Urinary B-cell-activating factor of the tumour necrosis factor family (BAFF) in systemic lupus erythematosus.

INTRODUCTION: We examined the clinical relevance of urinary concentrations of B-cell-activating factor of the tumour necrosis factor family (BAFF) and a proliferation-inducing ligand (APRIL) in systemic lupus erythematosus (SLE). METHODS: We quantified urinary BAFF (uBAFF) by enzyme-linked immunosorbent assay in 85 SLE, 28 primary Sjögren syndrome (pSS), 40 immunoglobulin A nephropathy (IgAN) patients and 36 healthy controls (HCs). Urinary APRIL (uAPRIL) and monocyte chemoattractant protein 1 (uMCP-1) were also quantified. Overall and renal SLE disease activity were assessed using the Systemic Lupus Erythematosus Disease Activity Index 2000. RESULTS: uBAFF was detected in 12% (10/85) of SLE patients, but was undetectable in HCs, IgAN and pSS patients. uBAFF was detectable in 28% (5/18) of SLE patients with active nephritis vs 5/67 (7%) of those without ( p = 0.03), and uBAFF was significantly higher in active renal patients ( p = 0.02) and more likely to be detected in patients with persistently active renal disease. In comparison, uAPRIL and uMCP-1 were detected in 32% (25/77) and 46% (22/48) of SLE patients, respectively. While no difference in proportion of samples with detectable uAPRIL was observed between SLE, HCs and IgAN patients, both uAPRIL and uMCP-1 were significantly detectable in higher proportions of patients with active renal disease. CONCLUSIONS: uBAFF was detectable in a small but a significant proportion of SLE patients but not in other groups tested, and was higher in SLE patients with active renal disease.

SHIP-1 deficiency in the myeloid compartment is insufficient to induce myeloid expansion or chronic inflammation.

SHIP-1 has an important role in controlling immune cell function through its ability to downmodulate PI3K signaling pathways that regulate cell survival and responses to stimulation. Mice deficient in SHIP-1 display several chronic inflammatory phenotypes including antibody-mediated autoimmune disease, Crohn's disease-like ileitis and a lung disease reminiscent of chronic obstructive pulmonary disease. The ileum and lungs of SHIP-1-deficient mice are infiltrated at an early age with abundant myeloid cells and the mice have a limited lifespan primarily thought to be due to the consolidation of lungs with spontaneously activated macrophages. To determine whether the myeloid compartment is the key initiator of inflammatory disease in SHIP-1-deficient mice, we examined two independent strains of mice harboring myeloid-restricted deletion of SHIP-1. Contrary to expectations, conditional deletion of SHIP-1 in myeloid cells did not result in consolidating pneumonia or segmental ileitis typical of germline SHIP-1 deficiency. In addition, other myeloid cell abnormalities characteristic of germline loss of SHIP-1, including flagrant splenomegaly and enhanced myelopoiesis, were absent in mice lacking SHIP-1 in myeloid cells. This study indicates that the spontaneous inflammatory disease characteristic of germline SHIP-1 deficiency is not initiated solely by LysM-positive myeloid cells but requires the simultaneous loss of SHIP-1 in other hematolymphoid lineages.

Ryk-deficient mice exhibit craniofacial defects associated with perturbed Eph receptor crosstalk.

Secondary palate formation is a complex process that is frequently disturbed in mammals, resulting in the birth defect cleft palate. Gene targeting has identified components of cytokine/growth factor signalling systems such as Tgf-alpha/Egfr, Eph receptors B2 and B3 (Ephb2 and Ephb3, respectively), Tgf-beta2, Tgf-beta3 and activin-betaA (ref. 3) as regulators of secondary palate development. Here we demonstrate that the mouse orphan receptor 'related to tyrosine kinases' (Ryk) is essential for normal development and morphogenesis of craniofacial structures including the secondary palate. Ryk belongs to a subclass of catalytically inactive, but otherwise distantly related, receptor protein tyrosine kinases (RTKs). Mice homozygous for a null allele of Ryk have a distinctive craniofacial appearance, shortened limbs and postnatal mortality due to feeding and respiratory complications associated with a complete cleft of the secondary palate. Consistent with cleft palate phenocopy in Ephb2/Ephb3-deficient mice and the role of a Drosophila melanogaster Ryk orthologue, Derailed, in the transduction of repulsive axon pathfinding cues, our biochemical data implicate Ryk in signalling mediated by Eph receptors and the cell-junction-associated Af-6 (also known as Afadin). Our findings highlight the importance of signal crosstalk between members of different RTK subfamilies.

Alterations in collagen production in mixed mononuclear leukocyte-fibroblast cultures.

The cell-cell interactions between fibroblasts and mononuclear leukocytes (MNL) which promote alterations in collagen accumulation were examined using a system of co-culture of human fibroblasts and peripheral blood MNL. The stimulation of collagen production was optimal after 48 h of co-culture and the increase in collagen correlated directly with the number of MNL added. The enhancement of collagen production was seen in both autologous and allogeneic co-cultures. Stimulation of non-collagenous protein was also noted. Co-culture supernatants contained soluble substances that were capable of stimulating collagen production, although they stimulated collagen production to a lesser degree than direct co-culture. Fractionation of these supernatants on Sephadex G-200 revealed a predominant area of stimulatory activity at 160,000 mol wt. Lesser areas of activity were noted at molecular weights of 80,000 and 25,000. Determination of the types of collagen produced by fibroblasts during co-culture with MNL showed that the ratio of type I:III collagen was decreased. These alterations in both the quantitative and qualitative accumulation of collagen mimic the changes often seen in wound healing and early inflammation suggesting that cellular interactions between fibroblasts and MNL may be important in the modulation of collagen production in normal and pathologic states.

Heterogeneity among human collagenases demonstrated by monoclonal antibody that selectively recognizes and inhibits human neutrophil collagenase.

The heterogeneity of human collagenases has been examined using a monoclonal antibody to neutrophil collagenase. This antibody inhibited collagenase activity and, when covalently coupled to Sepharose, bound both latent and active enzyme. Although human neutrophil collagenase was inhibited by the antibody, the activity of human skin and rheumatoid synovial collagenase was not significantly diminished in the presence of the antibody. Competitive inhibition studies also differentiated between these collagenases. Only human neutrophil collagenase effectively blocked the antibody in a competitive enzyme-linked immunosorbent assay while skin and rheumatoid synovial collagenase again failed to interact with the antibody. The unequivocal recognition of neutrophil collagenase as an immunologically distinct entity from other collagenases supports the hypothesis that neutrophil collagenase is a separate gene product from fibroblast or synovial collagenase.

Inhibition of the B cell by CD22: a requirement for Lyn.

Mice in which the Lyn, Cd22, or Shp-1 gene has been disrupted have hyperactive B cells and autoantibodies. We find that in the absence of Lyn, the ability of CD22 to become tyrosine phosphorylated after ligation of mIg, to recruit SHP-1, and to suppress mIg-induced elevation of intracellular [Ca2+] is lost. Therefore, Lyn is required for the SHP-1-mediated B cell suppressive function of CD22, accounting for similarities in the phenotypes of these mice.

The cytoplasmic domain of the integrin lymphocyte function-associated antigen 1 beta subunit: sites required for binding to intercellular adhesion molecule 1 and the phorbol ester-stimulated phosphorylation site.

We have defined the regions of the cytoplasmic domain of the leukocyte integrin lymphocyte function-associated antigen 1 (LFA-1) that are required for active binding of its extracellular domain to intercellular adhesion molecule 1 (ICAM-1). The NH2-terminal 28 amino acids in the cytoplasmic domain are dispensable, but a segment of 5 amino acids including three contiguous threonines (758-760) and Phe 766 in the COOH-terminal third of the cytoplasmic domain are required for binding to ICAM-1. Mutation and phosphoamino acid analysis show that Ser 756 is the major residue phosphorylated in response to phorbol ester. Furthermore, multiple mutations demonstrate that serine phosphorylation can be dissociated from phorbol ester-stimulated binding of LFA-1 to ICAM-1. The sites we have defined are previously unremarked, are well conserved in the beta 1, beta 3, and beta 7 integrin subunits, and may be of broad importance in regulating adhesiveness of integrins.

Distinct mutations in two patients with leukocyte adhesion deficiency and their functional correlates.

Two patients with leukocyte adhesion deficiency (LAD), one with a moderate phenotype (patient 14) and one with a severe phenotype (patient 2) who had been shown to have a normal sized beta subunit protein precursor, were analyzed in an attempt to determine the molecular basis for their disease. RNase mapping located possible mutations to two distinct but adjacent regions of the beta subunit cDNA. Sequencing of patient-derived cDNA clones in this region revealed a C for T difference at amino acid 149 in patient 14 which resulted in the substitution of a leucine for a proline, and an A for G substitution at amino acid 169 in patient 2 which mutated a glycine to an arginine. The mutated amino acids are in a region of the cDNA that is highly conserved between the beta subunits of the integrin family and are identical in all known integrin beta subunits. Co-transfection of the beta subunit cDNA containing the patient 2 mutation with the wild-type alpha subunit of LFA-1 in a mammalian expression system resulted in no expression of LFA-1. In the case of the mutation in patient 14 there was markedly diminished expression of LFA-1 with loss of function and loss of the epitope for a number of anti-beta mAbs. Normal half-life of the mutant beta subunits, and previous demonstration of a lack of alpha/beta complex formation during biosynthesis in patient cells, suggest a defect in association with the alpha subunit. Association with beta is required for expression of the alpha subunit of LFA-1. Loss of functional expression with both of these beta subunit mutations suggests that they lie in a site critical for association with the alpha subunit.

ICAM-1 (CD54): a counter-receptor for Mac-1 (CD11b/CD18).

While the leukocyte integrin lymphocyte function-associated antigen (LFA)-1 has been demonstrated to bind intercellular adhesion molecule (ICAM)-1, results with the related Mac-1 molecule have been controversial. We have used multiple cell binding assays, purified Mac-1 and ICAM-1, and cell lines transfected with Mac-1 and ICAM-1 cDNAs to examine the interaction of ICAM-1 with Mac-1. Stimulated human umbilical vein endothelial cells (HUVECs), which express a high surface density of ICAM-1, bind to immunoaffinity-purified Mac-1 adsorbed to artificial substrates in a manner that is inhibited by mAbs to Mac-1 and ICAM-1. Transfected murine L cells or monkey COS cells expressing human ICAM-1 bind to purified Mac-1 in a specific and dose-dependent manner; the attachment to Mac-1 is more temperature sensitive, lower in avidity, and blocked by a different series of ICAM-1 mAbs when compared to LFA-1. In a reciprocal assay, COS cells cotransfected with the alpha and beta chain cDNAs of Mac-1 or LFA-1 attach to immunoaffinity-purified ICAM-1 substrates; this adhesion is blocked by mAbs to ICAM-1 and Mac-1 or LFA-1. Two color fluorescence cell conjugate experiments show that neutrophils stimulated with fMLP bind to HUVEC stimulated with lipopolysaccharide for 24 h in an ICAM-1-, Mac-1-, and LFA-1-dependent fashion. Because cellular and purified Mac-1 interact with cellular and purified ICAM-1, we conclude that ICAM-1 is a counter receptor for Mac-1 and that this receptor pair is responsible, in part, for the adhesion between stimulated neutrophils and stimulated endothelial cells.

Regulation of adhesion of ICAM-1 by the cytoplasmic domain of LFA-1 integrin beta subunit.

Interactions between cytotoxic lymphocytes and their targets require the T cell antigen receptor (TCR) and the integrin lymphocyte function-associated molecule-1 (LFA-1, CD11a/CD18). LFA-1 is not constitutively avid for its counter-receptors, intercellular adhesion molecules (ICAMs)-1 and -2. Cross-linking of the TCR transiently converts LFA-1 to a high avidity state and thus provides a mechanism for regulating cellular adhesion and de-adhesion in an antigen-specific manner. Truncation of the cytoplasmic domain of the beta, but not the alpha, subunit of LFA-1 eliminated binding to ICAM-1 and sensitivity to phorbol esters. Thus, LFA-1 binding to ICAM-1 was found to be regulated by the cytoplasmic domain of the beta subunit of LFA-1.

Mechanisms for regulating expression of membrane isoforms of Fc gamma RIII (CD16).

Granulocyte and natural killer (NK) cell Fc receptors for immunoglobulin G (CD16) differ in only a few amino acids, yet have phosphatidylinositol glycan (PIG) or polypeptide membrane anchors, respectively. Mutagenesis shows that anchoring is regulated by a serine residue near the PIG anchor attachment site in the extracellular domain. The NK cell isoform was not expressed on the surface of COS cells unless cotransfected with a subunit that was expressed in NK cells and that was identical to the gamma subunit of the high affinity IgE Fc receptor (Fc epsilon RI). However, the CD16 sequence and not expression of the gamma subunit is dominant in regulating PIG reanchoring.

Human neutrophil gelatinase is a component of specific granules.

Previous investigators have proposed that gelatinase, a metalloproteinase found in neutrophils, is stored in a novel secretory compartment distinct from the two major granule populations, azurophilic and specific. To locate this proteinase in human neutrophils we reacted the cells for peroxidase and then applied monospecific polyclonal antibodies to human neutrophil gelatinase to immunolabel ultrathin frozen sections using an immunogold technique. Gelatinase was localized in a population of peroxidase-negative granules. Double-labeling experiments using antibodies against lactoferrin, a marker for specific granules, and gelatinase demonstrated colocalization of the two antigens in 80% of the specific granules. However, some granules immunostained with only the lactoferrin or gelatinase antibody. Similar techniques were used to examine precursor cells from bone marrow. In myelocytes both gelatinase and lactoferrin were present in large developing specific granules; however, some mature specific granules contained only lactoferrin. Thus, it is possible that lactoferrin synthesis begins earlier than gelatinase synthesis and that overlapping synthesis and segregation occurs during the myelocyte stage. These findings suggest that the main storage compartment of gelatinase is within the peroxidase-negative specific granules.

Expression of a metalloproteinase that degrades native type V collagen and denatured collagens by cultured human alveolar macrophages.

Human pulmonary alveolar macrophages obtained by bronchoalveolar lavage from both normal controls and smokers secreted in vitro a neutral proteinase that degraded denatured collagens. Optimal expression of the proteinase was detected after 3-5 d of culture. The proteinase could not be detected in the media of cultures that had been treated with 0.5 micrograms/ml of cycloheximide. The gelatinase had an Mr of 90,000 and was immunologically cross-reactive with human neutrophil gelatinase. When newly synthesized 35S-methionine-labeled proteins were analyzed, the proteinase appeared to be a major secretion product of alveolar macrophages. Chromatography on gelatin-Sepharose gave a single peak of activity that was predominantly composed of the 90,000-mol-wt proteinase. The proteolytic activity in the gelatin-Sepharose-purified material was inhibited by EDTA and 1,10-phenanthroline, but not by N-ethylmaleimide or phenylmethanesulfonyl fluoride, indicating that the proteinase was a metalloproteinase. The partially purified material was also capable of degrading native type V collagen and this degradation was inhibited in the presence of an antibody to neutrophil gelatinase. The data suggest that human alveolar macrophages in culture elaborate a metalloproteinase that degrades both native type V collagen and denatured collagens.

Constitutive expression of a 92-kD gelatinase (type V collagenase) by rheumatoid synovial fibroblasts and its induction in normal human fibroblasts by inflammatory cytokines.

Synovial fibroblasts freshly isolated from the rheumatoid joint are characterized by their marked connective tissue degradative ability. This phenotype includes the ability to secrete large amounts of the matrix-degrading metalloproteinases, collagenase, and stromelysin. We have found that another aspect of this phenotype is the constitutive expression at both protein and mRNA levels of a 92-kD gelatinolytic metalloproteinase, which is not secreted by normal dermal or lung fibroblasts and is immunologically cross-reactive with a type V collagenase expressed by activated macrophages and neutrophils. Expression of this 92-kD metalloproteinase confers upon the fibroblasts the capacity to degrade collagenase- and stromelysin-resistant interstitial elements, such as collagen types IV, V and XI. In contrast to the 92-kD metalloproteinase, a 68-kD gelatinase (type IV collagenase) was expressed by all fibroblast types studied, indicating that its regulation is distinct from that of the 92-kD gelatinase. To identify what cytokines may be important in the induction of the rheumatoid synovial phenotype, including expression of the 92-kD gelatinase, we exposed normal dermal fibroblasts to a number of cytokines including many known or considered likely to be present in rheumatoid synovial fluid and tissue. Although IL-1 beta, tumor necrosis factor-alpha, lymphotoxin, platelet-derived growth factor, and basic fibroblast growth factor were capable of stimulating fibroblasts to secrete collagenase, only tumor necrosis factor-alpha, lymphotoxin, and IL-1 beta were able to induce expression of the 92-kD gelatinase, demonstrating discordant regulation of the two metalloproteinases. Expression of the 68-kD gelatinase was independent of that of the 92-kD gelatinase, as demonstrated at the protein and mRNA levels. Late passage rheumatoid synovial fibroblasts, which no longer constitutively expressed the 92-kD gelatinase, displayed an accentuated response to IL-1 beta when compared to normal dermal fibroblasts. Thus, in addition to IL-1 beta, tumor necrosis factor-alpha or lymphotoxin may contribute to the expression of a specific rheumatoid synovial phenotype in vivo that is associated with progressive matrix destruction.

Transfection of cells from patients with leukocyte adhesion deficiency with an integrin beta subunit (CD18) restores lymphocyte function-associated antigen-1 expression and function.

Leukocyte adhesion deficiency (LAD) is an inherited immunodeficiency disease that is characterized by the deficient expression of the leukocyte adhesion glycoproteins lymphocyte function-associated antigen-1 (LFA-1), Mac-1, and p150,95. This loss of expression is attributed to heterogeneous defects in the common beta subunit shared by these glycoproteins. Here we demonstrate that expression of the LFA-1 alpha beta heterodimer in EBV-transformed B lymphoblastoid cells from LAD patients can be recovered after transfection with the beta subunit cDNA contained in an EBV-based vector. Four patients with differing severities of LAD comprising three distinct classes of mutations were studied. Flow cytometry analysis of stably transfected patient cells revealed near normal levels of expression of both the alpha and beta chains of LFA-1, and immunoprecipitation studies confirmed that fully processed alpha and beta chains were being expressed at the cell surface. In addition, Northern analysis of mRNA expression also demonstrated that the transfected LAD patient cells were expressing high quantities of exogenous beta subunit mRNA. Functional studies such as homotypic adhesion and adhesion to a purified counterreceptor for LFA-1, intracellular adhesion molecule-1, demonstrated that LFA-1 function had been restored in the stably transfected LAD patient cell lines. These studies unequivocally show that the defect in cells from patients with LAD is in the leukocyte integrin beta subunit.

Activation of the Ras/mitogen-activated protein kinase pathway by kinase-defective epidermal growth factor receptors results in cell survival but not proliferation.

Signalling by the epidermal growth factor (EGF) receptor (EGFR) has been studied intensively, but for most cell types the analysis is complicated by the fact that EGFR not only homodimerizes but can also form heterodimers with other EGFR family members. Heterodimerization is a particular problem in the study of EGFR mutants, where the true phenotype of the mutants is confounded by the contribution of the heterodimer partner to signal transduction. We have made use of the murine hemopoietic cell line BaF/3, which does not express EGFR family members, to express wild-type (WT) EGFR, three kinase-defective EGFR mutants (V741G, Y740F, and K721R), or a C-terminally truncated EGFR (CT957) and have measured their responses to EGF. We found that under the appropriate conditions EGF can stimulate cell proliferation of BaF/3 cells expressing WT or CT957 EGFRs but not that of cells expressing the kinase-defective mutants. However, EGF promotes the survival of BaF/3 cells expressing either of the kinase-defective receptors (V741G and Y740F), indicating that these receptors can still transmit a survival signal. Analysis of the early signalling events by the WT, V741G, and Y740F mutant EGF receptors indicated that EGF stimulates comparable levels of Shc phosphorylation, Shc-GRB-2 association, and activation of Ras, B-Raf, and Erk-1. Blocking the mitogen-activated protein kinase (MAPK) signalling pathway with the specific inhibitor PD98059 abrogates completely the EGF-dependent survival of cells expressing the kinase-defective EGFR mutants but has no effect on the EGF-dependent proliferation mediated by WT and CT957 EGFRs. Similarly, the Src family kinase inhibitor PP1 abrogates EGF-dependent survival without affecting proliferation. However blocking phosphatidylinositol-3-kinase or JAK-2 kinase with specific inhibitors does arrest growth factor-dependent cell proliferation. Thus, EGFR-mediated mitogenic signalling in BaF/3 cells requires an intact EGFR tyrosine kinase activity and appears to depend on the activation of both the JAK-2 and PI-3 kinase pathways. Activation of the Src family of kinases or of the Ras/MAPK pathway can, however, be initiated by a kinase-impaired EGFR and is linked to survival.

CD11b immunophenotyping identifies inflammatory profiles in the mouse and human lungs.

The development of easily accessible tools for human immunophenotyping to classify patients into discrete disease endotypes is advancing personalized therapy. However, no systematic approach has been developed for the study of inflammatory lung diseases with often complex and highly heterogeneous disease etiologies. We have devised an internally standardized flow cytometry approach that can identify parallel inflammatory alveolar macrophage phenotypes in both the mouse and human lungs. In mice, lung innate immune cell alterations during endotoxin challenge, influenza virus infection, and in two genetic models of chronic obstructive lung disease could be segregated based on the presence or absence of CD11b alveolar macrophage upregulation and lung eosinophilia. Additionally, heightened alveolar macrophage CD11b expression was a novel feature of acute lung exacerbations in the SHIP-1(-/-) model of chronic obstructive lung disease, and anti-CD11b antibody administration selectively blocked inflammatory CD11b(pos) but not homeostatic CD11b(neg) alveolar macrophages in vivo. The identification of analogous profiles in respiratory disease patients highlights this approach as a translational avenue for lung disease endotyping and suggests that heterogeneous innate immune cell phenotypes are an underappreciated component of the human lung disease microenvironment.

Crystal structure of Staphylococcus aureus tyrosyl-tRNA synthetase in complex with a class of potent and specific inhibitors.

SB-219383 and its analogues are a class of potent and specific inhibitors of bacterial tyrosyl-tRNA synthetases. Crystal structures of these inhibitors have been solved in complex with the tyrosyl-tRNA synthetase from Staphylococcus aureus, the bacterium that is largely responsible for hospital-acquired infections. The full-length enzyme yielded crystals that diffracted to 2.8 A resolution, but a truncated version of the enzyme allowed the resolution to be extended to 2.2 A. These inhibitors not only occupy the known substrate binding sites in unique ways, but also reveal a butyl binding pocket. It was reported that the Bacillus stearothermophilus TyrRS T51P mutant has much increased catalytic activity. The S. aureus enzyme happens to have a proline at position 51. Therefore, our structures may contribute to the understanding of the catalytic mechanism and provide the structural basis for designing novel antimicrobial agents.

Establishment of basal cell carcinoma in culture: evidence for a basal cell carcinoma-derived factor(s) which stimulates fibroblasts to proliferate and release collagenase.

The connective tissue adjacent to basal cell carcinomas (BCC) is frequently abnormal and contains increased numbers of fibroblasts and increased extractable collagenase. To determine whether BCC could produce these alterations by releasing mediators that regulated fibroblast function, we established BCC in culture and tested the ability of their culture supernatants to alter fibroblast proliferation and production of collagenase. Using tissue culture plates coated with type IV collagen and containing x-irradiated 3T3 feeder cells, we established epithelial colonies from 47% of the BCC cultured. The BCC-derived colonies differed from normal epidermal cell colonies in their morphology, growth rate, and keratin production. Culture supernatants from 4 out of 5 confluent BCC-derived colonies contained factors that stimulated fibroblasts to proliferate and release collagenase. These findings show that BCC-derived epidermal cell colonies release mediators which alter fibroblast functions and suggest that some of the connective tissue changes associated with BCC in vivo are the result of BCC-fibroblast interactions.

Lyn, a src-like tyrosine kinase.

Lyn is a member of the src family of non-receptor protein tyrosine kinases that is predominantly expressed in haematopoietic tissues. Like all members of the src family, lyn is thought to participate in signal transduction from cell surface receptors that lack intrinsic tyrosine kinase activity. It is associated with a number of cell surface receptors including the B cell antigen receptor and Fc epsilon RI. Lyn deficient mice develop autoimmune disease characterised by autoantibodies in serum and the deposition of immune complexes in the kidney, a pathology reminiscent of systemic lupus erythematosus. Lyn deficient mice also have impaired signalling involving Fc epsilon RI in mast cells, resulting in defective allergic responses.