Endothelial cells undergoing apoptosis become proadhesive for nonactivated platelets.

Under normal conditions, platelets do not adhere to endothelium. However, when platelets or endothelial cells are stimulated by thrombin or cytokines, respectively, platelets bind avidly to endothelium. Because there is accumulating evidence that endothelial cells may become apoptotic under certain proinflammatory or prothrombotic conditions, we investigated whether endothelial cells undergoing apoptosis may become proadhesive for nonactivated platelets. Human umbilical vein endothelial cells (HUVEC) were induced to undergo apoptosis by staurosporine, a nonspecific protein kinase inhibitor, or by culture in suspension with serum-deprivation. After treatment of HUVEC or platelets with different receptor antagonists, nonactivated, washed human platelets were allowed to adhere to HUVEC for 20 minutes. To exclude matrix involvement, platelet binding was measured in suspension by using flow cytometry. Independent of the method of apoptosis induction, there was a marked increase in platelet binding to apoptotic HUVEC. Although HUVEC exhibited maximal adhesiveness for platelets after 2 to 4 hours, complete DNA fragmentation of HUVEC occurred only several hours later. Adhesion assays after blockade of different platelet receptors showed only involvement of beta1-integrins. Platelet binding to apoptotic HUVEC was inhibited by more than 70% when platelets were treated with blocking anti-beta1 antibodies. Treatment of apoptotic HUVEC with blocking antibodies to different potential platelet receptors, including known ligands for beta1-integrins, did not affect platelet binding. As assessed by determination of beta-thromboglobulin and platelet factor 4 in the supernatants, platelets bound to apoptotic HUVEC became slightly activated. However, significant expression of platelet P-selectin (CD62P) was not found. These data provide further evidence that endothelial cells undergoing apoptosis may contribute to thrombotic events.

A novel beta 1 integrin-dependent mechanism of leukocyte adherence to apoptotic cells.

Adherence of leukocytes to cells undergoing apoptosis has been reported to be dependent on a variety of recognition pathways. These include alpha V beta 3 (CD51/CD61, vitronectin receptor), CD36 (thrombospondin receptor), macrophage class A scavenger receptor, phosphatidylserine translocated to the outer leaflet of apoptotic cell membranes, and CD14 (LPS-binding protein). We investigated the mechanism by which leukocytes adhere to apoptotic endothelial cells (EC). Peripheral blood mononuclear leukocytes and U937 monocytic cells adhered to human or bovine aortic EC induced to undergo apoptosis by withdrawal of growth factors, treatment with the promiscuous protein kinase inhibitor staurosporine, with the protein synthesis inhibitor and protein kinase activator anisomycin, or with the combination of cycloheximide and TNF-alpha. Expression of endothelial adherence molecules such as CD62E (E-selectin), CD54 (ICAM-1), and CD106 (VCAM-1) was not induced or increased by these treatments. A mAb to alpha V beta 3, exogenous thrombospondin, or blockade of phosphatidylserine by annexin V did not inhibit leukocyte adherence. Further, leukocyte binding to apoptotic EC was completely blocked by treatment of leukocytes but not EC with mAb to beta 1 integrin. These results define a novel pathway for the recognition of apoptotic cells.

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.

Retroviral-mediated gene transfer of the leukocyte integrin CD18 into peripheral blood CD34+ cells derived from a patient with leukocyte adhesion deficiency type 1.

Leukocyte adhesion deficiency or LAD is a congenital immunodeficiency disease characterized by recurrent bacterial infections in which the leukocytes from affected children fail to adhere to endothelial cells and migrate to the site of infection due to heterogeneous defects in the leukocyte integrin CD18 subunit. To assess the feasibility of human gene therapy of LAD, we transduced granulocyte colony-stimulating factor (G-CSF)-mobilized, CD34+ peripheral blood stem cells derived from a patient with the severe form of LAD using supernatant from the retroviral vector PG13/LgCD18. The highest transduction frequencies (31%) were found after exposure of the cells to retroviral vector on a substrate of recombinant fibronectin fragment CH-296 in the presence of growth factors interleukin-3 (IL-3), IL-6, and stem cell factor. When the phenotype of the transduced cells was monitored by fluorescence-activated cell sorting following in vitro differentiation with growth factors G-CSF and granulocyte-macrophage CSF (GM-CSF), CD11a surface expression was detected immediately after transduction. CD11b and CD11c were expressed at low levels immediately following transduction, but increased over 3 weeks in culture. Adhesion of the transduced cells was nearly double that of nontransduced cells in a cell adhesion assay using human umbilical vein endothelial cells. Transduced cells also demonstrated the ability to undergo a respiratory burst in response to opsonized zymosan, a CD11/CD18-dependent ligand. These experiments show that retrovirus-mediated gene transfer of the CD18 subunit complements the defect in LAD CD34+ cells resulting in CD11/CD18 surface expression, and that the differentiated myelomonocytic cells derived from the transduced LAD CD34+ cells display CD11/CD18-mediated adhesion function. These results indicate that ex vivo gene transfer of CD18 into LAD CD34+ cells, followed by re-infusion of the transduced cells, may represent a therapeutic approach to LAD.

Adhesion of activated platelets to endothelial cells: evidence for a GPIIbIIIa-dependent bridging mechanism and novel roles for endothelial intercellular adhesion molecule 1 (ICAM-1), alphavbeta3 integrin, and GPIbalpha.

Although it has been reported that activated platelets can adhere to intact endothelium, the receptors involved have not been fully characterized. Also, it is not clear whether activated platelets bind primarily to matrix proteins at sites of endothelial cell denudation or directly to endothelial cells. Thus, this study was designed to further clarify the mechanisms of activated platelet adhesion to endothelium. Unstimulated human umbilical vein endothelial cell (HUVEC) monolayers were incubated with washed, stained, and thrombin-activated human platelets. To exclude matrix involvement, HUVEC were harvested mechanically and platelet binding was measured by flow cytometry. Before the adhesion assay, platelets or HUVEC were treated with different receptor antagonists. Whereas blockade of platelet beta1 integrins, GPIbalpha, GPIV, P-selectin, and platelet-endothelial cell adhesion molecule (PECAM)-1 did not reduce platelet adhesion to HUVEC, blockade of platelet GPIIbIIIa by antibodies or Arg-Gly-Asp (RGD) peptides markedly decreased adhesion. Moreover, when platelets were treated with blocking antibodies to GPIIbIIIa-binding adhesive proteins, including fibrinogen and fibronectin, and von Willebrand factor (vWF), platelet binding was also reduced markedly. Addition of fibrinogen, fibronectin, or vWF further increased platelet adhesion, indicating that both endogenous platelet-exposed and exogenous adhesive proteins can participate in the binding process. Evaluation of the HUVEC receptors revealed predominant involvement of intercellular adhesion molecule (ICAM)-1 and alphavbeta3 integrin. Blockade of these two receptors by antibodies decreased platelet binding significantly. Also, there was evidence that a component of platelet adhesion was mediated by endothelial GPIbalpha. Blockade of beta1 integrins, E-selectin, P-selectin, PECAM-1, vascular cell adhesion molecule (VCAM)-1 and different matrix proteins on HUVEC did not affect platelet adhesion. In conclusion, we show that activated platelet binding to HUVEC monolayers is mediated by a GPIIbIIIa-dependent bridging mechanism involving platelet-bound adhesive proteins and the endothelial cell receptors ICAM-1, alphavbeta3 integrin, and, to a lesser extent, GPIbalpha.

Binding of human peripheral blood polymorphonuclear leukocytes to E-selectin (CD62E) does not promote their activation.

E-selectin (CD62E) is a cytokine-inducible endothelial cell adhesion molecule that tethers polymorphonuclear leukocytes (PMNs) and supports PMN rolling under conditions of flow. We examined whether interaction of PMNs with E-selectin also leads to activation of CD11b/CD18 (Mac-1, alphaMbeta2), an event that can promote firm adhesion. PMNs were added to monolayers of IL-1beta-activated HUVECs and Chinese hamster ovary (CHO) cells transfected with E-selectin cDNA. PMN activation was assessed by 1) increased CD11b/CD18 surface expression, 2) appearance of activation epitope on CD11b/CD18 (CD11b*) detected by mAb CBRM1/5, and 3) decreased L-selectin (CD62L) expression, as determined by flow cytometry. Both adherent and nonadherent supernatant PMNs became activated on IL-1beta-pretreated HUVECs. This activation was not affected by CD62E-blocking mAb P6E2. The activation state of PMNs adhered to CHO cells transfected with E-selectin cDNA was not increased over background and was similar to that of PMNs exposed to parent CHO cells. The findings were confirmed using confocal microscopy, which allowed staining of the cells for CD11b* in situ. In concert, the results suggest that PMN binding to E-selectin does not elicit inter-receptor signaling that could result in strengthening of PMN adhesion to endothelium.

Neutrophil adhesion in leukocyte adhesion deficiency syndrome type 2.

We have previously reported a newly discovered congenital disorder of neutrophil adhesion, leukocyte adhesion deficiency syndrome type 2 (LAD II). The clinical manifestations of this syndrome are similar to those seen in the classic leukocyte adhesion deficiency syndrome, now designated type 1 (LAD I), but the two syndromes differ in the molecular basis of their adhesion defects. LAD I is caused by a deficiency in the CD18 integrin adhesion molecules while LAD II patients are deficient in expression of sialyl-Lewis X (SLeX), a carbohydrate ligand for selectins. In this report we demonstrate that neutrophils from a LAD II patient bind minimally or not at all to recombinant E-selectin, purified platelet P-selectin, or P-selectin expressed on histamine-activated human umbilical vein endothelial cells, but have normal levels of L-selectin and CD11b/CD18 integrin, and adhere to and migrate across endothelium when CD11b/CD18 is activated. We compare LAD I and LAD II patient neutrophil function in vitro, demonstrating that integrin and selectin adhesion molecules have distinct but interdependent roles in neutrophil adhesion during an inflammatory response.

Cross-linking of sialophorin (CD43) induces neutrophil aggregation in a CD18-dependent and a CD18-independent way.

Normal human neutrophils bound an as yet unclustered mAb designated BS-1. The Ag immunoprecipitated with BS-1 was blotted by CD43 mAb (and vice versa), and is therefore identical to the large sialoglycoprotein. The CD43 Ag expression on the neutrophil surface is decreased upon neutrophil activation with the chemoattractant FMLP or with PMA. This can be (at least partially) explained by the release of CD43+ material with an altered electrophoretic mobility into the extracellular medium of the neutrophils upon activation. Cross-linking of the CD43 Ag with BS-1 also invoked neutrophil activation by itself: F(ab)2 fragments of BS-1-induced neutrophil aggregation, in contrast to F(ab) fragments. Neither respiratory burst activity nor a significant rise in intracellular Ca2+ level or actin polymerization were observed. The transient neutrophil aggregation response was largely CD18 dependent, especially in the initial phase of homotypic clustering. However, a significant CD18-independent mechanism contributed thereafter to the neutrophil aggregation, as was further substantiated by the use of cultured T (and EBV-transformed B) cell clones of a patient with a leukocyte adhesion deficiency. CD43 is the first molecule described on neutrophils able to induce adhesive properties in a dual fashion.

Benign cartilaginous tumors of the upper airway.

Benign cartilaginous neoplasms of the laryngotracheal apparatus are uncommon clinical entities. Two cases of cartilaginous lesions of the upper airway are reported. Resection with maintenance of upper airway structural integrity is the preferred treatment. Temporary tracheostomy is often necessary and can provide access for stenting of the tracheal repair.

Vascular cell adhesion molecule-1 mediates lymphocyte adherence to cytokine-activated cultured human endothelial cells.

The expression and function of a new cytokine-induced endothelial cell adhesion protein, vascular cell adhesion molecule-1 (VCAM-1), was characterized in vitro by using a monoclonal antibody, MoAb 4B9, which recognizes a functional epitope on this protein. As determined by enzyme-linked immunosorbent assay and radioimmunoprecipitation of metabolically labeled cells, VCAM-1 was minimally expressed on unstimulated human umbilical vein endothelium (HUVE), but was rapidly induced by recombinant human tumor necrosis factor-alpha (rhTNF-alpha), rh interleukin-1, and lipopolysaccharide. In contrast to intercellular adhesion molecule-1, VCAM-1 was not induced on dermal fibroblasts or arterial smooth muscle cells after stimulation with rhTNF, or on keratinocytes after stimulation with rh interferon-gamma. MoAb 4B9 significantly inhibited the adherence of peripheral blood lymphocytes (PBL) and lymphocytic cell lines, but not neutrophils, to rhTNF-activated HUVE. The inhibitory effect of MoAb 4B9 on PBL adherence to HUVE was additive to that produced by the CD18 MoAb 60.3. These results show that VCAM-1 mediates a CD18-independent pathway of peripheral blood lymphocyte adherence to cytokine-stimulated HUVE. We propose that lymphocyte binding to VCAM-1, induced on endothelium by cytokines, may be an important component of lymphocyte emigration at sites of inflammation or immune reaction.

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.

Phorbol ester causes down-regulation of CD11/CD18-independent neutrophil adherence to endothelium.

Neutrophils adhere to interleukin-1 (IL-1)-, tumour necrosis factor (TNF)- or lipopolysaccharide (LPS)-pretreated human umbilical vein endothelial cells (HEC) by CD11/CD18-dependent and independent mechanisms. We investigated CD11/CD18-independent neutrophil adherence to LPS-pretreated HEC by: (i) pretreating neutrophils with the anti-CD18 monoclonal antibody mAb 60.3; (ii) performing assays in the absence of Mg2; or (iii) using neutrophils isolated from a patient with leucocyte adhesion deficiency (CD11/CD18-deficiency). Under each of these conditions, CD11/CD18-independent neutrophil adherence to LPS-pretreated HEC was significantly greater than adherence to untreated HEC (15-18% versus 3-7%). In each case, however, stimulation of neutrophils with phorbol ester (PMA) abolished CD11/CD18-independent adherence to LPS-pretreated HEC (less than 5% adherence). Stimulation of neutrophils with bacterial chemotactic peptide (FMLP) or calcium ionophore (A23187) likewise reduced CD18-independent adherence to LPS-pretreated HEC. PMA also inhibited CD11/CD18-independent neutrophil adherence to HEC pretreated with IL-1 or TNF (80-90% inhibition). In contrast, PMA markedly enhanced CD11/CD18-dependent adherence to untreated or LPS-treated HEC. We conclude that stimulation of neutrophils with phorbol ester or other direct agonists down-regulates the CD11/CD18-independent mechanism of neutrophil adherence to IL-1, TNF- or LPS-pretreated HEC.

Continuous postoperative epidural analgesia in management of postoperative surgical pain.

In the last fifteen months we have used continuous postoperative epidural analgesia after open urologic surgery and herein report our experience with the first 64 patients. Incisional pain was completely eliminated in 96 percent of patients. Epidural analgesia diminished pain-related pulmonary complications without sedation. Complications were tolerable and manageable. Hypotension due to sympathetic blockade responds to intravenous fluid administration. Urinary retention is avoidable if the epidural infusion is discontinued prior to removing the urethral catheter. Itching is an undesirable consequence observed by 20 percent of patients when morphine is used.

Sulfhydryl reducing agents promote neutrophil adherence without increasing surface expression of CD11b/CD18 (Mac-1, Mo1).

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.

CD11/CD18-independent neutrophil adherence to inducible endothelial-leucocyte adhesion molecules (E-LAM) in vitro.

We examined the mechanisms involved in neutrophil adherence to cultured human umbilical vein endothelial cells (HEC) induced by direct stimulation of the neutrophils by phorbol myristate acetate (PMA), formylmethionyl-leucyl-phenylalanine (FMLP), or the calcium ionophore A23187 (neutrophil-dependent adherence), or by pretreatment of HEC with interleukin-1 (IL-1), tumour necrosis factor (TNF) or lipopolysaccharide (LPS) (endothelial-dependent adherence). Two distinct mechanisms for neutrophil adherence to HEC were demonstrated by performing adherence assays: (i) at 37 degrees versus 4 degrees; (ii) in the presence of Ca2+ only versus Mg2+ only; and (iii) in the presence or absence of monoclonal antibodies (mAb) to the CD11/CD18 adhesion complex of neutrophils. A CD11/CD18-dependent mechanism (i.e. inhibited by anti-CD18 mAb) was identified that was active in the presence of Mg2+ only but not of Ca2+ only, and at 37 degrees but not at 4 degrees. A CD11/CD18-independent mechanism (i.e. not inhibited by anti-CD18 mAb) was active at 4 degrees and at 37 degrees, and in the presence of Ca2+ only and of Mg2+ only. Neutrophil-dependent adherence induced by FMLP or PMA occurred solely via the CD11/CD18-dependent mechanism, whereas endothelial-dependent adherence induced by a 4-hr pretreatment with IL-1, TNF, or LPS involved both CD11/CD18-dependent and/independent mechanisms. CD11/CD18-deficient neutrophils isolated from a patient with leucocyte adherence deficiency (LAD) maintained the ability to adhere to LPS-pretreated HEC in the presence of Ca2+ only, indicating that this mechanism of adherence involves a receptor on the neutrophil distinct from CD11/CD18. Furthermore, the disappearance of the CD11/CD18-independent, but not of the CD11/CD18-dependent mechanism of adherence, in HEC treated with TNF for 24 hr suggests that the two mechanisms of neutrophil adherence also involve distinct inducible endothelial-leucocyte adhesion molecules (E-LAM).

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.

Cell proliferation in non-Hodgkin's lymphomas. Digital image analysis of Ki-67 antibody staining.

Ki-67 is a monoclonal antibody to a nuclear antigen present in cycling human cells but not in resting cells. The authors have performed immunoperoxidase on non-Hodgkin's lymphomas using Ki-67 antibody in order to correlate proliferation rates with tumor grade and type, and compare Ki-67 staining with S-phase content as determined by flow cytometry. Ki-67 staining of 109 sections was quantitated using a digital image analysis system (CAS 100). There was a significant difference among mean overall Ki-67 staining values in Working Formulation low (13.7%), intermediate (42.6%), and high grade tumors (57.9%, P less than 0.00001). The level of significance improved when a revised grading system was formulated based on proliferative activity, with the inclusion of diffuse large cell lymphomas in the high grade category. Within nodular and a few diffuse lymphomas, there were well-defined proliferation centers in which Ki-67 staining showed no correlation with grade. Flow cytometric DNA determination was performed on 74 specimens, and there was a positive correlation between Ki-67 positivity and S phase content (r = 0.66). It is concluded that Ki-67 staining of tissue sections is an alternative to flow cytometric quantitation of cell cycle activity in lymphomas, and provides the advantage of revealing histologic patterns of proliferation. By including G1 phase cells, Ki-67 staining allows a more complete determination of total cell cycle activity in lymphomas.

Expression of leukocyte adherence-related glycoproteins during differentiation of HL-60 promyelocytic leukemia cells.

We used the HL-60 human promyelocytic leukemia cell line to analyze the surface expression of a family of adherence-related leukocyte surface antigens during myeloid differentiation. These antigens are composed of discrete alpha subunits, designated alpha L, alpha M, and alpha X, that are each noncovalently associated with a common beta subunit. Monoclonal antibodies directed against the individual subunits served as markers in both indirect immunofluorescence studies and immunoprecipitations from HL-60 cells differentiated preferentially towards mature granulocytes (DMSO, retinoic acid) or monocyte/macrophages (PMA, vitamin D3). In undifferentiated HL-60 cells, the alpha L and alpha X subunits were constitutively expressed, whereas the alpha M subunit was not. Differentiation of HL-60 cells along the granulocytic pathway with DMSO resulted in a marked increase in alpha M and minimal increases in alpha L and alpha X. The phenotypic expression of these antigens on DMSO-treated HL-60 cells closely resembled that on normal circulating PMN. Differentiation along the monocyte/macrophage pathway when using PMA or vitamin D3 resulted in major increases in alpha L and alpha X expression, as well as alpha M. These changes resulted in a surface phenotype characteristic of that present on human monocyte-derived macrophages. Triggering of undifferentiated HL-60 cells with PMA caused no increase in subunit expression, whereas stimulation of DMSO-differentiated HL-60 cells with PMA produced more than a 1.5-fold enhancement of both the alpha M and alpha X subunits, and stimulation of human PMN with PMA increased the surface expression of alpha M more than fourfold and alpha X subunit twofold. Stimulation with PMA produced no change in expression of the alpha L subunit in any of the three cell populations. These results indicate that the alpha subunits of this glycoprotein family can be selectively regulated during in vitro differentiation of a human promyelocytic leukemia cell line. Second, DMSO-differentiated HL-60 cells and human PMN possessed an intracellular pool of alpha M and alpha X, but not alpha L, that could be translocated to the surface. Thus, despite structural and functional relationships among the alpha subunits in this glycoprotein family, they undergo disparate surface expression and intracellular regulation during differentiation.

Isolated congenital renal tubular immaturity in siblings.

This paper describes two cases of an unusual renal abnormality discovered in anuric siblings (one male, one female) who were born at 36 and 34 weeks of gestation and died of systemic complications secondary to severe pulmonary hypoplasia shortly after birth. Both gestations were complicated by marked oligohydramnios. Antenatal ultrasound examinations showed slightly enlarged kidneys in the first case and normal kidneys in the second case, with no evidence of hydronephrosis or cystic disease in either. With the exception of enlargement of the first infant's kidneys, autopsies revealed grossly unremarkable kidneys and ureters. Microscopy, however, demonstrated increased glomerulogenesis with normal glomeruli and global immaturity of renal tubules and ducts without concomitant features of dysplasia. Immunoperoxidase staining for epithelial membrane antigen revealed the immaturity or complete absence of proximal convoluted tubules. This precise constellation of findings had not been described previously. One other similar family has been documented in a report implicating genetic factors. In the present cases, the possibility of a cocaine-associated etiology is also addressed.

Monoclonal antibody-defined functional epitopes on the adhesion-promoting glycoprotein complex (CDw18) of human neutrophils.

We have evaluated the functional and immunochemical activities of three monoclonal antibodies (MoAbs) minimally reactive with adherence-defective neutrophils (PMN) from a patient with recurrent bacterial infections. In studies with normal PMN, MoAbs OKM1 and 60.1 both precipitate the same 165kd alpha-subunit (alpha M) within an alpha-beta heterodimer complex (CD11). The CD11 complex is part of a larger complex composed of four glycoproteins (CDw18) precipitated by MoAb 60.3, with properties suggesting that the CDw18 complex is equivalent to the Mac-1, LFA-1, p150, 95 glycoprotein family implicated in adherence-dependent leukocyte functions. PMN adherence to endothelium, spreading on surfaces, aggregation, and phagocytosis of zymosan particles were all inhibited in a dose-dependent fashion by MoAb 60.1 (analogous to previous studies with MoAb 60.3) while MoAb OKM1 had no effect. These findings unify previously disparate observations and suggest that a functionally active site on the adherence promoting glycoprotein complexes CD11 and CDw18 is distant from the alpha M epitope recognized by MoAb OKM1 but closely associated with the alpha M epitope recognized by MoAb 60.1 and the beta-epitope (or epitope created by alpha-beta quaternary structure) recognized by MoAb 60.3.