Publications

Protection against Leishmania major infection among inbred strains of mice is dependent upon successful expansion and activation of type 1 CD4+ effector (Th1) cells, a process that is aberrant in highly susceptible BALB strains. We sought to establish whether vaccination strategies using whole parasite lysates or a characterized immunodominant antigen, the Leishmania homolog of mammalian receptor for activated protein kinase C (LACK), would be capable of protecting subsequently infected BALB mice if given within a cytokine milieu capable of biasing the immune response toward Th1 cells. When given with neutralizing antibody to IL-4, but not when given alone, subcutaneously administered soluble Leishmania antigens mediated substantial protection to BALB/c mice against subsequent infection with parasites as assessed by size of the local lesion, enhanced Th1-type immune responses, and decreased parasite burdens. Similarly, when given with recombinant IL-12, LACK conferred substantial protection to cohorts of BALB.B, BALB/c, and BALB.K mice that was associated with reduction in serum IgE levels, consistent with effects on IL-4 production. Thus altering the cytokine milieu during administration of vaccine antigens by neutralizing IL-4 induced powerful Th1 recall responses during infection that were capable of mediating substantial levels of protection.

The cell migration that occurs during wound repair is dependent on modifications of the cell-matrix interaction in which extracellular matrix proteins and their receptors, the integrins, are involved. To study the interactions between airway epithelial cells and the extracellular matrix during the process of wound repair, we developed an in vitro wound model of human epithelial cells. Surface epithelial cells were dissociated from human nasal polyps and cultured on a type I collagen matrix. At confluency, a wound was made by the addition of 2 microliters of NaOH (1 N) to the cell culture. After the cell culture was washed, the wound area was recorded every 12 h for 96 h by a videomicroscopic technique. We calculated the wound-repair index that represents the decrease in the wound area per hour. Using immunofluorescence techniques, we first examined the localization, during wound repair, of fibronectin and of the beta 1-, alpha v-, alpha 2-, alpha 3-, and alpha 5-integrin subunits. Secondly, we carried out a series of wound-repair blocking experiments with the use of anti-integrin or anti-fibronectin antibodies diluted in the culture medium. We observed that fibronectin and the alpha 5- integrin subunit were exclusively expressed by the migratory cells in the wounded area. No difference in the localization of the alpha v-, alpha 2-, and alpha 3-integrin subunits was observed between the nonrepairing and repairing cells. The blocking experiments showed a significant decrease in the wound-repair index in the presence of either the anti-beta 1, -alpha 3, alpha 5, or the anti-fibronectin antibodies. Furthermore, the addition of fibronectin to the culture medium induced a significant increase in the wound repair index. These results suggest that fibronectin and the corresponding alpha 5 beta 1-integrin play an important role in the process of airway epithelium wound repair.

At least 10 different members of the integrin family have been reported to bind to fibronectin, and eight of these interact with the arginine-glycine-aspartic acid (RGD) site in the tenth type III repeat. However, studies utilizing recombinant fibronectin fragments have shown that for three of these, alpha 5 beta 1, alpha IIb beta 3, and alpha v beta 3, the structural requirements for binding to fibronectin differ. In the present study, we report that two additional integrins, alpha v beta 6, and alpha v beta 5 also demonstrate unique requirements for interaction with recombinant fibronectin fragments, alpha v beta 6, like alpha v beta 3, can support cell adhesion to the RGD-containing tenth repeat alone, and does not require the presence of a synergy site in the adjacent ninth repeat. In the cells used in this study, alpha v beta 5 only minimally supported adhesion to intact fibronectin, but did support adhesion to fragments composed of the eighth, ninth and tenth repeats or the tenth repeat, alone. Mutant fragments in which the eighth and tenth repeats were adjacent to one another enhanced adhesion mediated by alpha v beta 5, as well as adhesion mediated by alpha v beta 6. alpha v beta 5 and alpha v beta 6-mediated adhesion to all fibronectin fragments required interaction with the RGD site, as inferred by inhibition of adhesion with an RGD-containing peptide. These data suggest that each integrin that interacts with the RGD site in fibronectin has unique structural requirements for this interaction.

The pattern of integrin expression on human airway epithelium changes significantly in injury or inflammation. In particular, two integrins, the fibronectin receptor, alpha 5 beta 1 and the fibronectin/tenascin receptor alpha v beta 6, are expressed at low or undetectable levels in normal airways in vivo but are induced in response to airway epithelial injury. We investigated the effects of various growth factors known to be present in the airways on the expression of constitutively expressed and inducible airway epithelial integrins using flow cytometry. In primary cultures of human airway epithelial cells, transforming growth factor-beta 1 (TGF beta 1) dramatically increased expression of alpha v beta 6 and essentially did not affect the expression of any other integrin, including alpha 5 beta 1. In contrast, epidermal growth factor (EGF) upregulated surface levels of both alpha v beta 6 and alpha 5 beta 1. Together, TGF beta 1 and EGF had an additive effect on alpha v beta 6 and alpha 5 beta 1 expression while increasing levels of alpha 2 beta 1 and decreasing expression of alpha 3 beta 1- and alpha 6-containing integrins. In contrast, the transformed airway epithelial cell line, BEAS-2B, expressed a markedly different repertoire of integrins. Integrin expression on BEAS-2B cells was not affected by any of the growth factors tested in this study. These results demonstrate that, in primary cultures of human airway epithelial cells, the pattern of integrin expression can be dramatically altered by growth factors. The inducible integrins, alpha v beta 6, and alpha 5 beta 1 are most subject to regulation by growth factors and expression of each of these can be differentially regulated. The differential regulation of the two principal fibronectin receptors on airway epithelial cells suggests that they may mediate different cellular responses to fibronectin.