Changes in the motility, morphology, and F-actin architecture of human dendritic cells in an in vitro model of dendritic cell development.

An in vitro model has been developed for analyzing the two developmental phases of human dendritic cell (DC) migration. Employing the age of the culture and the addition of GM-CSF, IL-4, and serum to regulate cellular phenotype, and glass coated with acid-precipitated human plasma proteins to facilitate persistent DC translocation, the model produces three sequential in vitro phenotypes with the following suggested in vivo counterparts: (1) DCs recently isolated from blood, which are highly polar and motile, and reflect the behavior of "undifferentiated" DCs that must extravasate from the blood stream and migrate into peripheral tissue; (2) large, nonmotile, stellate DCs, which reflect the highly "differentiated" signature phenotype of DCs in peripheral tissue, whose function is to capture foreign antigens; and (3) the large, motile "dedifferentiated" DCs, which reflect the behavior of "veiled cells" that have captured an antigen, retracted dendritic processes, migrated out of peripheral tissue, and are in the process of transporting a captured antigen to a proximal draining lymph node for presentation to T cells. Computer-assisted motion analysis of the three sequential phenotypes and fluorescent staining of F-actin reveal three unique behavioral states and unique cellular architecture consistent with inferred in vivo function. This in vitro model should serve as a starting point for elucidating the cues and molecular mechanisms involved in the regulation of DC differentiation and motility.

HIV-induced T-cell syncytia release a two component T-helper cell chemoattractant composed of Nef and Tat.

Using a newly developed gradient chamber to provide independent measurements of chemokinesis (stimulated motility) and chemotaxis (stimulated motility up a concentration gradient) of individual T-helper cells, it was recently demonstrated that HIV-induced T-cell syncytia release two distinct chemotactic activities that are separable by their rates of diffusion. The molecular masses of the two chemoattractant activities were estimated to be 30 and 120 kDa. The higher molecular mass activity was demonstrated to be the viral glycoprotein gp120. In an attempt to identify the lower molecular mass activity, chemotaxis and chemokinesis of T-helper cells were analyzed in individual concentration gradients of the virally encoded proteins Rev, p24, Tat and Nef. None functioned alone as a chemoattractant, but both Tat and Nef alone functioned as chemokinetic stimulants. When Tat and Nef were used together to generate parallel gradients, they stimulated chemotaxis. Antibody to either Tat or Nef neutralized the lower molecular mass chemotactic activity released by syncytia. The addition of antibody to the CD4 receptor or the addition of soluble CD4 inhibited high molecular mass chemotactic activity but not the low molecular mass chemotactic activity in HIV-induced syncytium-conditioned medium, demonstrating that the former but not the latter activity is mediated through the CD4 receptor. These results identify the combination of Nef and Tat as the lower molecular mass T cell chemoattractant released by HIV-induced syncytia, and provide the first evidence suggesting that parallel concentration gradients of two proteins are necessary for chemotaxis.

T cell syncytia induced by HIV release. T cell chemoattractants: demonstration with a newly developed single cell chemotaxis chamber.

A chemotaxis chamber has been developed to analyze both the velocity and the directionality of individual T cells in gradients of high molecular mass molecules over long periods of time. Employing this chamber, it is demonstrated that syncytia induced by HIV in SUP-T1 cell cultures release two T cell chemoattractants with approximate molecular masses of 30 and 120 kDa. Neither uninfected single cells nor polyethylene glycol-induced syncytia release detectable chemoattractant, suggesting that these chemoattractants are linked to HIV infection. Soluble gp120 functions as a T cell chemoattractant and the addition of anti-gp120 antibody to syncytium-conditioned medium blocks the high molecular mass chemoattractant activity but not the low molecular mass activity. The addition of anti-CD4 antibody to syncytium-conditioned medium also blocks the high molecular mass chemoattractant activity but not the low molecular mass activity. These results demonstrate that HIV-induced T cell syncytia release a low and a high molecular mass T cell chemoattractant, and suggest that the high molecular mass factor is gp120 and that it functions through the CD4 receptor.

Overexpression of microfilament-stabilizing human caldesmon fragment, CaD39, affects cell attachment, spreading, and cytokinesis.

Previous studies have demonstrated that overexpression of the carboxyl-terminal fragment, CaD39, of human fibroblast caldesmon in Chinese hamster ovary cells protected endogenous tropomyosin from turnover and stabilized actin microfilament bundles [Warren et al., 1994: J. Cell Biol. 125:359-368]. To assess the consequences of having CaD39-stabilized microfilaments in living cell, we characterized the motile behaviors of stable CaD39-expressing lines. We here found that CaD39-expressing cells adhered faster to plastic, glass, fibronectin-coated glass, and collagen-coated glass than control cells. Moreover, the CaD39-expressing cells also exhibited enhanced spreading immediately after attachment. Despite these differences, overexpression of CaD39 had little effect on the velocity of intracellular granule movement, or the velocity and persistence of cellular translocation. However, CaD39-expressing cells were more elongate and encompassed less area than non-expressing cells during migration in a wound-healing assay. In interphase cells, the expressed CaD39 fragments were found associated with tropomyosin-enriched microfilaments. Like endogenous caldesmon, the CaD39 fragment was also modified at mitosis. Although a significant portion of CaD39 underwent only partial modification, the majority of the CaD39 was released from the microfilaments during mitosis. This is consistent with the finding that the CaD39-induced advantage for attachment and spreading was lost during mitosis. In CaD39-expressing cells, an incomplete release of the CaD39 from microfilaments at mitosis was found which may be responsible for the increase in the frequency of multinuclear cells in CaD39-expressing lines.

Ponticulin plays a role in the positional stabilization of pseudopods.

Ponticulin is a 17-kD glycoprotein that represents a major high affinity link between the plasma membrane and the cortical actin network of Dictyostelium. To assess the role of ponticulin in pseudopod extension and retraction, the motile behavior of two independently generated mutants lacking ponticulin was analyzed using computer-assisted two- and three-dimensional motion analysis systems. More than half of the lateral pseudopods formed off the substratum by ponticulin-minus cells slipped relative to the substratum during extension and retraction. In contrast, all pseudopods formed off the substratum by wild-type cells were positionally fixed in relation to the substratum. Ponticulin-minus cells also formed a greater proportion of both anterior and lateral pseudopods off the substratum and absorbed a greater proportion of lateral pseudopods into the uropod than wild-type cells. In a spatial gradient of cAMP, ponticulin-minus cells were less efficient in tracking the source of chemoattractant. Since ponticulin-minus cells extend and retract pseudopods with the same time course as wild-type cells, these behavioral defects in ponticulin-minus cells appear to be the consequence of pseudopod slippage. These results demonstrate that pseudopods formed off the substratum by wild-type cells are positionally fixed in relation to the substratum, that ponticulin is required for positional stabilization, and that the loss of ponticulin and the concomitant loss of positional stability of pseudopods correlate with a decrease in the efficiency of chemotaxis.

HIV-induced syncytia in peripheral blood cell cultures crawl by extending giant pseudopods.

It was previously demonstrated that HIV-induced syncytia of the immortalized T cell line SupT1 reorganize their cytoskeleton and form a spherical supernuclear complex, thus mimicking the organization, polarity, and morphology of a single SupT1 cell. Then, through extension of a single, giant pseudopod, these syncytia, which grow to more than 100 times the volume of a single SupT1 cell, translocate along a substratum. To verify that syncytium motility is not peculiar to the SupT1 cell line, we have analyzed the cytoskeletal organization and motile capabilities of HIV-induced syncytia formed in peripheral blood cell cultures containing more than 90% CD4-positive cells. The results demonstrate that although peripheral blood T cells differ from SupT1 cells in size and morphology, they are continuously motile and translocate along a substratum in a manner quite similar to that of SupT1 cells, and peripheral blood T cell syncytia induced by HIV-1LAI as well as two additional clinical isolates translocate by the extension of a giant anterior pseudopod in a fashion indistinguishable from that of HIV-induced SupT1 syncytia. Together, these results support the generalization that HIV-induced T cell syncytia are motile.

HIV-induced syncytia of a T cell line form single giant pseudopods and are motile.

The human immunodeficiency virus, HIV, induces syncytium formation in cultures of many T cell lines. These syncytia have previously been viewed as disorganized fusion products in the throes of death. Evidence is presented that in HIV1-infected SupT1 cultures, syncytia five times to over one hundred times larger than single cells organize their many nuclei into blastula-like balls, reorganize their cytoskeleton to mimic that of a single cell, and extend single, giant pseudopods in a polar fashion. Medium-sized syncytia are capable of translocation through extension of these giant pseudopods. The rate of translocation of syncytia is comparable to that of single cells. Single cell motility, syncytium motility and pseudopod extension also appear to play roles in the recruitment of cells into syncytia. Finally, condensation of F-actin at cell-syncytium and syncytium-syncytium adhesion sites suggests the involvement of the cytoskeleton in the adhesion and/or subsequent fusion event. These results suggest that the fusion events involved in HIV-induced syncytia formation involve both cell motility and reorganization of the cytoskeleton, and demonstrate that syncytia are highly organized, motile entities.