as either individual or subviral components to the specified assembly site and interaction among the viral components in an ordered fashion to initiate the budding process. Envelope glycoprotein(s) is believed to be the major determinant in selecting the viral budding site since the majority of the viral glycoproteins are directed to the budding site independent of other viral components. Influenza viruses bud from the apical surface of polarized epithelial cells and all three envelope proteins, hemagglutinin (HA), neuraminidase (NA), and M2, are also targeted independently to the apical surface. Since HA is the major viral envelope protein, we decided to test whether basolaterally expressed HA can make the virus bud from the basolateral surface. Accordingly, we introduced the tyrosine-based basolateral-sorting signal to the cytoplasmic tail of HA by changing Cys561 --> Tyr561 and generated a transfectant virus by reverse genetics. Compared to the parent WSN virus, the mutant virus (HAtyr virus) contained less HA on its envelope. While the wild-type (wt) HA was >95% apical, the mutated HA (HAtyr) was approximately 60% basolateral in both transfected and virus-infected polarized MDCK cells. Also, HAtyr protein exhibited a much higher rate of endocytosis than the wt HA, in both apical and basolateral surface of transfected as well as virus-infected cells. However, the HAtyr virus, similar to wt WSN virus, was seen to bud almost exclusively (>99%) from the apical side of polarized MDCK cells. This finding was confirmed by using neuraminidase to facilitate virus release, by treating the collected virus particles with trypsin to cleave HA0 --> HA1 and HA2, by protein analysis of released virus particles, and finally, by electron microscopy. Therefore HA, the major glycoprotein alone, does not determine the budding site, and other factor(s), possibly both viral and host, is responsible for selecting the budding site of influenza virus.     

Detergent-resistant membrane microdomains and apical sorting of GPI-anchored proteins in polarized epithelial cells

Detergent-insoluble microdomains or rafts play a crucial role in many cellular functions: membrane traffic, cell signalling and human diseases. In this work we investigate the role of rafts in the sorting of GPI-anchored proteins in polarized epithelial cells. In contrast to MDCK cells, the majority of endogenous GPI-anchored proteins are sorted to the basolateral surface of Fischer rat thyroid cells (Zurzolo et al., J. Cell Biol. 121, 1031-1039, 1993). We analyzed a set of transfected GPI proteins in order to understand the role of the GPI anchor and of association with rafts for apical sorting. We found that the GPI moiety is necessary but not sufficient for apical sorting of GPI proteins and that the ectodomain has a major role. We propose a new model in which the stabilization of proteins into rafts, probably mediated by interactions between protein ectodomains and a putative receptor, plays a crucial role in apical sorting.     

Mastoparan activates apical chloride and potassium conductances, decreases cell volume, and increases permeability of cultured epithelial cell monolayers.

Mastoparan is a tetradecapeptide. Mastoparan added to the apical surface of monolayers of Madin-Darby canine kidney (MDCK) epithelial cells, cultured on micropore filters, activated ion transport and increased the permeability of the paracellular pathway across the monolayers. In monolayers of similar MDCK cells in which the basolateral membrane was permeabilized with Staphylococcus aureus alpha toxin (Staph. alpha toxin), the effects of mastoparan on apical membrane ion conductances were dependent on the presence of guanosine triphosphate (GTP). Mastoparan and GTP increased apical membrane chloride conductance more than potassium conductance, with very little change in sodium conductance. In intact monolayers, addition of barium to the apical bath prevented mastoparan activation of ion transport and the increase in paracellular permeability. Increasing bath potassium to 130 mM also reduced ion transport and prevented the increase in paracellular permeability. We hypothesized that these observations could be linked by mastoparan activation of apical chloride and potassium conductances, with consequent decreases in cell volume and resultant increases in paracellular permeability. Addition of 270 mM mannitol to isosmotic media to decrease cell volume decreased MDCK monolayer transepithelial resistance. Addition of mastoparan to monolayers of MDCK cells grown on micropore filters decreased cell volume to the same extent as addition of 270 mM mannitol to isosmotic media. Addition of the potassium channel inhibitor, barium, prevented the decrease in cell volume in response to mastoparan. Mastoparan activates apical membrane chloride and potassium conductances in MDCK cells. The loss of these ions from the cells decreases cell volume, and the decrease in cell volume increases the permeability of the paracellular pathway.     

Polarized expression of Tamm-Horsfall protein by renal tubular epithelial cells activates human granulocytes

In renal bacterial infections granulocytes are of major importance in the primary immune defense against invading pathogens. However, the mechanisms of granulocytic activation in renal interstitial invasion have not been clarified. Renal tubular epithelial cell mechanisms inducing granulocytic activation and bacterial killing may include tubular cell expression of Tamm-Horsfall protein (THP), a urinary protein that is known to enhance cytokine expression in monocytes. We studied the role of THP in granulocytic activation. A strong binding of THP to human granulocytes was demonstrated by fluorescence-activated cell sorter analysis. Urinary THP and supernatants of THP-expressing cultured tubular epithelial cells (MDCK) enhanced interleukin-8 (IL-8) expression by human granulocytes. Renal tubular cells growing polarized on polycarbonate membranes were used to study apical versus basal THP expression. By electron microscopy THP immunoreactivity was exclusively found on the apical surfaces of tubular cells and was absent on the basolateral cell membrane. In the apical cell culture compartment we found significantly more stimulatory activity for granulocytic IL-8 expression. CD62L, a selectin less expressed in activated granulocytes, was decreased in granulocytes incubated with urinary THP and in supernatants of THP-producing renal tubular cells but not in supernatants from THP-negative cells. Again, the effect on CD62L expression was found only in apical culture media and was absent in the basal compartment. In summary our data give evidence that renal tubular cell THP expression may be relevant in kidney diseases since THP is a potent activator of human granulocytes. The regulation of apical versus basal THP expression and release in vivo may be crucial in the induction of the inflammatory response, e.g., in bacterial renal diseases.