Barbara R. Schwartz

Identification of surface proteins mediating adherence of CD11/CD18-deficient lymphoblastoid cells to cultured human endothelium.

Patients with the severe form of leukocyte adhesion deficiency syndrome do not express the CD11/CD18 adhesion complex on any of their leukocytes. Nevertheless, their lymphocytes, unlike their phagocytes, emigrate to extravascular sites of inflammation, demonstrating that surface proteins other than CD11/CD18 can mediate lymphocyte adherence to endothelium. Using a B-lymphoblastoid cell line (B-LCL) established from a CD11/CD18-deficient patient and cultured human umbilical vein endothelial cells (HEC), we investigated the CD11/CD18-independent mechanism(s) of lymphocyte adherence to endothelium. Monoclonal antibodies directed to the alpha 4 polypeptide (CD49d) and the beta 1 polypeptide (CD29) of the lymphocyte VLA-4 integrin receptor (CD49d/CD29), and to vascular cell adhesion molecule-1 (VCAM-1) on the endothelial cell significantly inhibited the adherence of the CD11/CD18-deficient B-LCL to untreated HEC and to HEC treated with recombinant human tumor necrosis factor-alpha. We suggest that the interaction of the lymphocyte receptor VLA-4 with the endothelial ligand VCAM-1 induced by cytokines at sites of inflammation or immune reaction represents a CD11/CD18-independent pathway of lymphocyte emigration.

Leukocyte Adhesion Deficiency Type II is a generalized defect of de novo GDP-fucose biosynthesis. Endothelial cell fucosylation is not required for neutrophil rolling on human nonlymphoid endothelium.

Leukocyte Adhesion Deficiency Type II (LAD II) is a recently described syndrome and the two patients with this defect lack fucosylated glycoconjugates. These glycoconjugates include the selectin ligand, sialyl LewisX, and various fucosylated blood group antigens. To date, the molecular anomaly in these patients has not been identified. We localized the defect in LAD II to the de novo pathway of GDP-fucose biosynthesis, by inducing cell-surface expression of fucosylated glycoconjugates after exposure of lymphoblastoid cell lines from the LAD II patients to exogenous fucose. This defect is not restricted to hematopoietic cells, since similar findings were elicited in both human umbilical vein endothelial cells (HUVEC) and fibroblasts derived from an affected abortus. We have used these LAD II endothelial cells to examine the consequence of fucosylation of endothelial cells on the rolling of normal neutrophils in an in vitro assay. Neutrophil rolling on LPS-treated normal and LAD II HUVEC was inhibited by an E-selectin monoclonal antibody at both high and low shear rates. LAD II HUVEC lacking fucosylated glycoproteins supported leukocyte rolling to a similar degree as normal HUVEC or LAD II cells that were fucose-fed. At low shear rates, an L-selectin antibody inhibited neutrophil rolling to a similar degree whether the LAD II cells had been fucose-fed or not. These findings suggest that fucosylation of nonlymphoid endothelial cells does not play a major role in neutrophil rolling and that fucose is not a critical moiety on the L-selectin ligand(s) on endothelial cells of the systemic vasculature.

Requirement for RhoA Kinase Activation in Leukocyte De-Adhesion

Leukocyte migration from bloodstream to tissue requires rapid coordinated regulation of integrin-dependent adhesion and de-adhesion. Whether de-adhesion is an active process mediated by a distinct signaling pathway(s) or a passive decay of initial adhesion remains undetermined. We found that blockade of RhoA with C3 exoenzyme or inhibition of RhoA kinase by the specific inhibitor Y-27632 enhanced phorbol ester-stimulated α4β1-dependent adhesion of Jurkat cells at 30 min. Similarly, Y-27632 treatment increased stimulated β2 integrin-dependent neutrophil adhesion at 30 min but not at 5 min. Because reduced de-adhesion could mimic augmentation of adhesion at later time points, we developed an assay to measure de-adhesion specifically. Treatment of phorbol ester—or bacterial chemoattractant peptide—but not Mn2+-stimulated neutrophils adherent to serum-coated plastic or endothelial cells with Y-27632 or C3 exoenzyme markedly reduced the rate of de-adhesion, while markedly increasing their spreading. RhoA kinase inhibitor effects on de-adhesion and spreading were reversed by treatment with the cytoskeletal-disrupting agent cytochalasin D. Treatment with Y-27632 influenced neither integrin activation epitope nor integrin clustering. We conclude that activation of RhoA kinase promotes leukocyte de-adhesion by inhibiting cytoskeletal-dependent spreading, and that these effects of RhoA kinase constitute a new mechanism for regulation of integrin receptor avidity.

The GTPase Rap1 Regulates Phorbol 12-Myristate 13-Acetate-stimulated but Not Ligand-induced β1Integrin-dependent Leukocyte Adhesion

Leukocyte migration from bloodstream to tissue requires rapid, coordinated regulation of integrin-dependent adhesion and de-adhesion. In a previous study we demonstrated that inhibition of protein geranylgeranylation inhibited phorbol ester-stimulated avidity modulation of β1 integrin in several leukocyte cell lines. Both RhoA and Rap1 require post-translational modification by geranylgeranylation for full function. In this report we identify Rap1, not RhoA, as a critical geranylgeranylated protein mediating phorbol ester-stimulated β1 and β2integrin-dependent adhesion of Jurkat cells. Overexpression of the Rap1-specific GTPase-activating protein, SPA-1, or inactivated form of Rap1 (N17Rap1) blocked phorbol ester-stimulated adhesion of Jurkat cells to fibronectin (α4β1) and ICAM-1 (αLβ2). With high concentrations of fibronectin as ligand, Jurkat cells adhered spontaneously without phorbol ester stimulation. Unlike the phorbol ester-stimulated adhesion, adhesion induced by high density ligand was not dependent upon Rap1 activation or actin cytoskeleton reorganization. Thus, the “inside-out” adhesion signal induced by phorbol ester and the “outside-in” signal induced by high density ligand involve different pathways.

Sulfhydryl reducing agents promote neutrophil adherence without increasing surface expression of CD11bCD18 (Mac-1, Mol)

The disulfide reducing agents dithioerythreitol and dithiothreitol, but not oxidized dithiothreitol, induced polymorphonuclear neutrophils to adhere to endothelial cells or to plastic. Adherence was inhibited by monoclonal antibodies 60.1 and 60.3, which are directed to functional epitopes on the CD11b and CD18 polypeptides of the neutrophil membrane adhesion complex (Mac-1, Mo1). The increased adherence induced by the sulfhydryl reducing agents was not accompanied by increased expression of CD11b/CD18. These studies demonstrate that a qualitative alteration in CD11b/CD18 is sufficient to promote neutrophil adherence.

Extracellular BCL2 proteins are danger-associated molecular patterns that reduce tissue damage in murine models of ischemia-reperfusion injury.

Background Ischemia-reperfusion (I/R) injury contributes to organ dysfunction in a variety of clinical disorders, including myocardial infarction, stroke, organ transplantation, and hemorrhagic shock. Recent investigations have demonstrated that apoptosis as an important mechanism of cell death leading to organ dysfunction following I/R. Intracellular danger-associated molecular patterns (DAMPs) released during cell death can activate cytoprotective responses by engaging receptors of the innate immune system. Methodology/Principal Findings Ischemia was induced in the mouse hind limb by tourniquet or in the heart by coronary artery ligation. Reperfusion injury of skeletal or cardiac muscle was markedly reduced by intraperitoneal or subcutaneous injection of recombinant human (rh)BCL2 protein or rhBCL2-related protein A1 (BCL2A1) (50 ng/g) given prior to ischemia or at the time of reperfusion. The cytoprotective activity of extracellular rhBCL2 or rhBCL2A1 protein was mapped to the BH4 domain, as treatment with a mutant BCL2 protein lacking the BH4 domain was not protective, whereas peptides derived from the BH4 domain of BCL2 or the BH4-like domain of BCL2A1 were. Protection by extracellular rhBCL2 or rhBCL2A1 was associated with a reduction in apoptosis in skeletal and cardiac muscle following I/R, concomitant with increased expression of endogenous mouse BCL2 (mBCL2) protein. Notably, treatment with rhBCL2A1 protein did not protect mice deficient in toll-like receptor-2 (TLR2) or the adaptor protein, myeloid differentiation factor-88 (MyD88). Conclusions/Significance Treatment with cytokine-like doses of rhBCL2 or rhBCL2A1 protein or BH4-domain peptides reduces apoptosis and tissue injury following I/R by a TLR2-MyD88-dependent mechanism. These findings establish a novel extracellular cytoprotective activity of BCL2 BH4-domain proteins as potent cytoprotective DAMPs.

Cyclin-Dependent Kinase Inhibitors Block Leukocyte Adhesion and Migration

Leukocyte trafficking is a tightly regulated process essential for an appropriate inflammatory response. We now report a new adhesion pathway that allows unstimulated leukocytes to adhere to and migrate through exposed endothelial matrix or high-density ligand, a process we have termed ligand-induced adhesion. This ligand-induced adhesion is integrin mediated, but in contrast to phorbol ester-stimulated adhesion, it is not dependent on the small GTPase Rap-1 activity. Instead, we show a critical role for cyclin-dependent kinase (Cdk) 4 in ligand-induced adhesion by three independent lines of evidence: inhibition by pharmacological inhibitors of Cdk, inhibition by dominant-negative construct of Cdk4, and inhibition by Cdk4 small interfering RNA. The major substrate of Cdk4, Rb, is not required for ligand-induced adhesion, suggesting the involvement of a novel Cdk4 substrate. We also demonstrate that Cdk4−/− mice have impaired recruitment of lymphocytes to the lung following injury. The finding that Cdk inhibitors can block leukocyte adhesion and migration may expand the clinical indications for this emerging class of therapeutics.

Extracellular Administration of BCL2 Protein Reduces Apoptosis and Improves Survival in a Murine Model of Sepsis

Background Severe sepsis and septic shock are major causes of morbidity and mortality worldwide. In experimental sepsis there is prominent apoptosis of various cell types, and genetic manipulation of death and survival pathways has been shown to modulate organ injury and survival. Methodology/Principal Findings We investigated the effect of extracellular administration of two anti-apoptotic members of the BCL2 (B-cell lymphoma 2) family of intracellular regulators of cell death in a murine model of sepsis induced by cecal ligation and puncture (CLP). We show that intraperitoneal injection of picomole range doses of recombinant human (rh) BCL2 or rhBCL2A1 protein markedly improved survival as assessed by surrogate markers of death. Treatment with rhBCL2 or rhBCL2A1 protein significantly reduced the number of apoptotic cells in the intestine and heart following CLP, and this was accompanied by increased expression of endogenous mouse BCL2 protein. Further, mice treated with rhBCL2A1 protein showed an increase in the total number of neutrophils in the peritoneum following CLP with reduced neutrophil apoptosis. Finally, although neither BCL2 nor BCL2A1 are a direct TLR2 ligand, TLR2-null mice were not protected by rhBCL2A1 protein, indicating that TLR2 signaling was required for the protective activity of extracellularly adminsitered BCL2A1 protein in vivo. Conclusions/Significance Treatment with rhBCL2A1 or rhBCL2 protein protects mice from sepsis by reducing apoptosis in multiple target tissues, demonstrating an unexpected, potent activity of extracellularly administered BCL2 BH4-domain proteins.

Neutrophil membrane sulfhydryl groups are involved in stimulated neutrophil adherence to endothelium.

We investigated the role of membrane sulfhydryl groups in adherence of stimulated polymorphonuclear neutrophils to cultured endothelial cells. Treatment of neutrophils with p‐chloromercuriphenyl sulfonate (PCMPS), a slowly penetrating sulfhydryl reagent, inhibited phorbol myristate acetate (PMA)‐ or calcium ionophore A23187‐stimulated adherence to cultured human or bovine endothelial cells. At concentrations which completely blocked PMA‐stimulated adherence, PCMPS did not cause release of lactic dehydrogenase, inhibit PMA‐mediated degranulation or hydrogen peroxide production, or prevent the PMA‐induced increased surface expression of CD11b/CD 18 (Mac‐1). Coincubation with a competing reduced sulfhydryl compound protected neutrophils from inhibition of PMA‐stimulated adherence by PCMPS, whereas coincubation with an oxidized sulfhydryl compound did not. Monobromotrimethylammoniobimane, a nonpenetrating sulfhydryl reagent that is structurally unrelated to PCMPS, also inhibited stimulated neutrophil adherence to endothelium cultures. We conclude that stimulated neutrophil adherence to endothelium involves neutrophil membrane protein sulfhydryl groups.

Role of Cdk4 in lymphocyte function and allergen response

We recently described a new adhesion pathway in lymphocytes that is dependent on Cyclin-dependent kinase (Cdk) 4 activity and mediates lymphocyte interactions with endothelial matrix. We showed that Cdk4-/- mice had impaired recruitment of lymphocytes following bleomycin model of acute lung injury. In this study, we characterized the development and function of hematopoietic cells in Cdk4-/- mice and assessed the response of Cdk4-/- mice to allergen challenge. Cdk4-/- mice had hypoplastic thymuses with decreased total thymocyte cell numbers and increased CD4/CD8 double negative cells. Cdk4-/- bone marrow (BM) chimeric mice showed similar findings. Thymocytes from either Cdk4-/- or Cdk4-/- BM chimeric mice proliferated equally well as wild type controls in response to IL-2 activation. However Cdk4-/- thymocytes had decreased adhesion to both endothelial cell matrix and fibronectin compared to wildtype (WT) controls, whereas Cdk4-/- and WT splenocytes had similar adhesion. When Cdk4-/- BM chimeric mice and wild type BM chimeric mice were sensitized and challenged by intranasal administration of ovalbumin, we found no differences in allergic responses in the lung and airways between the two groups, as measured by inflammatory cell infiltrate, airway hyperreactivity, IgE levels and cytokine levels. In summary, we show that Cdk4 plays a previously unrecognized role in thymocyte maturation and adhesion, but is not required for thymocyte proliferation. In addition, Cdk4 is not required for lymphocyte trafficking to the lung following allergen sensitization and challenge.