We studied the postulated involvement of the protein kinase C beta 1 (PKC beta 1) isoform in the regulation of endothelial permeability using human dermal microvascular endothelial cell line (HMEC-1). We overexpressed the recombinant PKC beta 1 gene via retroviral-mediated transduction in these cells. PKC beta 1 gene transfer was stable, and PKC beta 1 protein production was persistent for at least 1 month posttransduction. Addition of 2 x 10(-9) M and 2 x 10(-8) M phorbol 12-myristate 13-acetate (PMA) to the control (nontransduced) HMEC-1 cells increased the transendothelial 125I-albumin clearance rate (an index of endothelial permeability) from 2.5 +/- 0.2 x 10(-2) microliters/min to 5.4 +/- 1.2 x 10(-2) microliters/min and 16.8 +/- 3.1 x 10(-2) microliters/min, respectively. However, addition of 2 x 10(-9) M PMA to PKC beta 1-overexpressing HMEC-1 cells produced a maximal increase in the transendothelial 125I-albumin clearance rate of 15.9 +/- 2.0 x 10(-2) microliters/min. Challenge of these cells with 2 x 10(-8) M PMA did not further augment the increase in permeability. Activation with PMA was associated with the translocation of the PKC beta 1 from the cytosol to the membrane. These data show that PKC beta 1 overexpression augments the increase in endothelial permeability in response to PKC activation, suggesting an important function for the PKC beta 1 isoform in the regulation of endothelial barrier.

We studied the basis of inhibition of polymorphonuclear leukocyte (PMN) adhesion induced by neutrophil inhibitory factor (NIF), a 41-kDa CD11/CD18 beta(2) integrin-binding protein isolated from the canine hookworm (Ancylostoma caninum). NIF blocked PMN adhesion in a concentration-dependent manner with complete blockade occurring at approximately 10 nM NIF. Because CD11a and CD11b beta(2) integrins are functionally active on stimulated PMNs, and yet NIF is postulated to inhibit only CD11b integrin by binding to its I domain, we evaluated the contributions of CD11a and CD11b beta(2) integrins in the mechanism of inhibition of PMN adhesion to endothelial cells. We observed an additive inhibitory effect (>90% inhibition) of PMN adhesion to endothelial cells when NIF was used in combination with anti-CD11b monoclonal antibodies, which alone at saturating concentrations reduced PMN adhesion by only 50%. NIF also prevented aggregation of phorbol ester-stimulated JY lymphoblastoid cells that expressed only the functionally active CD11a, suggesting that NIF also can inhibit CD11a-dependent response. We transduced the NIF cDNA into human dermal microvessel endothelial cells in which NIF synthesis and release prevented PMN adhesion to the transduced human dermal microvessel endothelial cells. These data indicated that the potent antiadhesive effect of NIF may be the result of inhibition of CD11a and CD11b beta(2) integrins on PMNs. Moreover, the strategy of NIF release from transduced endothelial cells suggests the feasibility of blocking the CD11a- and CD11b beta(2) integrin-dependent PMN adhesion and PMN migration responses specifically at sites of endothelial cell activation.

We investigated the function of the Ca2+-dependent protein kinase C (PKC) beta1 in the regulation of endothelial barrier property. Human dermal microvascular endothelial cells (HMEC-1) were transduced with full-length PKCbeta1 antisense (AS) cDNA or control pLNCX vector to generate stable cell lines (HMEC-AS and HMEC-pLNCX, respectively). Analyses indicated that HMEC-AS expressed the antisense PKCbeta1 transcript with decreased PKCbeta protein level (without a change in PKCalpha or PKCepsilon). The baseline transendothelial 125I-albumin clearance rates of HMEC-1, HMEC-pLNCX, and HMEC-AS were 5.0+/-0.5 x 10(-2), 6.8+/-0.4 x 10(-2), and 6.9+/-0.6 x 10(-2) microl/min, respectively. Activation of HMEC-1 and HMEC-pLNCX with phorbol 12-myristate 13-acetate (PMA) increased the rates to the respective 14.5+/-1.7 x 10(-2) microl/min and 16.9+/-2.8 x 10(-2) microl/min (corresponding to 191% and 149% increases over baseline). However, in HMEC-AS, PMA increased the rate to 9.8+/-1.0 x 10(-2) microl/min (42%). When HMEC-1 and HMEC-pLNCX were activated with thrombin, the rates increased to 10.8+/-1.4 x 10(-2) and 14.0+/-1.9 x 10(-2) microl/min, respectively (116% and 106%). In contrast, thrombin stimulation of HMEC-AS more than doubled the increase to 27.2+/-3.5 x 10(-2) microl/min (294%). Furthermore, the thrombin-induced peak increase in the [Ca2+]i in HMEC-AS was greater than in control cells. Fluorescence-activated cell sorter analysis of thrombin receptor expression indicated that the augmented thrombin-induced responses were not attributable to altered receptor density in HMEC-AS. These results indicate that PKCbeta functions in a negative feedback manner to inactivate thrombin-generated signals and thereby modulates the endothelial permeability increase. Because decreased PKCbeta expression significantly reduced the PMA-induced permeability increase, PKCbeta may downregulate thrombin receptor function upstream of PKC activation (i.e., Ca2+).