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The U.S. physician-scientist (PS) workforce is invaluable to the nation's biomedical research effort. It is through biomedical research that certain diseases have been eliminated, cures for others have been discovered, and medical procedures and therapies that save lives have been developed. Yet, the U.S. PS workforce has both declined and aged over the last several years. The resulting decreased inflow and outflow to the PS pipeline renders the system vulnerable to collapsing suddenly as the senior workforce retires. In November 2015, the Alliance for Academic Internal Medicine hosted a consensus conference on the PS workforce to address issues impacting academic medical schools, with input from early-career PSs based on their individual experiences and concerns. One of the goals of the conference was to identify current impediments in attracting and supporting PSs and to develop a new set of recommendations for sustaining the PS workforce in 2016 and beyond. This Perspective reports on the opportunities and factors identified at the conference and presents five recommendations designed to increase entry into the PS pipeline and nine recommendations designed to decrease attrition from the PS workflow.

Purpose: In 1995, the American Board of Internal Medicine (ABIM) formalized an integrated residency curriculum including both clinical and research training (the Research Pathway), designed to develop careers of physician-scientists. Individuals who completed Pathway training between 1995 and 2007 were surveyed to determine the extent to which graduates established research-oriented careers., Method: In 2012, the authors used a Web-based, 56-question, multiple-choice electronic survey of 813 participants in Research Pathway programs who completed their residency training between the years of 1995 and 2007. Survey questions addressed source and type of funding, research productivity, and job title/content. Descriptive and inferential analyses were performed., Results: Forty-seven percent of solicited Pathway graduates participated in the survey. Ninety-seven percent of the respondents completed Pathway training. Ninety-one percent reported some research effort, with a group average of 58.6% of total professional effort spent in research. Seventy-two percent currently hold positions in academic medicine; 8.6% in the biomedical industry; and 2.1% in government medical service. Over 85% reported extramural research funding, with 81.4% receiving research support from federal sources. Among the variables positively correlated with the highest level of research engagement were previous graduate-level research training, any first-author publications arising from the Pathway research experience, and the receipt of extramural career development funding supporting the Pathway research., Conclusions: On the basis of a very high level of active research engagement reported by 385 ABIM Research Pathway graduates, this special research training track appears to be effectively meeting its goal of training biomedical scientists.

The author was privileged to be an early contributor to the concept that cell adhesion molecules, the leukocyte (β2) integrins, play a pivotal role in the acute inflammatory process. For the author, this began with the development of a monoclonal antibody (anti-Mo1) that identified a differentiation antigen on the surface of human myeloid cells (including neutrophils, monocytes, and natural killer (NK) cells). Serendipitously, it was discovered that the Mo1 antigen was the heterodimeric glycoprotein (gp155,95) absent from the surface of neutrophils isolated from patients with adhesion defects in vitro and a syndrome characterized by chronic, life-threatening infections in vivo (a syndrome now termed leukocyte adhesion deficiency, type 1) (LAD-1). Collaborative efforts with other investigators (including members of the ACCA) revealed that patients with LAD-1 exhibited genetic mutations on chromosome 21 resulting in absent or diminished expression of a class of 4 surface adhesion molecules (now termed CD11a/CD18, CD11b/CD18, CD11c/CD18, and CD11d/CD18) known as the leukocyte or β2 family of integrins. Knowledge of the role of the β2 integrins in the acute inflammatory response led to the development of effective gene therapy strategies to treat LAD-1 in preclinical animal models and to the comprehensive testing of anti-integrin antibodies as anti-inflammatory agents to prevent organ damage as a complication of acute inflammation. This retrospective provides one illustration of the potential of bench-to-bedside research to generate new knowledge of clinical significance.

A survey of directors of adult and pediatric hematology/oncology subspecialty training programs in the United States and Canada was conducted to assess the environment in which recruitment and training is conducted in these medical disciplines. A total of 107 program directors responded to the survey, representing 66% of internal medicine and 47% of pediatric subspecialty programs in hematology or hematology/oncology. Specific areas covered in the web-based questionnaire included the type and demographics of the training program, profile of the training program director, characteristics of the applicant pool and existing trainee recruits, characteristics of the training program environment and curricula, research productivity of trainees, and the career pathways taken by recent training program graduates (including dominant areas of clinical interest). The results of this survey show considerable heterogeneity in the recruiting practices and the environment in which subspecialty training occurs, leading the authors to recommend improvements in or a heightened attention to issues, including recruitment of minority trainees, flexibility to recruit international medical school graduates, timing of trainee acceptance, maintaining the financial support of Medicare graduation medical education (GME), training of physician scientists, organization of the continuity clinic experience, visibility of nonmalignant hematology as a career path, and level of training program director support.

Previous studies have shown that the urokinase-type plasminogen activator receptor (uPAR) is localized to the adherence sites of leukocytes and tumor cells suggesting that pericellular proteolysis may accompany focal activation of adherence. To assess for focused pericellular proteolytic activity, we prepared two-dimensional substrates coated with FITC-casein or Bodipy FL-BSA. These molecules are poorly fluorescent, but become highly fluorescent after proteolytic degradation. Fluorescent peptide products were observed at adherence sites of stationary human neutrophils and at lamellipodia of polarized neutrophils. During cell migration, multiple regions of proteolysis appeared sequentially beneath the cell. Similarly, proteolytic action was restricted to adherence sites of resting HT1080 tumor cells but localized to the invadopodia of active cells. Using an extracellular fluorescence quenching method, we demonstrate that these fluorescent peptide products are extracellular. The uPA/uPAR system played an important role in the observed proteolytic activation. Plasminogen activator inhibitor-1 significantly reduced focal proteolysis. Sites of focal proteolysis matched the membrane distribution of uPAR. When uPA was dissociated from uPAR by acid washing, substantially reduced pericellular proteolysis was found. uPAR-negative T47D tumor cells did not express significant levels of substrate proteolysis. However, transfectant clones expressing uPAR (for example, T47D-26) displayed high levels of fluorescence indicating proteolysis at adherence sites. To provide further evidence for the role of the uPA/uPAR system in pericellular proteolysis, peritoneal macrophages from uPA knock-out (uPA-/-) and control (uPA+/+) mice were studied. Pericellular proteolysis was dramatically reduced in uPA-negative peritoneal macrophages. Thus, we have: (1). developed a novel methodology to detect pericellular proteolytic function, (2). demonstrated focused activation of proteolytic enzymatic activity in several cell types, (3). demonstrated its usefulness in real-time studies of cell migration, and (4). showed that the uPA/uPAR system is an important contributor to focal pericellular proteolysis.

Urokinase-type plasminogen activator receptor (uPAR) binding by the mannose 6-phosphate/insulin-like growth factor II receptor (Man-6-P/IGF2R) is considered important to Man-6-P/IGF2R tumor suppressor function via regulation of cell surface proteolytic activity. Our goal was to map the uPAR binding site of the Man-6-P/IGF2R by analyzing the uPAR binding characteristics of a panel of minireceptors containing different regions of the Man-6-P/IGF2R extracytoplasmic domain. Coimmunoprecipitation assays revealed that soluble recombinant uPAR (suPAR) bound the Man-6-P/IGF2R at two distinct sites, one localized to the amino-terminal end of the Man-6-P/IGF2R extracytoplasmic domain (repeats 1-3) and the other to the more carboxyl-terminal end (repeats 7-9). These sites correspond with the positions of the two Man-6-P binding domains of Man-6-P/IGF2R. Indeed, the suPAR-Man-6-P/IGF2R interaction was inhibited by Man-6-P, and binding-competent su-PAR species represented a minor percentage (8-30%) of the suPAR present. In contrast, Man-6-P/IGF2R binding of endogenous, full-length uPAR solubilized from plasma membranes of the prostate cancer cell line, PC-3, was not inhibited by Man-6-P. Further studies showed that very little (<5%) endogenous uPAR was Man-6-P/IGF2R binding-competent. We conclude that, contrary to previous reports, the interaction between uPAR and Man-6-P/IGF2R is a low percentage binding event and that suPAR and full-length uPAR bind the Man-6-P/IGF2R by different mechanisms.

Age-related macular degeneration (ARMD), proliferative vitreoretinopathy (PVR) and uveitis are characterized by RPE motility through the ECM of retinal lesions. The purpose of this study was to test the hypothesis that multiple proteolytic systems are functionally intact at the HRPE surface and peri-cellular region and that these activities are differentially modulated by IL-1beta. HRPE cells were evaluated: (1). as individual cells or cell extracts, (2). during migration across three-dimensional ECM-like layers and (3). in tissue sections. The urokirase plasminogen activator receptor (uPAR; CD87) was detected on HRPE cells as well as its functional activity. Although uPAR was associated with CD11b (CR3) on live resting cells, polarized migratory HRPE cells were found to dissociate uPAR from CR3; uPAR then translocated to anterior pole of the cell, where it enhanced PAI-1-inhibitable local proteolytic activity. The relative contribution of uPAR and collagenase in HRPE migration was evaluated using three-dimensional gelatin matrices. Interestingly, uPAR/uPA was found to play a key role in migration across these layers. IL-1 upregulated uPAR, collagenase, and elastase activities, suggesting that cytokines may affect the invasive program of HRPE cells in vivo. Immunohistochemistry for uPAR was performed in sections of human retina. Immunoreactive uPAR was present along the HRPE basolateral membrane in retinal sections and in sections of diseased retinal tissue at an enhanced level. Our results suggest that multiple proteolytic systems are present in association with HRPE and that the uPAR/uPA system may be particularly important.

A lectin function within CD11b mediates both cytotoxic priming of Mac-1/complement receptor type 3 (CR3) by beta-glucan and the formation of transmembrane signaling complexes with GPI-anchored glycoproteins such as CD16b (FcgammaRIIIb). A requirement for GPI-anchored urokinase plasminogen activator receptor (uPAR; CD87) in neutrophil adhesion and diapedesis has been demonstrated with uPAR-knockout mice. In this study, neutrophil activation conditions generating high-affinity (H-AFN) or low-affinity (L-AFN) beta(2) integrin adhesion were explored. A role for the Mac-1/CR3 lectin domain and uPAR in mediating H-AFN or L-AFN adhesion was suggested by the inhibition of Mac-1/CR3-dependent adhesion to ICAM-1 or fibrinogen by beta-glucan or anti-uPAR. The formation of uPAR complexes with Mac-1/CR3 activated for L-AFN adhesion was demonstrated by fluorescence resonance energy transfer. Conversely, Jurkat cell LFA-1 H-AFN-adhesion to ICAM-1 was not associated with uPAR/LFA-1 complexes, any requirement for GPI-anchored glycoproteins, or inhibition by beta-glucan. A single CD11b lectin site for beta-glucan and uPAR was suggested because the binding of either beta-glucan or uPAR to Mac-1/CR3 selectively masked two CD11b epitopes adjacent to the transmembrane domain. Moreover, treatment with phosphatidylinositol-specific phospholipase C that removed GPI-anchored proteins increased CD11b-specific binding of (125)I-labeled beta-glucan by 3-fold and this was reversed with soluble recombinant uPAR. Conversely, neutrophil activation for generation of Mac-1/CR3/uPAR complexes inhibited CD11b-dependent binding of (125)I-labeled beta-glucan by 75%. These data indicate that the same lectin domain within CD11b regulates both the cytotoxic and adhesion functions of Mac-1/CR3.

Integrins participate in many aspects of immunologic and inflammatory responses, especially those involving cell migration, adherence, and activation. Although leukocyte integrins such as complement receptor type 3 (CR3) are known to carry out certain functions without the intervention of other plasma membrane receptors, many plasma membrane proteins are now known to physically interact and functionally cooperate with integrins. Several of these interactions are highly dynamic within cell membranes; thus integrin-partner protein interactions change during certain physiological processes. This allows an extraordinary adaptability of the system to prime and promote proinflammatory signaling. Since our discovery of the CR3-FcgammaRIIIB interaction, the plasma membrane protein repertoire of beta1, beta2, and beta3 integrins has grown to include: FcgammaRIIA (CD32), uPAR (urokinase-type plasminogen activator receptor; CD87), CD14, voltage-gated K+channels (Kv1.3), integrin-associated protein (IAP), CD98, tetraspans (TM4SF), insulin receptors, and PDGFbeta receptors. In this article we will highlight certain features of this growing field of research, especially with regard to their relevance in immunology and inflammation.

Signaling motifs located within the cytoplasmic domain of certain receptors contribute to lysosome fusion. Most studies have described lysosome fusion with respect to endocytic receptors. Phagolysosome fusion has not been extensively studied. To test the hypothesis that the tail of FcgammaRIIA participates in phagolysosomal fusion, a "reverse" genetic complementation system was used. It was previously shown that complement receptor type 3 (CR3) can rescue the phagocytic activity of a mutant FcgammaRIIA lacking its cytoplasmic domain (tail-minus form). This system has allowed us to study Fcgamma receptor-dependent phagocytosis and phagolysosome fusion in the presence and absence of the cytoplasmic domain of FcgammaRIIA. Fluorescent dextran was used to label lysosomes. After target internalization, wild-type FcgammaRIIA-mediated phagolysosome formation was observed as indicated by colocalization of fluorescent dextran and the phagosome. In addition, when studying mutants of FcgammaRIIA containing a full-length cytoplasmic tail with the 2 ITAM tyrosines mutated to phenylalanine, (1) phagocytosis was abolished, (2) CR3 restored phagocytosis, and (3) lysosomal fusion was similar to that observed with the wild-type receptor. In contrast, in the presence of CR3 and the tail-minus form of FcgammaRIIA, internalized particles did not colocalize with dextran. Electron microscopy revealed that the lysosomal enzyme acid phosphatase colocalized with immunoglobulin G-coated targets internalized by wild-type FcgammaRIIA but not by tail-minus FcgammaRIIA and CR3. Thus, the tail of FcgammaRIIA contributes to phagolysosome fusion by a mechanism that does not require a functional ITAM sequence.

The cellular localization of human cytokeratin 1 (CK1), urokinase plasminogen activator receptor (uPAR), and gC1qR, high-molecular-weight kininogen (HK)-binding proteins on endothelial cells, was determined. CK1 was found on the external membrane of nonpermeabilized endothelial cells by immunoperoxidase staining, immunofluorescence, and transmission electron microscopy using immunogold. Human umbilical vein endothelial cells (HUVECs) had 7.2 +/- 0.2 x 10(4) specific CK1 membrane sites/cell by (125)I-F(ab')(2) anti-CK1 antibody binding. Flow cytometry studies confirmed the presence of CK1, uPAR, and gC1qR on HUVECs. On laser scanning confocal microscopy and transmission electron microscopy, CK1 and uPAR, but not gC1qR, colocalized on the cell surface of HUVECs. The HUVEC surface distribution of these proteins was distinctly different from that for von Willebrand factor. In competitive inhibition experiments, anti-CK1, anti-uPAR, or anti-gC1qR blocked both biotin-HK binding and prekallikrein (PK) activation on HUVECs with an inhibitory concentration of 50% (IC(50)) of 300 to 350 nM, 50 to 60 nM, or 35 to 100 nM, respectively. Also, antibodies to uPAR and gC1qR each inhibited 86% of kallikrein-mediated, 2-chain urokinase plasminogen activation, whereas antibodies to CK1 only inhibited 24% of plasminogen activation. On HUVECs, CK1 and uPAR, but not gC1qR, colocalized to be a multiprotein receptor complex for HK binding, PK activation, and 2-chain urokinase plasminogen activation.

Purpose: High-dose radiation may improve outcomes in non-small-cell lung cancer (NSCLC). By using three-dimensional conformal radiation therapy and limiting the target volume, we hypothesized that the dose could be safely escalated., Materials and Methods: A standard phase I design was used. Five bins were created based on the volume of normal lung irradiated, and dose levels within bins were chosen based on the estimated risk of radiation pneumonitis. Starting doses ranged from 63 to 84 Gy given in 2.1-Gy fractions. Target volumes included the primary tumor and any nodes >or= 1 cm on computed tomography. Clinically uninvolved nodal regions were not included purposely. More recently, selected patients received neoadjuvant cisplatin and vinorelbine., Results: At the time of this writing, 104 patients had been enrolled. Twenty-four had stage I, four had stage II, 43 had stage IIIA, 26 had stage IIIB, and seven had locally recurrent disease. Twenty-five received chemotherapy, and 63 were assessable for escalation. All bins were escalated at least twice. Although grade 2 radiation pneumonitis occurred in five patients, grade 3 radiation pneumonitis occurred in only two. The maximum-tolerated dose was only established for the largest bin, at 65.1 Gy. Dose levels for the four remaining bins were 102.9, 102.9, 84 and 75.6 Gy. The majority of patients failed distantly, though a significant proportion also failed in the target volume. There were no isolated failures in clinically uninvolved nodal regions., Conclusion: Dose escalation in NSCLC has been accomplished safely in most patients using three-dimensional conformal radiation therapy, limiting target volumes, and segregating patients by the volume of normal lung irradiated.

This year the Hematology Grants Workshop, chaired by Dr. Todd, includes a comprehensive listing of available National Institutes of Health, Department of Veterans Affairs, and non-federal grants applicable to fellows and junior faculty as well as to established investigators. In Section II, Dr. Miller discusses the essential principles of successful grant writing with a special emphasis on the young investigator. He highlights the best strategies to take and the common mistakes to avoid. In Section III, Dr. Silverstein outlines the structure of the current NIH Integrated Review Group (IRG) system and the study sections of the most relevance to hematology. He traces the path that a grant takes from review to funding including the way in which grants are reviewed at NIH Study Section Meetings and provides advice in the preparation of revised applications.

Exposure to environmental heavy metals has been reported to affect the immune system. Here, we tested the hypothesis that Hg(+2), acting through membrane proteins, disrupts metabolic dynamics and downstream cell functions in human neutrophils. We found that HgCl(2) inhibited: (1) polarization and (2) immunoglobulin (Ig)G-mediated phagocytosis of sheep erythrocytes in a dose-dependent manner from 2.5 to 10 microM. Because these activities have been linked with pro-inflammatory signalling, we also studied the effects of HgCl(2) on intracellular signalling by measuring protein tyrosine phosphorylation. HgCl(2) at doses = 1 microM increased tyrosine phosphorylation. We also studied the effect of HgCl(2) on neutrophil metabolism by measuring NAD(P)H autofluorescence as an indicator of intracellular NAD(P)H concentration. After HgCl(2) treatment, we found that normal sinusoidal NAD(P)H oscillations became incoherent. We recently reported that the NAD(P)H oscillation frequency is affected by cell migration and activation, which can in turn be regulated by integrin-mediated signalling. Therefore, we examined the effects of HgCl(2) on cell surface distribution of membrane proteins. After exposure to environmentally relevant concentrations of HgCl(2) we found that CR3, but not other membrane proteins (e.g. uPAR, Fc gamma RIIA and the formyl peptide receptor), became clustered on cell surfaces. We suggest that HgCl2 disrupts integrin signalling/functional pathways in neutrophils.

Numerous opportunities exist for successful careers in the fields of hematology and oncology. Trainees must choose between dedicated training in one or both disciplines, and between careers in private practice or academic medicine. Individuals who favor academic careers must decide between patient-oriented clinical investigation and laboratory research. This article addresses the factors that influence these decisions and inform trainees about critical elements of the training experience that foster successful career development in academic medicine and private practice. Counsel on employment opportunities is provided along with advice on evaluating the merits of employment offers.

Leukocytes use urokinase receptors (uPAR; CD87) in adhesion, migration, and proteolysis of matrix proteins. Typically, uPAR clusters at cell-substratum interfaces, at focal adhesions, and at the leading edges of migrating cells. This study was undertaken to determine whether uPAR clustering mediates activation signaling in human polymorphonuclear neutrophils. Cells were labeled with fluo-3/AM to quantitate intracellular Ca2+ ([Ca2+]i) by spectrofluorometry, and uPAR was aggregated by Ab cross-linking. Aggregating uPAR induced a highly reproducible increase in [Ca2+]i (baseline to peak) of 295 +/- 37 nM (p = 0.0002). Acutely treating cells with high m.w. urokinase (HMW-uPA; 4000 IU/ml) produced a response of similar magnitude but far shorter duration. Selectively aggregating uPA-occupied uPAR produced smaller increases in [Ca2+]i, but saturating uPAR with HMW-uPA increased the response to approximate that of uPAR cross-linking. Cross-linking uPAR induced rapid and significant increases in membrane expression of CD11b and increased degranulation (release of beta-glucuronidase and lactoferrin) to a significantly greater degree than cross-linking control Abs. The magnitude of degranulation correlated closely with the difference between baseline and peak [Ca2+]i, but was not dependent on the state of uPA occupancy. By contrast, selectively cross-linking uPA-occupied uPAR was capable of directly inducing superoxide release as well as enhancing FMLP-stimulated superoxide release. These results could not be duplicated by preferentially cross-linking unoccupied uPAR. We conclude that uPAR aggregation initiates activation signaling in polymorphonuclear neutrophils through at least two distinct uPA-dependent and uPA-independent pathways, increasing their proinflammatory potency (degranulation and oxidant release) and altering expression of CD11b/CD18 to favor a firmly adherent phenotype.

In this study we have re-examined the molecular mechanisms involved in activation of T cells by dendritic cells (DC). Human peripheral blood DC (PBDC) were derived by 2 h adhesion followed by 7 day culture in a combination of granulocyte macrophage colony stimulating factor and IL-4, and depletion of residual T and B cells. These PBDC were used to induce autologous T cell proliferation in a CD3-dependent response, and antibodies against CD11a/18 and CD86 were used as control inhibitors of accessory function. Antibodies against five of the cell surface molecules that we have recently identified on the surface of DC, CD13, CD87, CD98, CD147 and CD148, and an antibody which recognizes a molecule that has not as yet been identified, all inhibited the CD3-induced T cell proliferation. These findings were observed not only when antibodies were present throughout the culture, but also when they were prepulsed on to the surface of the DC, suggesting the inhibition was mediated via the antigen-presenting cells rather than the T cell. The same set of antibodies also inhibited an allospecific mixed lymphocyte reaction, confirming that the inhibitory effect was not dependent on the use of a CD3 antibody as the stimulating agent. All the antibodies of known specificity inhibited both CD4 and CD8 T cells equally. Unlike CD87, CD98 and CD147 antibodies, which inhibited activation of both CD45RA (naive) T cells and CD45RO (memory) T cells, CD13 and CD148 appeared to be involved in activation of naive cells only. The molecules identified in this study have not previously been demonstrated to play a role as accessory molecules on DC, the cells that are pivotal for immune induction. Therefore they may provide new potential targets for modulation of the immune response at the APC level.

Previous studies have shown that Ebola virus' secretory glycoprotein (sGP) binds to Fc gamma RIIIB (CD16b) and inhibits L-selectin shedding. In this study, we test the hypothesis that sGP interferes with the physical linkage between CR3 and Fc gamma RIIIB. Neutrophils were stained with rhodamine-conjugated anti-CD16b mAb (which does not inhibit sGP binding) and fluorescein-conjugated anti-CR3 mAb reagents and then incubated in media with or without sGP. Physical proximity between fluorochrome-labeled CR3 and Fc gamma RIIIB on individual cells was measured by resonance energy transfer (RET) imaging, quantitative RET microfluorometry, and single-cell imaging spectrophotometry. Cells incubated with control supernatants displayed a significant RET signal, indicative of physical proximity (<7 nm) between CR3 and Fc gamma RIIIB. In contrast, cells exposed to sGP showed a significant reduction in the CR3-Fc gamma RIIIB RET signal using these methods. Interestingly, colocalization and cocapping of CR3 and Fc gamma RIIIB were not affected, suggesting that the proximity of these two receptors is reduced without triggering dissociation. Thus, sGP alters the physical linkage between Fc gamma RIIIB and CR3.

This year the Hematology Grants Workshop, chaired by Dr. Robert Todd, includes a comprehensive listing of available NIH, VA, and non-federal grants applicable to fellows and junior faculty as well as to established investigators. In Section II, Dr. Donald Miller discusses the essential principles of successful grant writing with a special emphasis on the young investigator. He highlights the best strategies to take and the common mistakes to avoid. In Section III, Dr. Roy Silverstein outlines the structure of the current NIH Integrated Review Group (IRG) system and the study sections of the most relevance to hematology. He traces the path that a grant takes from review to funding including the way in which grants are reviewed at NIH Study Section Meetings and provides advice in the preparation of revised applications.

Leukocytes utilize urokinase receptors (uPAR; CD87) in adhesion, migration, and matrix proteolysis. uPAR aggregate at cell-substratum interfaces and at leading edges of migrating cells, so this study was undertaken to determine whether uPAR aggregation is capable of initiating activation signaling. Monocyte-like U937 cells were labeled with fluo-3-acetoxymethyl ester to quantitate intracellular Ca2+ concentrations ([Ca2+]i) by spectrofluorometry, and uPAR was aggregated by mAb cross-linking. uPAR aggregation induced highly reproducible increases in [Ca2+]i of 103.0 +/- 10.9 nM (p < 0.0001) and >3-fold increases in cellular d-myoinositol 1,4,5-trisphosphate (Ins(1,4,5)P3) levels. Similar increases in [Ca2+]i were also elicited by uPAR aggregation in human monocytes, but cross-linking a control IgG2a had no effect on [Ca2+]i. Selectively cross-linking uPA-occupied uPAR with an anti-uPA mAb produced smaller increases in [Ca2+]i, but fully saturating uPAR with exogenous uPA enhanced the [Ca2+]i response to equal the effect of aggregating uPAR directly. Increased [Ca2+]i was inhibited by thapsigargin, herbimycin A, and U73122, but only partially reduced by low extracellular [Ca2+], indicating that uPAR aggregation increases [Ca2+]i by activating phospholipase C through a tyrosine kinase-dependent mechanism, generating Ins(1,4,5)P3 and releasing Ca2+ from Ins(1,4, 5)P3-sensitive intracellular stores. Cross-linking the beta2 integrin CR3 could not duplicate the effect of uPAR cross-linking, and uPAR-triggered Ca2+ mobilization was not blocked by anti-CR3 mAbs. These results indicate that uPAR aggregation initiates phosphoinositide hydrolysis by mechanisms that are not strictly dependent on associated uPA or CR3.

Urokinase-type plasminogen activator receptor (UPA-R-CD87) is a GPI-anchored membrane protein which promotes the generation of plasmin on the surface of many cell types, probably facilitating cellular extravasation and tissue invasion. A flow cytometric quantitative analysis of expression levels for UPA-R was performed on fresh blast cells from patients with acute myeloid leukaemia (AML, n = 74), acute lymphoblastic leukaemia (ALL, n = 24), and biphenotypic leukaemia (BAL, n = 3) using two CD87 monoclonal antibodies (McAbs) (3B10 and VIM5). Peripheral blood and bone marrow (BM) cells from 15 healthy adults served as controls. Using 3B10 McAb, UPA-R was expressed (>99%) by blood monocytes, neutrophils, and BM myelomonocytic precursors in controls, whereas resting T and B lymphocytes, and CD34+ cells were UPA-R negative. We also attempted to clarify whether UPA-R has a role in mediating neutrophil functions. Oriented locomotion induced by different chemotaxins and lysozyme release by granules stimulated with fMLP or PMA were significantly decreased when UPA-R was neutralized by CD87 McAb. In contrast, the anti-UPA-R McAb had no effect on superoxide anion generation of normal neutrophils. Blasts from AML showed a heterogenous pattern of expression for the UPA-R McAbs, with reactivity strictly dependent on FAB subtype. The highest UPA-R expression was seen in the M5 group: all patients tested (n = 20) showed strong positivity for the UPA-R McAb whereas only 12% (3/24) of ALL patients were CD87 positive, and 2/3 of BAL patients showed a dim expression for CD87. The number of receptors expressed by blast cells in 6/74 (8.1%) AML patients was higher than those of normal samples: in addition, since co-expression of UPA-R and CD34 was not found in normal haemopoietic cells, it may be postulated that CD87 can be used alone (when overexpressed) or in combination with CD34 for the detection of minimal residual disease. Results also indicated that patients with UPA-receptors >12 x 10(3) ABC/cell, irrespective of FAB subtype, had a greater tendency for cutaneous and tissue infiltration and a higher frequency of chromosome abnormalities, thus suggesting the concept that cellular UPA-R content positively correlates with the invasive potential of AML cells. The combination of higher UPA-R positivity, abnormalities of chromosome 11, and M5 FAB morphology may identify a peculiar subset of AML, characterized by a more aggressive clinical course.

Having previously associated metabolic oscillations with cell locomotion, we hypothesized that patients with abnormalities in neutrophil trafficking may display aberrant intracellular oscillations. A pyoderma gangrenosum patient exhibiting aberrant leukocyte trafficking in vivo and skin ulceration without infection was identified. This patient's neutrophils constitutively overexpressed and clustered the leukocyte integrins CR3 and CR4 and failed to display appropriate integrin-to-GPI receptor interactions. Increased levels of tyrosine phosphorylation were observed. NAD(P)H oscillations, which are sinusoidal in normals, were chaotic with multiple frequency components in this patient's neutrophils. Normal cell shape and sinusoidal NAD(P)H oscillations were restored by providing a pulsed electric field to drive metabolic oscillations and by temperature reduction. N-acetyl-D-glucosamine disrupted CR3 clusters and sinusoidal NAD(P)H oscillations returned. Anecdotal reports suggest that local hypothermia is clinically useful for this patient. These data define the first metabolic oscillation-associated disease and suggest that pyoderma gangrenosum can be classified as a dynamical disease at the cellular level.

The receptor for urokinase plasminogen activator (uPAR; CD87) is a 50- to 65-kDa glycosylphosphatidylinositol (GPI)-anchored glycoprotein expressed by leukocytes and tumor cells where it facilitates uPA-dependent, plasmin-mediated pericellular proteolysis during cellular invasion. Because uPAR is inducibly shed into culture supernatants and human body fluids, we tested the hypothesis that soluble uPAR (suPAR) can bind to the plasma membrane of hematopoietic cells where it might modulate their invasive phenotype. As measured by flow cytometry, recombinant biotinylated-suPAR (B-suPAR) bound in a specific fashion to THP-1 leukemia cells and blood PMNs and monocytes (but not to lymphocytes). B-suPAR also demonstrated specific binding to a variety of leukemic lines, including cells that are positive or negative for membrane uPAR expression. Binding of B-suPAR to THP-1 cells was enhanced four- to sevenfold by 24-h exposure of cells to PMA or by co-incubation with uPA ligand (but not its isolated catalytic and binding fragments). Conversely, binding of B-suPAR to PMNs was unaffected by brief exposure to fMLP, and was inhibited by co-incubation with uPA. B-suPAR biding to PMA-differentiated THP-1 cells in the presence of uPA was further enhanced by acid washing (removing endogenous uPA) but was partially inhibited by treatment of cells with trypsin. Pretreatment of PMA-differentiated THP-1 cells and unstimulated PMNs with soluble sugars, calcium chelators, and antibodies specific for integrins or extracellular matrix proteins failed to consistently block the binding of B-suPAR. Whereas the binding of suPAR did not measurably affect cell-associated plasmin activation, suPAR did competitively inhibit the binding of exogenous uPA to membrane-associated uPAR. These observations support the hypothesis that suPAR can bind specifically to trypsin-sensitive receptors expressed by certain normal and neoplastic hematopoietic cells where its binding is variably influenced by uPA ligand.

Adhesion to extracellular matrices is known to modulate leukocyte activation, although the mechanisms are not fully understood. Mononuclear phagocytes are exposed to fibrinous provisional matrix throughout migration into inflammatory foci, so this study was undertaken to determine whether fibrinogen triggers activation of NF-kappa B transcription factors. U937 cells differentiated with PMA in nonadherent culture were shown to express two fibrinogen-binding integrins, predominately CD11b/CD18, and to a lesser extent, CD11c/CD18. Cells stimulated with fibrinogen (10-100 microg/ml)/Mn2+ (50 microM) for 2 h were examined by electrophoretic mobility shift assay. NF-kappa B activation, minimal in unstimulated cells, was substantially up-regulated by fibrinogen. Fibrinogen also caused activation of AP-1, but not SP1 or cAMP response element-binding protein (CREB) factors. Blocking mAbs against CD18 and CD11b abrogated fibrinogen-induced NF-kappa B activation. To determine the effects on transcriptional regulation, U937 cells were transfected with a plasmid containing the HIV-1 enhancer (bearing two NF-kappa B sites) coupled to a chloramphenicol acetyltransferase (CAT) reporter. Cells were subsequently stimulated with 1) PMA for 24 h, inducing CAT activity by 2.6-fold, 2) fibrinogen/Mn2+ for 2 h, inducing CAT activity by 3.2-fold, or 3) costimulation with fibrinogen and PMA, inducing 5.7-fold the CAT activity induced by PMA alone. We conclude that contact with fibrinogen-derived proteins may contribute to mononuclear phagocyte activation by signaling through CD11b/CD18, resulting in selective activation of transcriptional regulatory factors, including NF-kappa B.

We investigated the relationship between the respiratory burst in neutrophils and the membrane distribution of the IgG receptor, Fc gamma RIII. Fc gamma RIII receptors were labeled with a fluoresceinated antibody that does not block binding of immune complexes. The respiratory burst was detected using covalently bound rosamine stain previously described for flow cytometric applications. This method allows visualization of intracellular oxidant production in fixed cells using attenuated illumination with a laser. Strong cytosolic oxidation of rosamine was observed only in those cells that displayed prominent receptor endocytosis upon interaction with insoluble immune complexes. Soluble immune complexes or insoluble complexes in the presence of cytochalasin B did not stimulate endocytosis of Fc gamma RIII and induced no rosamine oxidation. Extracellular superoxide production measured by the cytochrome c test did not correlate with intracellular rosamine oxidation: it was maximal in cytochalasin-treated cells and did not require any visible receptor rearrangement. Our results demonstrate the utility of the rosamine stain as an intracellular marker of the oxidative burst, support the role of Fc gamma RIII in neutrophil activation and emphasize the compartmental regulation of the oxidative burst.

We have previously shown that the beta2 integrins CR3 and CR4 physically and functionally interact with urokinase receptors (uPAR) on neutrophil plasma membranes in an oscillatory fashion. In this study we have analyzed neutrophils from patient SC, a 34 y old African American female, with aberrant skin window results and recurrent perianal abscesses and pretibial lesions diagnosed as pyoderma gangrenosum. Although untreated migrating normal neutrophils exhibited 20 s sinusoidal oscillations in CR4-uPAR proximity, neutrophils from SC demonstrated a faster oscillation (10 s) in the form of a flyback sawtooth wave. This waveform mimicked that observed for normal neutrophils treated with subsaturating doses of the kinase inhibitors staurosporine, genistein, and erbstatin. As beta2 integrins are regulated by phosphorylation, we tested the hypothesis that the aberrant CR4-uPAR proximity oscillations seen in SC's neutrophils are due to defective kinase activity that might be balanced by a decrease in phosphatase activity. When SC's cells are exposed to subsaturating concentrations of the phosphatase inhibitor pervanadate, this caused the CR4-uPAR oscillations to become sinusoidal in shape with a 20 s period, as seen in normal migrating neutrophils. Although SC's neutrophils were deficient in spontaneous and N-formyl-methionyl-leucyl-phenylalanine-induced polarization, 0.5 microM pervanadate returned cell polarization to nearly normal levels, thus paralleling the acquisition of normal receptor interactions. Inasmuch as SC's cellular phenotype is mimicked by kinase inhibitors and corrected by phosphatase inhibitors, we suggest that a mutation(s) affecting the kinetics of intracellular signaling enzymes, but not blocking the pathway per se, may be responsible for this clinical state.

Migrating neutrophils utilize beta2 integrins for substrate attachment and urokinase receptors (uPAR) to focus pericellular proteolysis. Our studies show that CR3 associates with uPAR on resting cells, whereas uPAR associates with CR4 at lamellipodia of migrating cells. Using resonance energy transfer (RET) microscopy, we show that the molecular proximity between CR4 and uPAR oscillates on migrating cells, thus suggesting that CR4 molecules periodically bind/release uPAR. Cell contact with fibrinogen, endothelial cells, chemotactic factors and indomethacin, and treatment with sub-optimal doses of signal transduction inhibitors, affect the oscillations' period, amplitude, and/or waveform. The oscillations were indistinguishable in period and 180 degrees out-of-phase with cytosolic NAD(P)H autofluorescence oscillations. Thus, CR4 and CR3 identify a neutrophil's axis of migration and CR4 may restrain uPAR at lamellipodia. Oscillations in signal transduction and energy metabolism may coordinate cell adherence, local proteolysis, oxidant release, actin assembly, and cell extension.

Although glycosylphosphatidyl-inositol (GPI) linked membrane proteins do not possess transmembrane or cytosolic sequences they elicit transmembrane signals. Using microscopic fluorescence imaging and resonance energy transfer (RET) techniques we have shown that certain pro-inflammatory GPI-linked membrane proteins can interact with leukocyte beta 2 integrins (complement receptor type 3 (CR3) and 4 (CR4) and the leukocyte function-associated antigen-1 (LFA-1)). For example, physical associations between CR3 and Fc gamma RIIIB, CR3 and urokinase receptors, and CR3 and CD14 (lipopolysaccharide receptor) have been found. Although Fc gamma RIIIB appears to be constitutively associated with CR3, urokinase receptors and CD14 associations with CR3 are influenced by their ligation status and cell function (e.g. adherence and locomotion). CR3-to-urokinase receptor interactions have been confirmed by immunoprecipitation techniques. Immunoprecipitation of CR3 from Brij-58 lysates after biotinylation of neutrophil membranes revealed proteins of M(r) = 40,000, 50,000, 74,000 and 120,000, in addition to bands corresponding to the integrin alpha and beta chains. Cell functions such as transmembrane signaling and superoxide release/priming have been linked to these interactions. Importantly, reagents that affect the lectin-like site of CR3, such as N-acetyl-D-glucosamine, alpha-methyl-D-mannoside and beta-glucan alter these interactions and, in parallel, leukocyte functions. Thus, the interactions of GPI-linked proteins and integrins can be highly dynamic events linked to cell activities. Our studies suggest that it may be possible to develop new drugs directed at the lectin-like site of beta 2 integrins that block GPI-linked protein-to-integrin coupling thereby controlling inflammatory cell processes including cell adherence, locomotion and activation. Such drugs may be useful in clinical conditions such as ischemia-reperfusion injury, sepsis, arthritis and others.

Fig. 1 depicts our current thinking about the ways in which Mo1 and p150,95 form cis interactions with other leukocyte receptors. With respect to the associations of Mo1 with Fc gamma RIIIB and uPAR, the inhibitory effect of saccharides such as NADG suggests a lectin-carbohydrate interaction that may involve the recognition of Mo1's beta-glucan site for N-linked carbohydrates4 that are expressed by both Fc gamma RIIIB and uPAR. This hypothesis is supported by the results of Stockl et al., who showed that the binding of C-terminal-specific mAb VIM12 to Mo1, which enhances the phospholipase C-mediated release of Fc gamma RIIIB, was inhibited by NADG. However, unlike the sample lectin-carbohydrate interaction that appears to govern the association between Mo1 and Fc gamma RIIIB, effective Mo1-dependent uPAR signaling also depends on the binding of intact uPA to uPAR (the receptor-binding ATF of uPA proving insufficient to prime neutrophils for an enhanced burst response to FMLP). We speculate that ATF (residues 6-135) binds to uPAR while the carboxyl terminal fragment (residues 136-411), which includes a glycosylation site at residue 144, binds to the lectinlike site of Mo1, thus fostering the linkage between the two receptors. In support of this model is the fact that exposure of neutrophils to ATF reduced the degree of molecular proximity between Mo1 and uPAR (the latter probably occupied by endogenous intact uPA) and increased the molecular association between Mo1 and Fc gamma RIIIB (both as detected by quantitative RET). This hypothesis is analogous to the concept proposed by Nykjaer et al in which plasminogen activator inhibitor-1 initially binds to uPA to form a complex that secondarily binds to the alpha 2 macroglobulin receptor, leading to internalization of the complex. Whereas the contribution of intact uPA to the interaction between Mo1 and uPAR remains speculative (based on the indirect data available), no such ambiguity exists for the role of the LPS/LBP ligand in regulating the association between Mo1 and CD14. In this circumstance, no physical linkage exists between the two receptors without the ligand complex. This observation is consistent with the previously described affinity of the beta 2 integrins for LPS, leading to the notion that the LPS portion of the LPS/LPB complex binds to Mo1, serving to link it with LPS/LBP bound to CD14. The observed reversibility of the interactions between the integrin glycoproteins and uPAR or CD14 illustrates the fact that these associations can be highly dynamic and tied to cellular processes that include directed motility (Mo1-uPAR), adherence to substrates (Mo1-CD14), and energy metabolism (p150,95-uPAR). We speculate that the GPI-anchored receptor proteins serve as rapidly diffusible, expendable "scouts" for the beta 2 integrins, which serve to expand their ligand binding repertoire in a cis-acting fashion.

We have shown previously that the urokinase-type plasminogen activator receptor (uPAR) physically associates with beta2 integrins on human leukocyte membranes. We now report that uPAR associates with certain members of the beta1 and beta3 integrin families expressed by a nonhematopoietic fibrosarcoma cell line (HT1080) when adherent to certain extracellular matrix molecules. Flow cytometry studies indicated that HT1080 cells expressed uPAR and beta1 and beta3 integrins. Double staining immunofluorescence was used to label uPAR and beta1 and beta3 integrins. The staining patterns of uPAR and beta1 integrins were strikingly similar when attached to fibronectin, laminin, or vitronectin but not polylysine-coated substrates. Resonance energy transfer (RET) between uPAR and beta1 integrins was observed, especially at focal adhesion plaques; this indicates that these molecules are within about 7 nm of each other on these cell membranes. uPAR and beta3 integrin coclustering and RET were also observed on tumor cells adherent to vitronectin but not to fibronectin, laminin, or polylysine-coated surfaces. Because N-acetyl-D-glucosamine was found previously to inhibit beta2 integrin-uPAR association, we tested the effect of saccharides on the beta1-uPAR and beta3-uPAR colocalization and RET. Colocalization and RET between uPAR and beta1 or beta3 integrins were effectively inhibited by N-acetyl-D-glucosamine on extracellular matrix-coated surfaces. To better define which members of beta1 and beta3 integrin families associate with uPAR, we studied the association of several alpha subunits with uPAR on tumor cells. We found that: (a) alpha5 colocalizes with uPAR on cells attached to fibronectin-coated surfaces; (b) alpha5 and alpha(v) colocalize with uPAR on cells adherent to vitronectin; and (c) alpha3 and alpha6 associate with uPAR on cells attached to laminin. In further support of physical associations between integrins and uPAR on tumor cells, uPAR was found to coimmunoprecipitate with beta1 integrins in Brij-58 lysates of HT1080 cells (as detected by anti-uPAR Western blotting of material isolated from an anti-beta1 integrin immunoaffinity column). Thus, uPAR may laterally associate with integrins of tumor cells when attached to specific extracellular matrix elements to enable directional proteolysis for tumor cell migration and invasion.

We report a novel method of flow cytometric detection of the oxidative burst in human neutrophils. The cells were covalently labeled on the plasma membrane by incubation with 4-carboxydihydrotetramethylrosamine succinimidyl ester on ice. Activation of neutrophils resulted in an increase in red fluorescence at 575 nm using 514 nm excitation. The sensitivity of this assay in detecting the response to chemotactic peptide N-formyl-met-leu-phe (FMLP) and phorbol 12-myristate 13-acetate (PMA) was comparable to that observed with the cytochrome c oxidation test. We compared the new method to previously described methods using intracellular fluorescent stains. Our method showed the lowest nonspecific oxidation and the best sensitivity to FMLP-induced activation. It was equally or slightly less efficient than the green fluorescent stain dihydrorhodamine 123 in detection of neutrophil activation by immune complexes and PMA but much more sensitive than red fluorescent stains hydroethidine and dihydrotetramethylrosamine. It can be successfully used in kinetic experiments and yields a fluorescence signal that remains stable for at least 1.5 h after formaldehyde fixation with antioxidant added.

Previous studies have suggested that complement receptors cooperate with Fc receptors to mediate Ab-dependent effector functions. In the present study, we tested the capacity of complement receptor type 3 (CR3; alphaM beta2; CD11b/CD18) to participate in Fc gammaRIIA (CD32)-mediated phagocytosis. To test this hypothesis, we transfected a phagocytosis-defective tail-minus mutant of Fc gammaRIIA into fibroblasts that did or did not express CR3. As controls, cells exposed to the transfection protocol but not expressing either CR3 or Fc gammaRIIA constructs were also selected. Expression of cell surface receptors was confirmed by both flow cytometry and fluorescence microscopy. Cells were tested for their ability to bind and internalize IgG-opsonized erythrocytes and for surface proximity of CR3 and Fc gammaRIIA using resonance energy transfer techniques. Resonance energy transfer studies showed a physical proximity between CR3 and Fc gammaRIIA on cell surfaces. Cells expressing only the tail-minus mutant of Fc gammaRIIA were unable to internalize erythrocytes but showed significant binding ability. In contrast, cells expressing both mutant Fc gammaRIIA and CR3 internalized opsonized erythrocytes. These results show that CR3 can complement the phagocytic function of defective Fc gammaRIIA. These data suggest that CR3 is capable of transducing a phagocytic signal generated by Fc gammaRIIA recognition events that mediates internalization of IgG-opsonized targets.

Recent studies suggest physical and functional interactions of glycosylphosphatidylinositol (GPI)-linked proteins (CD14, CD16b and CD87) with leukocyte beta 2 integrins. As discussed in this article, it now appears that beta 2 integrins relay proinflammatory information from GPI-anchored membrane receptors to the cytoplasm via exodomain interactions.

Urokinase receptors (uPAR; CD87) from complexes with complement receptor 3 (CR3) (CD11b/CD18), a beta2 integrin. In this study, we sought to determine if this association modulates the adhesive function of CR3. Both CR3 and uPAR concentrate at the ventral surface of fibrinogen-adherent human monocytes, and CR3-uPAR coupling increases substantially upon adhesion to fibrinogen. Pretreatment with anti-uPAR monoclonal antibody reduced adhesion to CR3 counterligands (fibrinogen and keyhole limpet hemocyanin) by 50%, but did not affect adhesion to fibronectin, a beta1 integrin counterligand. Antisense (AS) oligonucleotides were used to determine if selectively suppressing uPAR expression also modulates CR3 adhesive function. AS-uPAR oligo reduced CR3-dependent adhesion by 43+/-9% (P<0.01), but did not affect CR3-independent adhesion. To determine if the effects of uPAR are mediated through its ligand, monocytes were pre-treated with AS oligo to block uPA expression. Unlike the effects of blocking uPAR expression, AS-uPA oligo increased adhesion by 46% (P<0.005), and exogenous intact uPA, but not uPA fragments, reversed this effect. We conclude that complex formation with uPAR facilitates the adhesive functions of CR3. This function of uPAR is not dependent upon its occupancy with uPA, which negatively influences adhesion.

CD14, a glycosylphosphatidyl inositol (GPI)-linked membrane protein, is a key membrane binding site for LPS (endotoxin). Although CD14 lacks transmembrane and cytoplasmic sequences, it activates CR3-mediated leukocyte adhesion and cytokine release. Since CR3 has been shown to interact with other GPI-linked membrane proteins, we tested the hypothesis that CD14 can physically associate with CR3. Using qualitative and quantitative resonance energy transfer microscopy, we show that LPS in the presence of serum or LPS binding protein triggers formation of CD14-CR3 complexes. Kinetic studies show that CD14-CR3 complexes dissociate as neutrophils attach to substrates. We speculate that LPS-charged CD14 enhances CR3-mediated adhesion by directly binding to CR3.

Previous studies have shown that the leukocyte integrin CR3 (CD11b/CD18) is physically associated with the urokinase-type plasminogen activator receptor (uPAR;CD87), a glycosyl-phosphatidylinositol (GPI)-linked protein, in resting neutrophil membranes. We now show that uPAR-to-CR3 interactions are reversible, correlating with cell shape. Neutrophils were first labeled with fluorescein conjugates of anti-CR3 F(ab')2 fragments followed by capping using a second-step F(ab')2 directed against murine F(ab')2s. Cells were then probed using rhodamine-conjugated anti-uPAR F(ab')2s. Although uPAR co-caps with CR3 on resting cells, uPAR was found to dissociate or "uncap" coincident with spontaneous cell polarization for migration. CR3 caps transformed into uropods while uPAR accumulated at lamellipodia of polarized cells. Capping was unnecessary for the observed distribution of CR3 and uPAR since the anti-CR3 and anti-uPAR F(ab')2s traffic to the uropod and lamellipodium, respectively, during polarization of uncapped cells. These receptors reassociate when cells return to a spherical morphology. In contrast to uPAR, Fc gamma RIIIB did not dissociate from CR3 caps during cell polarization. Resonance energy transfer (RET) microscopy was used to image the spatial distribution of RET and to follow the kinetics of association and dissociation. Initial levels of RET dramatically fell during cell polarization, but did not change on cells fixed with paraformaldehyde. Receptor reassociation was a biphasic process with initial reassociation about the perimeter of a cap, followed by a plateau and a slower rise in RET within a cap. We suggest that cells regulate receptor-receptor associations depending upon their physiologic activities.

Human cell lines of myelo/monocytic origin express the cellular receptor for urokinase-type plasminogen activator (uPA-R). The receptor localizes urokinase-type plasminogen activator (uPA) to the surface of the cell, where it can convert plasminogen to the active serine proteinase plasmin. Plasmin may subsequently account for proteolysis of pericellular proteins. We demonstrated the expression of the uPA-R by freshly isolated neutrophilic granulocytes by using a specific mAb. In freshly isolated granulocytes we detected only a weak occupation of the uPA-R; further uPA binding by granulocytes was saturable and proceeded in a dose-dependent manner. Receptor-bound uPA retained its enzymatic activity. Saturation of isolated granulocytes with exogenous uPA enhanced cellular infiltration into a fibrin matrix in vitro. uPA-dependent infiltration was inhibited by an anti-catalytic monoclonal anti-uPA antibody. The findings show that circulating neutrophilic granulocytes express the cell surface uPA-R and suggest that surface-binding of uPA may facilitate the infiltration of granulocytes into a fibrin clot, a process that might add to thrombolysis in vivo.

During recruitment, leukocytes respond to chemotaxins and traverse matrix barriers. Urokinase-type plasminogen activator (uPA), bound to its receptor (uPAR; CD87) facilitates plasmin formation, which promotes matrix proteolysis. Polymorphonuclear leukocytes (PMNs) are critical to the inflammatory response and express both uPA and CD87. To determine whether uPA and CD87 are required for PMN chemotaxis, PMNs were pretreated with an anti-CD87 monoclonal antibody (mAb), a neutralizing anti-uPA mAb, or uPA. PMN chemotaxis was profoundly suppressed by the anti-CD87 mAb but was unaffected by anti-uPA mAb or uPA. The role CD87 plays in chemotaxis may be related to its ability to associate with CR3. CD87/CR3 coupling can be disrupted by specific saccharides. The same saccharides that disrupt CD87/CR3 coupling (NADG, D-mannose, and mannoside) inhibit PMN chemotaxis. We conclude that CD87 plays a crucial role in PMN chemotaxis in vitro that is independent of uPA enzyme activity but may be related to the ability of CD87 to interact with CR3.

Cultured keratinocytes resemble migrating keratinocytes under conditions of reepithelialization during wound healing. Such keratinocytes express urokinase-type plasminogen activator (uPA) and its specific receptor (uPA receptor). Receptor-bound uPA activates plasminogen, thus providing plasmin for pericellular proteolysis. uPA is regulated by the plasminogen activator inhibitors PAI-1 and PAI-2. As indicated by immunohistology, neither uPA nor uPA receptor is expressed in normal epidermis. Thus, the down-regulation of uPA and uPA-receptor expression in keratinocytes appears to be an important event in epidermal healing and restoration of a normal epidermal tissue architecture. We have addressed this matter by using a culture and differentiation system for keratinocytes in vitro. Keratinocytes were grown in organotypic cocultures for 4, 7, and 14 days. Frozen sections were analyzed with indirect immunofluorescence staining and overlay zymography, the latter detecting activity of plasminogen activators. While tPA and PAI-1 stainings were consistently negative over the entire observation period, uPA and uPA receptor were expressed by basal keratinocytes at Days 4 and 7, but not at Day 14. Accordingly, overlay zymography revealed uPA activity at Days 4 and 7. PAI-2 was found throughout the entire observation period, but with varying distribution: at Days 4 and 7 all suprabasal keratinocytes stained positive for PAI-2. At Day 14, PAI-2-specific stainings were confined to the uppermost cells of the stratum spinosum. Our data demonstrate that uPA and uPA receptor, which are up-regulated in cultured keratinocytes, are down-regulated upon restoration of an epidermis-like structure. The distribution of PAI-2 varied over the observation period and at Day 14 resembled the distribution of PAI-2 in normal epidermis. Taken together, keratinocytes in organotypic coculture behave like keratinocytes in healing wounds in vivo with respect to the expression of the plasminogen activator system.

To explore potential inter-receptor interactions between Fc gamma RIIIB, a GPI-linked protein, and the leukocyte integrin CR3, we have prepared transfected 3T3 fibroblast cell lines expressing Fc gamma RIIIB, CR3, or both Fc gamma RIIIB and CR3. We test the hypothesis that Fc gamma RIIIB and CR3 are physically associated in membranes using fluorescence recovery after photobleaching (FRAP) and resonance energy transfer (r.e.t.) microscopy. Cells expressing Fc gamma RIIIB alone displayed a diffusion coefficient (D) of 3.4 x 10(-9) (+/- 2.9 x 10(-9) cm2/second and a mobile fraction (m.f.) of 0.73 (+/- 0.10). In contrast, Fc gamma RIIIB exhibited D = 2.5 x 10(-9) (+/- 1.4 x 10(-9) cm2/second (n.s.) and a m.f. of 0.48 (+/- 0.08) (p < 0.01) on cells expressing both Fc gamma RIIB and CR3, thus indicating that co-expression of CR3 constrains the lateral diffusion of Fc gamma RIIIB. To further test for a direct physical interaction between these gene products, (r.e.t.) microscopy was performed. Donor-labeled anti-CR3 and acceptor-labeled anti-Fc gamma RIIIB on cells expressing both receptors yielded a r.e.t. photon count rate of 8.9(+/- 6.4) kilocounts/second (kC/s), whereas CR3-to-CR3 measurements gave 1.6(+/- 0.6) kC/s (p < 0.01). Moreover, the addition of exogenous agents such as N-acetyl-D-glucosamine, but not indomethacin, diminished the magnitude of these interactions in transfectant membranes. These data support the notion that a subpopulation of Fc gamma RIIIB is physically associated with CR3 and that this association can be affected by exogeneous compounds.

Urokinase-type plasminogen activator (uPA), which binds to cells via a specific receptor (uPAR), participates in pericellular proteolysis during leukocyte migration. Previous studies have indicated that uPAR is physically associated with CR3 (CD11b/CD18). To test the functional interactions of CR3 and uPAR, we have examined the ability of uPA to elicit changes in cytosolic calcium levels of normal neutrophils, neutrophils from a leukocyte adhesion deficiency (LAD) patient, and 3T3 transfectants expressing CR3, uPAR, or both. We found that calcium levels of neutrophils increased from 106 +/- 6 nM in untreated cells to 199 +/- 25 nM in the presence of uPA. In contrast, no significant change in calcium was observed when neutrophils from an leukocyte adhesion deficiency patient were examined. The uPA-dependent calcium rise was inhibited by mAb directed against either CR3 or uPAR and required intact uPA. To substantiate further these findings, we prepared transfectants expressing genes encoding uPAR, CR3, and both receptors; only cells expressing both receptors experienced a rise in intracellular calcium. Although uPA's calcium signal is insufficient to trigger superoxide production, FMLP dose-dependent superoxide production was greatly enhanced by incubating neutrophils with intact, but not fragmented, uPA. Flow cytometry experiments utilizing an FMLP analogue exclude the possibilities that urokinase binds to the FMLP receptor or up-regulates its expression. We suggest that calcium is a second messenger of uPA, that this message is mediated in a CR3-dependent fashion, and that this signal primes neutrophils for superoxide production.