Anti-PR3 immune responses induce segmental and necrotizing glomerulonephritis.

Wegener's granulomatosis (WG) is a life-threatening autoimmune vasculitis that affects lungs, kidneys and other organs. A hallmark of WG is the presence of classic anti-neutrophil cytoplasmic antibodies (c-ANCA) against self-proteinase 3 (PR3). Little is known about the role of these antibodies and PR3-specific immune responses in disease development. In this study, we demonstrate that PR3-specific autoimmune responses are pathogenic in non-obese diabetic (NOD) mice with an impaired regulatory arm of the immune response. Immunization of autoimmunity prone NOD mice with rmPR3 (recombinant mouse PR3) in complete Freund's adjuvant (CFA) resulted in high levels of c-ANCA, without detectable disease development. However, when splenocytes from these immunized mice were transferred into immunodeficient NOD-severe combined immunodeficiency (SCID) mice, the recipient mice developed vasculitis and severe segmental and necrotizing glomerulonephritis. No disease developed in NOD-SCID mice that received splenocytes from the CFA-alone-immunized donors (controls), indicating that disease development depends upon PR3-specific immune responses. In contrast to the pathology observed in NOD-SCID mice, no disease was observed when splenocytes from rmPR3-immunized C57BL/6 mice were transferred into immunodeficient C57BL/6-RAG-1(-/-) mice, suggesting that complex and probably multi-genetic factors play a role in the regulation of disease development.

Recombinant integrin CD11b A-domain blocks polymorphonuclear cells recruitment and protects against skeletal muscle inflammatory injury in the rat.

The beta2 integrin CD11b/CD18 (CR3) is a major adhesion receptor of neutrophils, normally utilized to fend off infections. This receptor contributes, however, to multiple forms of non-infectious inflammatory injury when dysregulated as shown in gene knock-outs and through the use of blocking monoclonal antibodies. The major ligand recognition site of CR3 has been mapped to the A-domain in the CD11b subunit (CD11bA). The recombinant form of this domain exhibits a ligand binding profile similar to that of the holoreceptor. To assess the potential anti-inflammatory activity of CD11bA as a competitive antagonist of CR3 in vivo, we assessed its effects on a developed animal model of traumatic skeletal muscle injury in the rat. Recombinant soluble rat CD11bA-domain fused to glutathione-S-transferase (GST) was administered intravenously in a single dose at 1 mg/kg to nine groups of Wistar rats, five in each group, 30 min before inducing traumatic skeletal muscle injury. Control animals received either a function-blocking anti-CD11b/CD18 monoclonal antibody (1 mg/kg), non-functional mutant forms of the CD11bA (D140GS/AGA, T209/A, D242/A), recombinant GST or buffer alone. In control animals, the wounded muscle showed oedema, erythrocyte extravasation and myonecrosis both within and outside the immediate wounded area (5-10 mm zone) and influx of neutrophils was detected 30 min post-wound, followed by a second wave 3 hr later. Wild-type CD11bA- or anti-CD11b monoclonal antibody (mAb)-treated rats showed a comparable and significant decrease in the number of infiltrating PMN (78 + 4%, n = 70 and 86 +/- 2%, n = 50, respectively) and preservation of the muscular fibres outside the immediate zone of necrosis (75 + 4%, n = 70, 84 +/- 1%, n = 50, respectively), compared to controls. These data demonstrate that CD11bA can be an effective tissue-preserving agent in acute inflammatory muscular injury.

Integrin structure, allostery, and bidirectional signaling.

Alphabeta heterodimeric integrins mediate dynamic adhesive cell-cell and cell-extracellular matrix (ECM) interactions in metazoa that are critical in growth and development, hemostasis, and host defense. A central feature of these receptors is their capacity to change rapidly and reversibly their adhesive functions by modulating their ligand-binding affinity. This is normally achieved through interactions of the short cytoplasmic integrin tails with intracellular proteins, which trigger restructuring of the ligand-binding site through long-range conformational changes in the ectodomain. Ligand binding in turn elicits conformational changes that are transmitted back to the cell to regulate diverse responses. The publication of the integrin alphaVbeta3 crystal structure has provided the context for interpreting decades-old biochemical studies. Newer NMR, crystallographic, and EM data, reviewed here, are providing a better picture of the dynamic integrin structure and the allosteric changes that guide its diverse functions.

Integrins, cations and ligands: making the connection.

Integrins are cell adhesion receptors that couple extracellular divalent cation-dependent recognition events with intracellular mechanical and biochemical responses and vice versa, thus affecting every function of nucleated cells. The structural basis of this bidirectional signaling and its dependency on cations has been the focus of intensive study over the past three decades. Significant progress made recently in elucidating the three-dimensional structure of the extracellular and cytoplasmic segments of integrins is giving valuable new insights into the tertiary and quaternary changes that underlie activation, ligand recognition and signaling by these receptors.

Polycystin-1 interacts with intermediate filaments.

Polycystin-1, the protein defective in a majority of patients with autosomal dominant polycystic kidney disease, is a ubiquitously expressed multi-span transmembrane protein of unknown function. Subcellular localization studies found this protein to be a component of various cell junctional complexes and to be associated with the cytoskeleton, but the specificity and nature of such associations are not known. To identify proteins that interact with the polycystin-1 C-tail (P1CT), this segment was used as bait in a yeast two-hybrid screening of a kidney epithelial cell library. The intermediate filament (IF) protein vimentin was identified as a strong polycystin-1-interacting partner. Cytokeratins K8 and K18 and desmin were also found to interact with P1CT. These interactions were mediated by coiled-coil motifs in polycystin-1 and IF proteins. Vimentin, cytokeratins K8 and K18, and desmin also bound directly to P1CT in GST pull-down and in in vitro filament assembly assays. Two observations confirmed these interactions in vivo: (i) a cell membrane-anchored form of recombinant P1CT decorated the IF network and was found to associate with the cytoskeleton in detergent-solubilized cells and (ii) endogenous polycystin-1 distributed with IF at desmosomal junctions. Polycystin-1 may utilize this association for structural, storage, or signaling functions.

Crystal structure of the extracellular segment of integrin alpha Vbeta3.

Integrins are alphabeta heterodimeric receptors that mediate divalent cation-dependent cell-cell and cell-matrix adhesion through tightly regulated interactions with ligands. We have solved the crystal structure of the extracellular portion of integrin alphaVbeta3 at 3.1 A resolution. Its 12 domains assemble into an ovoid "head" and two "tails." In the crystal, alphaVbeta3 is severely bent at a defined region in its tails, reflecting an unusual flexibility that may be linked to integrin regulation. The main inter-subunit interface lies within the head, between a seven-bladed beta-propeller from alphaV and an A domain from beta3, and bears a striking resemblance to the Galpha/Gbeta interface in G proteins. A metal ion-dependent adhesion site (MIDAS) in the betaA domain is positioned to participate in a ligand-binding interface formed of loops from the propeller and betaA domains. MIDAS lies adjacent to a calcium-binding site with a potential regulatory function.

Polycystin-2, the protein mutated in autosomal dominant polycystic kidney disease (ADPKD), is a Ca2+-permeable nonselective cation channel.

Defects in polycystin-2, a ubiquitous transmembrane glycoprotein of unknown function, is a major cause of autosomal dominant polycystic kidney disease (ADPKD), whose manifestation entails the development of fluid-filled cysts in target organs. Here, we demonstrate that polycystin-2 is present in term human syncytiotrophoblast, where it behaves as a nonselective cation channel. Lipid bilayer reconstitution of polycystin-2-positive human syncytiotrophoblast apical membranes displayed a nonselective cation channel with multiple subconductance states, and a high perm-selectivity to Ca2+. This channel was inhibited by anti-polycystin-2 antibody, Ca2+, La3+, Gd3+, and the diuretic amiloride. Channel function by polycystin-2 was confirmed by patch-clamping experiments of polycystin-2 heterologously infected Sf9 insect cells. Further, purified insect cell-derived recombinant polycystin-2 and in vitro translated human polycystin-2 had similar ion channel activity. The polycystin-2 channel may be associated with fluid accumulation and/or ion transport regulation in target epithelia, including placenta. Dysregulation of this channel provides a mechanism for the onset and progression of ADPKD.

Molecular genetics and pathogenesis of autosomal dominant polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) is a common and systemic disease characterized by formation of focal cysts. Of the three potential causes of cysts, downstream obstruction, compositional changes in extracellular matrix, and proliferation of partially dedifferentiated cells, evidence strongly supports the latter as the primary abnormality. In the vast majority of cases, the disease is caused by mutations in PKD1 or PKD2, and appears to be recessive at the cellular level. Somatic second hits in the normal allele of cells containing the germ line mutation initiate or accelerate formation of cysts. The intrinsically high frequency of somatic second hits in epithelia appears to be sufficient to explain the frequent occurrence of somatic second hits in the disease-causing genes. PKD1 and PKD2 encode a putative adhesive/ion channel regulatory protein and an ion channel, respectively. The two proteins interact directly in vitro. Their cellular and subcellular localization suggest that they may also function independently in a common signaling pathway that may involve the membrane skeleton and that links cell-cell and cell-matrix adhesion to the development of cell polarity.

An isoleucine-based allosteric switch controls affinity and shape shifting in integrin CD11b A-domain.

In response to cell activation signals, integrins switch from a low to a high affinity state. Physiologic ligands bind to integrins through a von Willebrand Factor A-type domain. Crystallographic studies revealed two conformations of this domain, "closed" and "open." The latter crystallizes in complex with a pseudoligand or ligand, suggesting that it represents the high affinity state; data linking structure and activity are lacking however. In this communication, we expressed stable low and high affinity forms of integrin CD11b A-domain and determined their binding isotherms and crystal structures. The low affinity form, generated by deleting an N-terminal extension extrinsic to the domain, did not bind to physiologic ligands, and crystallized in the closed conformation. The high affinity form was generated by either deleting or substituting an invariable C-terminal Ile(316), wedged into a hydrophobic socket in the closed form, but displaced from it in the open structure. Both mutants crystallized in the open conformation, and the Ile(316) --> Gly-modified integrin displayed high affinity. Structural differences between the low and high affinity forms were detected in solution. These data establish the structure-function correlates for the CD11b A-domain, and define a ligand-independent isoleucine-based allosteric switch intrinsic to this domain that controls its conformation and affinity.

Divergent response to LPS and bacteria in CD14-deficient murine macrophages.

Gram-negative bacteria and the LPS constituent of their outer membranes stimulate the release of inflammatory mediators believed to be responsible for the clinical manifestations of septic shock. The GPI-linked membrane protein, CD14, initiates the signaling cascade responsible for the induction of this inflammatory response by LPS. In this paper, we report the generation and characterization of CD14-null mice in which the entire coding region of CD14 was deleted. As expected, LPS failed to elicit TNF-alpha and IL-6 production in macrophages taken from these animals, and this loss in responsiveness is associated with impaired activation of both the NF-kappaB and the c-Jun N-terminal mitogen-activated protein kinase pathways. The binding and uptake of heat-killed Escherichia coli, measured by FACS analysis, did not differ between CD14-null and wild-type macrophages. However, in contrast to the findings with LPS, whole E. coli stimulated similar levels of TNF-alpha release from CD14-null and wild-type macrophages at a dose of 10 bioparticles per cell. This effect was dose dependent, and at lower bacterial concentrations CD14-deficient macrophages produced significantly less TNF-alpha than wild type. Approximately half of this CD14-independent response appeared to be mediated by CD11b/CD18, as demonstrated by receptor blockade using neutrophil inhibitory factor. An inhibitor of phagocytosis, cytochalasin B, abrogated the induction of TNF-alpha in CD14-deficient macrophages by E. coli. These data indicate that CD14 is essential for macrophage responses to free LPS, whereas other receptors, including CD11b/CD18, can compensate for the loss of CD14 in response to whole bacteria.

CD43 gene expression is mediated by a nuclear factor which binds pyrimidine-rich single-stranded DNA.

CD43 is a leukocyte-specific surface molecule which plays an important role both in adhesion and signal transduction. We have identified a site spanning nucleotides +18 to +39 within the human CD43 gene promoter which in vitro is hypersensitive to cleavage by nuclease S1. Repeats of this region are sufficient to activate expression of a heterologous promoter in CD43-positive cell lines. Two nuclear factors, PyRo1 and PyRo2, interact with the hypersensitive site. PyRo1 is a single-stranded DNA-binding protein which binds the pyrimidine-rich sense strand. Mutation analysis demonstrates that the motif TCCCCT is critical for PyRo1 interaction. Replacement of this motif with the sequence CATATA abolishes PyRo1 binding and reduces expression of the CD43 promoter by 35% in Jurkat T lymphocytic cells and by 52% in the pre-erythroid/pre-megakaryocytic cell line K562. However, this same replacement failed to affect expression in U937 monocytic cells or in CEM T lymphocytic cells. PyRo1, therefore, exhibits cell-specific differences in its functional activity. Further analysis demonstrated that PyRo1 not only interacts with the CD43 gene promoter but also motifs present within the promoters of the CD11a, CD11b, CD11c and CD11d genes. These genes encode the alpha subunits of the beta2 integrin family of leukocyte adhesion receptors. Deletion of the PyRo1 binding site within the CD11c gene reduced promoter activity in T lymphocytic cells by 47%. However, consistent with our analysis of the CD43 gene, the effect of this same deletion within U937 monocytic cells was less severe. That PyRo1 binds preferentially to single-stranded DNA and sequences within the CD43 and CD11 gene promoters suggests that expression of these genes is influenced by DNA secondary structure.

Polycystin 1 is required for the structural integrity of blood vessels.

Autosomal dominant polycystic kidney disease (ADPKD), often caused by mutations in the PKD1 gene, is associated with life-threatening vascular abnormalities that are commonly attributed to the frequent occurrence of hypertension. A previously reported targeted mutation of the mouse homologue of PKD1 was not associated with vascular fragility, leading to the suggestion that the vascular lesion may be of a secondary nature. Here we demonstrate a primary role of PKD1 mutations in vascular fragility. Mouse embryos homozygous for the mutant allele (Pkd1(L)) exhibit s.c. edema, vascular leaks, and rupture of blood vessels, culminating in embryonic lethality at embryonic day 15.5. Kidney and pancreatic ductal cysts are present. The Pkd1-encoded protein, mouse polycystin 1, was detected in normal endothelium and the surrounding vascular smooth muscle cells. These data reveal a requisite role for polycystin 1 in maintaining the structural integrity of the vasculature as well as epithelium and suggest that the nature of the PKD1 mutation contributes to the phenotypic variance in ADPKD.

Interaction of purified human proteinase 3 (PR3) with reconstituted lipid bilayers.

Proteinase 3 (PR3), the major target autoantigen in Wegener's granulomatosis is a serine proteinase that is normally stored intracellularly in the primary granules of quiescent neutrophils and monocytes. Upon cell activation, a significant portion of this antigen is detected on the cell surface membrane. The nature of the association of PR3 with the membrane and its functional significance are unknown. We investigated the interaction of purified human PR3 with mixtures of zwitterionic (dimyristoyl-L-alpha-phosphatidylcholine, DMPC) and anionic (dimyristoyl-L-alpha-phosphatidylglycerol, DMPG) phospholipids in reconstituted lipid bilayers using differential scanning calorimetry and lipid photolabeling, and measured the affinity of this interaction using spectrophotometry. Two other primary granule constituents, human neutrophil elastase (HNE) and myeloperoxidase (MPO) were investigated for comparison. In calorimetric assays, using lipid vesicles of mixed DMPC/DMPG, increasing PR3 concentrations (protein/lipid molar ratio from 0 to 1 : 110) induced a significant decrease of the main chain transition enthalpy and a shift in chain melting temperatures which is indicative of partial insertion of PR3 into the hydrophobic region of the lipid membranes. This was confirmed by hydrophobic photolabeling using liposomes containing trace amounts of the photoactivable [125I]-labeled phosphatidylcholine analog TID-PC/16. The molar affinity of PR3, HNE, and MPO to lipid vesicles of different DMPC/DMPG ratios was then determined by spectrophotometry. At a DMPC/DMPG ratio of 1 : 1, molar affinities of PR3, Kd = 4.5 +/- 0.3 microm; HNE, 14.5 +/- 1.2 microm; and MPO, 50 +/- 5 microm (n = 3) were estimated. The lipid-associated PR3 exhibited two-fold lower Vmax and Km values, and its enzyme activity was slightly more inhibited (Ki) by the natural alpha1-proteinase inhibitor (alpha1-PI) or an autoantibody to PR3.

Two functional states of the CD11b A-domain: correlations with key features of two Mn2+-complexed crystal structures.

In the presence of bound Mn2+, the three- dimensional structure of the ligand-binding A-domain from the integrin CR3 (CD11b/CD18) is shown to exist in the "open" conformation previously described only for a crystalline Mg2+ complex. The open conformation is distinguished from the "closed" form by the solvent exposure of F302, a direct T209-Mn2+ bond, and the presence of a glutamate side chain in the MIDAS site. Approximately 10% of wild-type CD11b A-domain is present in an "active" state (binds to activation-dependent ligands, e.g., iC3b and the mAb 7E3). In the isolated domain and in the holoreceptor, the percentage of the active form can be quantitatively increased or abolished in F302W and T209A mutants, respectively. The iC3b-binding site is located on the MIDAS face and includes conformationally sensitive residues that undergo significant shifts in the open versus closed structures. We suggest that stabilization of the open structure is independent of the nature of the metal ligand and that the open conformation may represent the physiologically active form.

Outside-in signaling by lipopolysaccharide through a tailless integrin.

Ligand binding to integrins activates intracellular signaling pathways that coordinate and regulate a variety of cellular responses. There is evidence to suggest that the cytoplasmic tails play a key role in several of these signaling events. We sought to determine whether the beta2 integrin complement receptor type 3 (CR3; CD11b/CD18), a receptor for LPS, could initiate an intracellular signal in the absence of its cytoplasmic domains. Expression of full length CR3 in a Chinese hamster ovary-K1 fibroblast line enabled serum-independent translocation of nuclear factor-kappaB in response to binding LPS. Unexpectedly, a cell line expressing a mutated form of CR3 deficient in the cytoplasmic domains was also competent for transmitting a signal in response to LPS. In contrast, phagocytosis of whole Gram-negative bacteria and iC3b-coated erythrocytes took place only with a full length receptor. Thus, while full length CR3 is necessary for productive phagocytic signals, LPS activation does not require the cytoplasmic domains. CR3 may function to activate cells by presenting LPS to a downstream signal transducer.

Emerging paradigms of integrin ligand binding and activation.

Adhesion of cells to each other or to the extracellular matrix provides essential signals that regulate many cellular functions including cell migration, proliferation, differentiation and apoptosis. The integrin superfamily orchestrates many of these complex adhesive events through regulated interactions with a large variety of ligands. Crystallization of some ligands and of a ligand-binding integrin domain, reviewed here, together with extensive mutagenesis studies are beginning to shed light on the inner workings of these receptors.