Candida albicans stimulates cytokine production and leukocyte adhesion molecule expression by endothelial cells.

Endothelial cells have the potential to influence significantly the host immune response to blood-borne microbial pathogens, such as Candida albicans. We investigated the ability (of this organism to stimulate endothelial cell responses relevant to host defense in vitro. Infection with C. albicans induced endothelial cells to express mRNAs encoding E-selectin, intercellular adhesion molecule 1, vascular cell adhesion molecule 1, interleukin 6, interleukin 8, monocyte chemoattractant protein 1, and inducible cyclooxygenase (cox2). All three leukocyte adhesion molecule proteins were expressed on the surfaces of the endothelial cells after 8 h of exposure to C. albicans. An increase in secretion of all three cytokines was found after 12 h of infection. Cytochalasin D inhibited accumulation of the endothelial cell cytokine and leukocyte adhesion molecule mRNAs in response to C. albicans, suggesting that endothelial cell phagocytosis of the organism is required to induce this response. Live Candida tropicalis, Candida glabrata, a nongerminating strain of C. albicans, and killed C. albicans did not stimulate the expression of any of the cytokine or leukocyte adhesion molecule mRNAs. These findings indicate that a factor associated with live, germinating C. albicans is required for induction of endothelial cell mRNA expression. Furthermore, since endothelial cells phagocytize killed C. albicans, phagocytosis is likely necessary but not sufficient for this organism to stimulate mRNA accumulation. In conclusion, the secretion of proinflammatory cytokines and expression of leukocyte adhesion molecules by endothelial cells in response to C. albicans could enhance the host defense against this organism by contributing to the recruitment of activated leukocytes to sites of intravascular infection.     

Gamma interferon protects endothelial cells from damage by Candida albicans by inhibiting endothelial cell phagocytosis.

Once Candida albicans comes in contact with endothelial cells, it induces cellular injury. This endothelial cell injury may be a mechanism by which blood-borne organisms escape from the intravascular compartment and invade the tissue parenchyma during hematogenous infection. We have been investigating the ability of cytokines to modulate endothelial cell injury caused by C. albicans. Previously we reported that pretreatment of endothelial cells with gamma interferon (IFN-gamma) protects these cells from candidal injury in vitro. In the current study, we examined potential mechanisms of the cytoprotective effects of IFN-gamma. Time course experiments demonstrated that maximal reduction in candidal injury of endothelial cells occurred after the endothelial cells had been exposed to IFN-gamma for at least 72 h. In other studies, we determined that IFN-gamma reduced endothelial cell phagocytosis of C. albicans by 41.3% compared with that of untreated endothelial cells (P < 0.01). Since endothelial cell phagocytosis of C. albicans is required for damage to occur, inhibition of phagocytosis is likely a mechanism by which IFN-gamma protects endothelial cells from candidal injury. We also found that the cytoprotective effect of IFN-gamma is not mediated by reducing access of the organisms to intracellular endothelial cell iron or by upregulating the synthesis of reactive oxygen intermediates (which could potentially reduce the ability of C. albicans to injure endothelial cells). Thus, inhibiting endothelial cell phagocytosis of C. albicans may be a mechanism by which IFN-gamma augments the host defense against hematogenously disseminated candidal infections.     

Centrosomes, microtubules, and microfilaments in the reendothelialization and remodeling of double-sided in vitro wounds.

The maintenance of endothelial monolayer integrity is an important function of aortic endothelial cells. Our study was designed to test two hypotheses related to the repair of the wounded endothelial monolayer. First, that the reappearance of the highly ordered cobblestone monolayer after wound closure is associated with specific sequential changes in the cytoskeletal system. Second, that there are different patterns of reendothelialization depending on whether microfilaments or microtubules are disrupted. One and a half millimeter wide wounds were created down the middle of confluent endothelial monolayers so that there were two wound edges facing each other. During the initiation of repair, the centrosomes of the cells on both sides of the wound reorientated to the front of the cell. The dense peripheral band of actin microfilaments disappeared, the cells elongated and migrated as a uniform sheet with wound closure occurring within 60 hours. The rate of closure remained constant until the migrating fronts met. The cytoskeletal changes observed and the rate of closure were similar to those we reported in a single edge wound. At closure, however, there was a transient piling up of cells which disappeared after 24 to 36 hours. Within 36 to 48 hours after closure, the centrosomes became randomly distributed around the nucleus. By 40 to 48 hours, the dense peripheral band started to reappear and the cells returned to a cobblestone appearance 72 to 96 hours after closure. Thus, the remodeling of the confluent monolayer after wound closure occurs in association with a specific series of cytoskeletal changes. When the microfilaments were disrupted with cytochalasin B, cell migration still occurred but it took up to four times longer for closure. These cells were initially flatter than normal and contained only a few microfilament fibers; however, the microtubule system was intact and centrosome reorientation occurred, but at a slower rate. However, when the microtubules were disrupted with colchicine, either at the onset of wounding or during repair, neither centrosome reorientation nor cell migration occurred. Thus wound closure of small-sized wounds require the presence of intact microtubules, whereas the additional presence of microfilaments results in a more rapid and efficient system of reendothelialzation.     

The effect of ethchlorvynol on cultured endothelial cells. A model for the study of the mechanism of increased vascular permeability.

Ethchlorvynol (ECV), an agent which produces reversible pulmonary edema, was studied for its effects on cultured bovine pulmonary artery endothelial cell (BPAE) and human umbilical vein endothelial cell (HUVE) monolayers. Endothelial cell monolayers 6 days post-confluent were treated with 1 mg/ml ECV for time intervals of from 5 minutes to 15 hours. ECV treatment caused a mild endothelial cell retraction evident at 10 minutes which increased in severity with increasing duration of exposure to ECV. Retraction of endothelial cells resulted in the formation of irregularly delineated gaps between cells, which remained attached to one another by slender filamentous processes. Despite the severity of the endothelial cell lesion, no cell lysis or cell detachment from the substratum occurred. Furthermore, removal of ECV from cell cultures resulted in the reversal of the endothelial cell lesion. Cytochemical distribution of actin microfilaments in control monolayers localized to a dense peripheral band of actin filaments and to a set of interconnected central microfilaments oriented in general parallel to the long axis of the cell. Endothelial cells treated with ECV for as little as 10 minutes showed a loss of F-actin from the dense peripheral band of microfilaments progressing until the dense peripheral band was entirely lost after 4 hours' exposure to ECV. By 4 hours central microfilaments had reorganized into a prominent series of microfilament bundles aligned parallel to each other and to the long axis of the cell. For investigation of a possible loss of attachment sites of actin filaments as the basis for the lesion, the localization of vinculin was examined in control and ECV-treated BPAE monolayers. After 2 hours' exposure to ECV, vinculin localization within monolayers was affected little, if at all. No effects of ECV on intermediate filaments were observed either. It is proposed that the dense peripheral band of actin bundles is important in maintaining well-spread endothelial cells in monolayers and that ECV acts to destroy the integrity of this structure. It is further proposed that a reaction of endothelial cells to ECV in vivo analogous to that seen in tissue culture accounts for the production of pulmonary edema by creating gaps between cells.     

Microtubule-dependent intracellular transport of murine polyomavirus

The mechanisms used by murine polyomavirus for intracellular migration are yet to be clarified. In this work we selectively depolymerized microtubules or actin fibers and then studied the progression of polyomavirus infection in cultured cells. Our results demonstrate that microtubule depolymerization prevents polyomavirus migration toward the nucleus and from the nucleus to the cell surface, being also involved in viral release, while disruption of the actin microfilaments appears to have no detrimental effect on the virus ability to reach the nucleus. The ultrastructural observation of polyomavirus nonenveloped particles interacting with the free end and the lateral sides of microtubules together with the coimmunoprecipitation of tubulin and viral VP-1 further supports the idea that polyomavirus intracellular migration seems to be mediated by the interaction of polyomavirus major capsid protein VP-1 with tubulin.     

LPS对体外枯否细胞吞噬功能的影响

目的:探讨LPS对体外培养枯否细胞吞噬功能的影响和机制。方法:胶原酶灌流消化肝脏, 密度梯度离心分离枯否细胞。在不同剂量脂多糖作用不同时间, 分别用聚苯乙烯乳珠吞噬实验、荧光染色法、荧光光度法和流式细胞分析检测枯否细胞的吞噬功能、微丝和微管表达及凋亡情况。结果:小剂量短时间作用时, LPS显著增强体外培养枯否细胞吞噬能力, 但随作用时间延长和剂量的增加, 吞噬能力反而下降;LPS使枯否细胞微丝和微管表达增强, 肌动蛋白含量和微管荧光灰度显著增加, 但剂量过大表达反而减弱;大剂量LPS可导致枯否细胞凋亡。结论:LPS可以增强体外培养枯否细胞的吞噬能力, 但剂量过大、时间过长反而抑制其吞噬能力。这可能与枯否细胞微丝、微管的变化和凋亡有关。     

Endothelial Cell Injury Caused by Candida albicans Is Dependent on Iron

Although it is known that Candida albicans causes endothelial cell injury, in vitro and in vivo, the mechanism by which this process occurs remains unknown. Iron is critical for the induction of injury in many types of host cells. Therefore, we investigated the role of iron in Candida-induced endothelial cell injury. We found that pretreatment of endothelial cells with the iron chelators phenanthroline and deferoxamine protected them from candidal injury, even though the organisms germinated and grew normally. Loading endothelial cells with iron reversed the cytoprotective effects of iron chelation. Moreover, chelation of endothelial cell iron significantly reduced phagocytosis of C. albicans by these cells, while candidal adherence to chelator-treated endothelial cells was slightly enhanced. Since endothelial cell phagocytosis of C. albicans is required for endothelial cell injury to occur, inhibition of phagocytosis is likely the principal mechanism of the cytoprotective effects of iron chelation. The production of toxic reactive oxygen intermediates by host cells is known to be inhibited by iron chelation. Therefore, we investigated whether treating endothelial cells with antioxidants could mimic the cytoprotective effects of iron chelation. Neither extracellular nor membrane-permeative antioxidants reduced candidal injury of endothelial cells. Furthermore, depleting endothelial cells of the endogenous antioxidant glutathione did not render them more susceptible to damage by C. albicans. These results suggest that candidal injury of endothelial cells is independent of the production of reactive oxygen intermediates and that the cytoprotective effects of iron chelation are not due to inhibition of the synthesis of these toxic intermediates.During the process of hematogenous dissemination, Candida albicans must first cross the endothelial cell lining of the vasculature to invade the tissue parenchyma. One mechanism by which the organism may escape from the vascular compartment is by causing endothelial cell injury. This injury likely results in exposure of the subendothelial cell basement membrane, which may enhance candidal adherence and facilitate tissue invasion (23). We have been investigating the mechanisms by which C. albicans injures endothelial cells in vitro. Previously, we found that phagocytosis of the organism by endothelial cells is required for endothelial cell damage to occur (8, 9). This phagocytosis requires both intact endothelial cell microfilaments and microtubules. In addition, although endothelial cells are able to phagocytize killed organisms (35), only the phagocytosis of live, germinating organisms causes endothelial cell injury (8).After endothelial cells phagocytize C. albicans, the organism may injure the endothelial cells by several potential mechanisms. It is possible that phospholipases and/or proteinases secreted by C. albicans injure host cells (6, 18, 38). Another possibility is that C. albicans causes endothelial cell injury by an iron-dependent process. For example, endothelial cells are known to synthesize and release superoxide anions during phagocytic activity (11, 14). Iron is required for the assembly of enzymes, such as xanthine oxidase (33) and cytochromes (20), that catalyze the synthesis of these reactive oxygen intermediates. In addition, iron serves as a cofactor, which converts these anions to highly reactive hydroxyl radicals (21) which cause cellular damage (10, 24). Finally, iron is required for the function of nitric oxide synthase, an enzyme that can catalyze the synthesis of cytotoxic concentrations of nitric oxide in some cell types (31).In this study, we used the iron chelators phenanthroline and deferoxamine to examine the role of iron in endothelial cell injury caused by C. albicans. We found that chelation of endothelial cell iron protected these cells from injury by C. albicans. The cytoprotective effects of iron chelation were likely due to reduced phagocytosis of this fungus by endothelial cells. Furthermore, we found that C. albicans damages endothelial cells by a process that is likely independent of the production of reactive oxygen intermediates.(This work was presented in part at the 35th Interscience Conference on Antimicrobial Agents and Chemotherapy, September 1995, San Francisco, Calif.)     

Melatonin modulates cytoskeletal organization in the rat brain hippocampus

Melatonin concentration in plasma reaches high levels during the night and synchronizes body rhythms with the photoperiod. Previous evidence obtained in cultured cells suggests that melatonin synchronizes cytoskeletal re-arrangements at nocturnal plasma concentration. In this study, we determined the amount of microtubules and microfilaments in the rat hippocampus as an index of cytoskeletal organization in rats submitted to a photoperiodic regime. Additionally, these parameters were determined in control rats, sham rats, pinealectomized rats, and rats that were pinealectomized and treated with melatonin for 1 week. The results showed an increase in both the amount of microfilaments in the hippocampus of rats sacrificed in the dark phase, and in melatonin levels. In addition, a decrease in both microfilament and microtubule amounts occurred in pinealectomized rats. In contrast, melatonin treatment partially reestablished actin and tubulin proportions organized in microfilaments and microtubules, respectively. The results indicate that actin organization in microfilaments was associated with both the photoperiod and with melatonin levels. Together, the data support that cytoskeletal organization is regulated rhythmically by melatonin in synchrony with the photoperiod.     

Role of microtubules in the adaptive response to low phosphate of Na/Pi cotransport in opossum kidney cells

The role of microtubules and actin microfilaments in adaptive changes of the apical Na-dependent transport of phosphate (P_i) was investigated in opossum kidney (OK) cells. Up-regulation of Na/P_i cotransport was achieved by incubating OK cells in a medium containing 0.1 mM P_i; down-regulation of Na/P_i cotransport was provoked by refeeding adapted cells with 2 mM P_i. Up-regulation of Na/P_i cotransport was found to be inhibited by approximately 50% after a pretreatment of the cells with the microtubule disrupting agents nocodozole and colchicine; indirect immunofluorescence indicated complete depolymerization of the microtubular network. No inhibition of the adaptive response was observed after treatment of the cells with cytochalasin B to depolymerize actin microfilaments. In adapted cells, depolymerization of microtubules by nocodozole led to a reversibility of Na/P_i cotransport similar to that observed after refeeding adapted cells with 2 mM P_i. No effects of the microtubule disrupting drugs were observed on Na/l-glutamic acid transport. Depolymerization of microtubules did not prevent parathyroid-hormone-mediated inhibition of Na/P_i cotransport. It is concluded that microtubules are (at least in part) involved in the correct insertion of newly synthesized apical Na/P_i cotransport systems and that microtubules are not involved in the internalization of Na/P_i cotransport systems.     

Mechanisms by which Candida albicans induces endothelial cell prostaglandin synthesis.

One strategy for improving resistance to opportunistic pathogens is to determine host cellular responses during the invasion process and upregulate those responses that are relevant to host defense mechanisms. Within this context, we have shown previously that invasion of endothelial cells by Candida albicans in vitro causes increased production of prostaglandins. As a prerequisite for modulating endothelial cell prostaglandin production, we now characterize the mechanisms through which this process occurs. Endothelial cell invasion by C. albicans appeared to stimulate the conversion of arachidonic acid into prostaglandins by upregulating the synthesis of endothelial cell cyclooxygenase and increasing the activity of the endothelial cell phospholipase. The enhanced activities of these two enzymes were independent of calphostin C-sensitive protein kinase C and resulted in the increased production and extracellular secretion of prostaglandin I2 (PGI2), PGF2 alpha, and PGE2. The secretion of these prostaglandins had no effect on the amount of endothelial cell injury induced by C. albicans. The role of the increased prostaglandin secretion by endothelial cells is likely related to modulation of the leukocyte response at the candida-leukocyte-endothelial cell interface.     

Inhibition of hydrophobic protein-mediated Candida albicans attachment to endothelial cells during physiologic shear flow

Adhesion interactions during hematogenous dissemination of Candida albicans likely involve a complex array of host and fungal factors. Possible C. albicans factors include changes in cell surface hydrophobicity and exposed antigens that have been shown in static adhesion assays to influence attachment events. We used a novel in vitro shear analysis system to investigate host-pathogen interactions and the role of fungal cell surface hydrophobicity in adhesion events with human endothelial cells under simulated physiologic shear. Endothelial monolayers were grown in capillary tubes and tested with and without interleukin-1 beta activation in buffered medium containing human serum. Hydrophobic and hydrophilic stationary-phase C. albicans yeast cells were infused into the system under shear flow and found to adhere with widely varying efficiencies. The average number of adherent foci was determined from multiple fields, sampled via video microscopy, between 8 and 12 min after infusion. Hydrophobic C. albicans cells demonstrated significantly more heterotypic binding events (Candida-endothelial cell) and greater homotypic binding events (Candida-Candida) than hydrophilic yeast cells. Cytokine activation of the endothelium significantly increased binding by hydrophobic C. albicans compared to unactivated host cells. Preincubation of hydrophobic yeast cells with a monoclonal antibody against hydrophobic cell wall proteins significantly blocked adhesion interactions with the endothelial monolayers. Because the antibody also blocks C. albicans binding to laminin and fibronectin, results suggest that vascular adhesion events with endothelial cells and exposed extracellular matrix may be blocked during C. albicans dissemination. Future studies will address the protective efficacy of blocking or redirecting blood-borne fungal cells to favor host defense mechanisms.     

Ability of Escherichia coli isolates that cause meningitis in newborns to invade epithelial and endothelial cells.

Escherichia coli isolates that cause meningitis in newborns are able to invade the circulation and subsequently cross the blood-brain barrier. One mechanism for traversing the blood-brain barrier might involve transcytosis through the endothelial cells. The ability of the meningitis isolate E. coli IHE3034, of serotype 018:K1:H7, to invade epithelial (T24) and endothelial (EA-hy926) cells was investigated by the standard gentamicin survival assay and by electron microscopy. Human bladder epithelial and endothelial cells were efficiently invaded by strain IHE3034, whereas epithelial human colon Caco-2 cells, canine kidney MDCK cells, and the opossum [correction of opposum] epithelial kidney cell line OK were not invaded. The ability to invade human epithelial cells of the bladder could also be demonstrated for several other newborn meningitis E. coli strains and one septicemic E. coli strain. Studies utilizing inhibitors which act on eukaryotic cells revealed a dependence on microfilaments as well as on microtubules in the process of E. coli IHE3034 entry into T24 and EA-hy926 cells. These results indicated that cell cytoskeletal rearrangements are involved in bacterial uptake and suggest that there are either two pathways (microtubule dependent and microfilament dependent) or one complex pathway involving both microtubules and microfilaments. The intracellular IHE3034 organisms were contained in a host-membrane-confined compartment mainly as single microorganisms. Intracellular replication of 1HE3034 was not detected, nor did the number of intracellular bacteria decrease significantly during a 48-h period. The ability of E. coli O18:K1 to invade and survive within certain eukaryotic cells may be another virulence factor of meningitis-associated E. coli.     

Mechanisms of the proinflammatory response of endothelial cells to Candida albicans infection

Endothelial cells can influence significantly the host inflammatory response against blood-borne microbial pathogens. Previously, we found that endothelial cells respond to in vitro infection with Candida albicans by secreting interleukin 8 (IL-8) and expressing E-selectin, intercellular adhesion molecule 1 (ICAM-1), and vascular cell adhesion molecule 1 (VCAM-1). We have now examined the mechanisms mediating this endothelial cell response. We determined that C. albicans stimulated endothelial cells to synthesize tumor necrosis factor alpha (TNF-alpha), which in turn induced these infected cells to secrete IL-8 and express E-selectin by an autocrine mechanism. Expression of VCAM-1 was mediated not only by TNF-alpha but also by IL-1alpha and IL-1beta, all of which were synthesized by endothelial cells in response to C. albicans. These three cytokines remained primarily cell associated rather than being secreted. Candidal induction of ICAM-1 expression was independent of TNF-alpha, IL-1alpha, and IL-1beta. These observations demonstrate that different proinflammatory endothelial cell responses to C. albicans are induced by distinct mechanisms. A clear understanding of these mechanisms is important for therapeutically modulating the endothelial cell response to C. albicans and perhaps other opportunistic pathogens that disseminate hematogenously.     

Microtubule-actin interactions may regulate endothelial integrity and repair.

An important mechanism for the initiation and progression of atherosclerosis is the loss of endothelial integrity, which is required for normal blood vessel function. The important components of the endothelial cell cytoskeleton system that regulate endothelial integrity include actin microfilaments and microtubules, which are both associated with protein complexes that regulate cell-cell and cell-substratum adhesion. To date, studies have shown that microfilaments are essential in maintaining the structural integrity of the endothelium while microtubules regulate the directional cell migration during repair. When microtubules are disrupted at the onset of wounding, neither centrosome reorientation, which is essential for efficient endothelial cell wound repair, nor cell migration occurs. Disruption of microfilaments is also associated with inefficient endothelial cell migration and repair. How then might these systems be associated with one another? Linker proteins, which may facilitate interaction between microtubules and actin microfilaments, have recently been identified in nonendothelial systems. It is likely that microtubule-microfilament interactions are important in the complex regulation of endothelial integrity and repair especially as they relate to atherosclerotic plaque formation.     

Candida albicans metabolite affects the cytoskeleton and phagocytic activity of murine macrophages

Candida albicans is the most common opportunistic fungal pathogen of humans, causing systemic disease in immunocompromised patients. Host resistance to C. albicans infections is mediated predominantly by neutrophils and monocytes/macrophages. We have previously shown that exposure of a human epithelial cell line (HEp2) to C. albicans or to a culture filtrate of C. albicans caused actin rearrangement in the HEp2 cells. Since shifting of actin from the filamentous to the globular form may be crucial to the activity of phagocytes, we assessed in the present study the effect of the C. albicans metabolite (lyophilized culture filtrate) on the cytoskeleton of murine peritoneal macrophages and on their phagocytic activity. Our results showed a significant decrease in phagocytosis of C. albicans, ranging from 53-63% and a 25% reduction for C. glabrata cells. Using confocal laser scanning microscopy an actin rearrangement in the macrophages could be demonstrated that may be associated with the decrease of phagocytosis. We also tested the effect of mannan and of the secreted aspartic proteinase (Sap) inhibitor--pepstatin, on the activity of the metabolite in order to define the putative component and found no influence. In conclusion, our data indicate that a C. albicans metabolite affects phagocytic activity of macrophages, probably by alterations in their cytoskeleton.     

Cross-Reactivity Between Antigens of Coccidioides immitis, Histoplasma capsulatum, and Blastomyces dermatitidis in Lymphocyte Transformation Assays

The kinetics of phagocytosis and killing of four fungal forms with varying virulence by two types of phagocytic cells was examined. Human monocytes ingested Saccharomyces cerevisiae, Candida tropicalis, and the blastospores of Candida albicans more rapidly than did human neutrophils. There was no difference in the rate of phagocytosis of C. albicans pseudohyphae by these two cell types. Intracellular killing of each of the four fungal forms was consistently and significantly more rapid by monocytes than by neutrophils. Neutrophils were unable to destroy ingested C. albicans pseudohyphae. These experiments suggest that the monocyte plays an important role in host defenses against fungal diseases and that the relative virulence of the pathogenic yeasts in human disease may be related to the ability of these organisms to survival after being ingested by circulating phagocytes.     

Optimal development of Chlamydophila psittaci in L929 fibroblast and BGM epithelial cells requires the participation of microfilaments and microtubule-motor proteins

The cytoskeleton is involved in several cellular activities, including internalization and transport of foreign particles. Although particular functions to each cytoskeleton component have been described, interactions between those components seem to occur. The involvement of the different host cell cytoskeletal components in uptake and development of Chlamydophila psittaci is incompletely understood. In this study, the participation of the microfilament network along with the kinesin and dynein microtubule motor proteins in the internalization and further development of Chlamydophila psittaci were investigated in L929 fibroblast and BGM epithelial cells. Cytochalasin D disruption of actin filaments, and blockage of the motor proteins through the introduction of monoclonal antibodies into the host cells were carried out, either single or combined, at different moments around bacterial inoculation, and Chlamydophila infectivity determined 24 h post- inoculation by direct immunofluorescence. Our results show that, although Chlamydophila Ipsittaci can make use of both microfilament-dependent and independent entry pathways in both cell types, Chlamydophila internalization and development in the fibroblast cells mainly concerned processes mediated by microfilaments while in the epithelial cells mechanisms that require microtubule motor proteins were the ones predominantly involved. Evidence that mutual participation of the actin and tubulin networks in both host cells are required for optimal growth of Chlamydophila psittaci is also presented.     

CD44, alpha(4) integrin,and fucoidin receptor-mediated phagocytosis of apoptotic leukocytes

Various types of phagocytes mediate the clearance of apoptotic cells. We previously reported that human and murine high endothelial venule (HEV) cells ingest apoptotic cells. In this report, we examined endothelial cell fucoidin receptor-mediated phagocytosis using a murine endothelial cell model mHEV. mHEV cell recognition of apoptotic leukocytes was blocked by fucoidin but not by other phagocytic receptor inhibitors such as mannose, fucose, N-acetylglucosamine, phosphatidylserine (PS), or blocking anti-PS receptor antibodies. Thus, the mHEV cells used fucoidin receptors for recognition and phagocytosis of apoptotic leukocytes. The fucoidin receptor-mediated endothelial cell phagocytosis was specific for apoptotic leukocytes, as necrotic cells were not ingested. This is in contrast to macrophages, which ingest apoptotic and necrotic cells. Endothelial cell phagocytosis of apoptotic cells did not alter viable lymphocyte migration across these endothelial cells. Antibody blocking of CD44 and alpha4 integrin on the apoptotic leukocyte inhibited this endothelial cell phagocytosis, suggesting a novel function for these adhesion molecules in the removal of apoptotic targets. The removal of apoptotic leukocytes by endothelial cells may protect the microvasculature, thus ensuring that viable lymphocytes can successfully migrate into tissues.     

Microtubule and microfilament populations of cell processes in the dental pulp

An attempt has been made to characterize the nature of the unidentified cell processes participating in gap junctions in the odontoblast layer. In peripheral and pulpal nerves, there is a strong relationship between axon caliber and microfilament and microtubule populations. This characteristic, together with the ratio of microtubules to microfilaments, has been measured and compared for four types of cell processes found in the dental pulp, including those participating in gap junctions. The processes taking part in the gap junctions cannot be distinguished from pulpal axons on the basis of microtubule-to-microfilament ratio nor on the relationship between microtubule and microfilament population and process caliber. While these findings do not prove that the “gap members” are nerve fibers, it does support the hypothesis that the processes taking part in gap junctions in the peripheral dental pulp are nerve fibers.