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Julie M Williams

Publications and source records attributed to Julie M Williams.

10 recordsLinked to original sources
PubMed2007

Chimeric antibodies to proteinase 3 of IgG1 and IgG3 subclasses induce different magnitudes of functional responses in neutrophils.

BACKGROUND: Antineutrophil cytoplasmic antibodies (ANCA) are associated with small-vessel vasculitis and have been implicated in its pathogenesis. The subclass distribution of ANCA IgG deviates from normal patterns, and it has been suggested that the IgG3 subclass may have pathogenic potential over the IgG1 subclass and may be more likely to be associated with active disease and renal involvement. OBJECTIVE: To deal with potential pathogenicity, chimeric antibodies were constructed of IgG1 and three subclasses with human IgG1 or three constant regions and a murine-derived variable region that binds an epitope within the ANCA antigen proteinase 3 (PR3) that is recognised by human autoantibodies. METHODS: The antibodies were characterised for binding to PR3, including affinity and avidity, before being used as tools to explore their ability to activate human neutrophils for superoxide release, cytokine release, degranulation and ability to induce neutrophil adhesion under flow. RESULTS: Both subclass antibodies elicited similar neutrophil responses for superoxide release, degranulation and interleukin (IL) 8 production, although quantitative responses showed that the IgG1 subclass favoured degranulation and the IgG3 subclass favoured IL8 production. Both antibodies were able to convert neutrophils from selectin-dependent rolling adhesion to integrin-dependent stationary adhesion in a flow assay. CONCLUSIONS: These findings indicate that humanised antibodies directed against a single epitope of PR3 can recapitulate the effects of polyclonal human ANCA, which recognises multiple PR3 epitopes. Further, PR3-ANCA of both IgG1 and IgG3 subclasses can activate neutrophils, although the more potent IL8 response by IgG3 PR3-ANCA may encourage further neutrophil recruitment and amplify injury.

Animals↗
PubMed2005

Effect of verocytotoxins (Shiga-like toxins) on human neutrophils in vitro.

Neutrophil activation occurs in diarrhoea-associated HUS and correlates with disease severity, implying a role in pathogenesis. Verocytotoxin (Shiga-like toxin) has been shown to stimulate endothelium to release chemokines and express leukocyte adhesion molecules that would lead to indirect neutrophil-endothelial interaction. A direct action of verocytotoxin (VT) on neutrophils has been proposed, although in vitro studies of this are controversial. In this report we examine the effect of verocytotoxin-1 (Shiga-like toxin-1) (VT1) and verocytotoxin-2 (VT2) on human neutrophils in vitro with regard to priming, the release of superoxide and elastase, and chemotaxis. Neutrophils were incubated with VT1 or VT2 and superoxide and elastase release was measured over 120 and 45 minutes respectively. Priming was investigated by pre-treating the neutrophils with VT1 or VT2, exposing them to formyl-met-leu-phe (fMLP) or phorbol myristic acid (PMA) and measuring superoxide release. Neutrophil chemotaxis towards fMLP was assessed with and without pre-incubation with VT1 and VT2. We found that neither of the toxins induced superoxide or elastase release. Priming with VT1 significantly reduced superoxide release when neutrophils were stimulated with fMLP or PMA. VT2 priming gave a reduced superoxide release with PMA but not fMLP. Heat-inactivation of the toxins gave similar results. Pre-treatment of neutrophils with VT1 or VT2 did not affect chemotaxis towards fMLP after a 2-hour incubation period. In conclusion, VT1 and VT2 do not activate primed neutrophils in vitro. Nor do they affect chemotaxis towards fMLP. They may impair neutrophil priming.

Animals↗
PubMed2005

Translating basic science into patient therapy for ANCA-associated small vessel vasculitis.

ANCA (anti-neutrophil cytoplasm antibody)-associated small vessel vasculitis is an inflammatory condition associated with the production of autoantibodies to neutrophil cytoplasmic components. The disorder results in destruction of the microvasculature, infiltration of neutrophils into tissues, which is followed later by mononuclear cells, leading to injury and the formation of granulomatous lesions. Initiators for the disease are undetermined but a pro-inflammatory environment is required. Other influencing factors may include environmental triggers, genetic propensity or infectious agents. The primary cellular event in the condition involves the neutrophils, which are likely to be responsible for the majority of tissue injury. Binding of the autoantibody to neutrophils initiates cell activation via a complex intracellular signalling cascade, culminating in the release of pro-inflammatory mediators, proteolytic enzymes and reactive oxygen species. Adhesion of neutrophils to endothelial cells is observed in vitro and more investigations in this area may explain the focussing of the disease to certain vessels/tissues. Current treatment regimens have substantial toxicity. Although newer developments are an improvement there is still a pressing need for more targeted therapies, which could be provided by extrapolating information emerging from basic scientific research.

Antibodies, Antineutrophil Cytoplasmic↗
PubMed2005

Potential immunosuppressive therapeutics to target small vessel vasculitis of the kidney.

Small vessel vasculitis of the kidney is an inflammatory disorder that results in glomerulonephritis. Current treatment regimes have proved effective, with patient survival rates of 80% at five years. Of the 20% that die, a significant number of deaths are due to the side effects of therapies, and immunosuppression. A more tailored approach is required that targets the cells involved rather than generally restraining the immune system. This can only be achieved by increasing our knowledge of disease pathogenesis.

Animals↗
PubMed2004

Characterization of the regulation and functional consequences of p21ras activation in neutrophils by antineutrophil cytoplasm antibodies.

Antineutrophil cytoplasm antibodies (ANCA) are implicated in the pathogenesis of systemic vasculitis. ANCA are directed against antigens expressed on the surface of cytokine-primed neutrophils. It was shown previously that whole IgG ANCA and its fraction antigen binding [F(ab')(2)] fragment can activate the GTPase p21(ras). This study shows a functional involvement of this molecule in the ANCA activation of neutrophils by inhibiting the production of superoxide with farnesylthiosalicylic acid. Using the ras activation assay, farnesylthiosalicylic acid inhibits p21(ras) binding to its substrate at comparable concentrations to those seen for superoxide inhibition. It is also shown that activation of p21(ras) by ANCA is transient, peaking at 5 to 10 min and returning to baseline by 30 min. The use of ras isoform-specific antibodies in Western blots established, for the first time, that Harvey-ras is not present in human neutrophils, but both Kirsten-ras (K-ras) and Neuronal-ras are. Stimulation with ANCA is able to differentially activate K-ras without effects on neuronal-ras. The activation of p21(ras) by ANCA and its F(ab')(2) is prevented by inhibition of both Src kinases and phosphatidylinositol-3-kinase, indicating a cooperative role for both molecules in the G protein pathway activated by ANCA F(ab')(2) upstream of p21(ras). It is concluded that ANCA selectively activates K-ras during induction of a respiratory burst via pathways involving multiple upstream kinases.

Antibodies, Antineutrophil Cytoplasmic↗
PubMed2004

Identification and importance of N-glycosylation of the human 5-hydroxytryptamine3A receptor subunit.

We investigated the presence and potential role of N-glycosylation of the human (h) 5-hydroxytryptamine3 (5-HT3A) receptor subunit expressed in COS-7 cells. Incubation of cells with the N-glycosylation inhibitor, tunicamycin, reduced the molecular weight of the predominant immunoreactive h5-HT3A subunit species (from approximately 59 to 45 kDa) indicating that the h5-HT3A subunit is normally N-glycosylated. Site-directed mutagenesis studies individually substituting four identified N-terminal asparagines (N5, N81, N147, N163) demonstrated that each expressed mutant displayed a reduced molecular weight (by approximately 3 kDa) suggesting that each asparagine residue was subject to N-glycosylation. In addition, 5-HT3 receptor binding studies indicated that prevention of N-glycosylation, by individual amino acid substitution at each of the four asparagine residues, either prevented (N81, N147, N163) or greatly reduced (N5) the production of a 5-HT3 receptor binding site. Corresponding with the radioligand binding studies, immunocytochemical studies demonstrated that substitution of each asparagine either prevented (N81, N147, N163) or reduced considerably (N5) mutant protein expression within the cell membrane. The present study demonstrates an important role for N-glycosylation at multiple identified asparagine residues in the N-terminus of the h5-HT3A receptor subunit.

Amino Acid Sequence↗
PubMed2004

ANCA induces beta2 integrin and CXC chemokine-dependent neutrophil-endothelial cell interactions that mimic those of highly cytokine-activated endothelium.

Antineutrophil cytoplasm antibodies (ANCA) activate neutrophils to undergo a series of coordinated interactions, leading to transendothelial migration, eventually culminating in vascular destruction. The molecular events underlying neutrophil recruitment in ANCA-associated vasculitis need to be defined to enable effective therapeutic manipulation. A flow-based adhesion assay was used to investigate the role of beta2 integrins (CD11a/CD18 and CD11b/CD18) and chemokine receptors [CXC chemokine receptor (CXCR)1 and CXCR2] in neutrophil migration through the endothelium. Two endothelial models were used: a highly activated model stimulated with 100 U/ml tumor necrosis factor alpha (TNF-alpha) and a minimally activated model stimulated with 2 U/ml TNF-alpha and in which ANCA was present as a secondary neutrophil stimulus. CD11a/CD18, CD11b/CD18, and CXCR2 contributed to adhesion and transendothelial migration in both models. However, when the endothelium was minimally activated with TNF-alpha, CD11b/CD18 played an important role in stabilizing adhesion induced by ANCA immunoglobulin G (IgG). Analysis of beta2 integrins and chemokine receptors demonstrated that ANCA IgG had no effect on expression levels at the neutrophil surface but enabled an active conformational change of CD11b/CD18. Similar molecular mechanisms control neutrophil adhesion and migration through highly or minimally TNF-alpha-activated endothelium. However, the direct ANCA-mediated neutrophil stimulation is needed to drive migration through minimally activated endothelium, and CD11b/CD18 is recruited for greater stability of adhesion during this process and can undergo an activatory, conformational change in response to ANCA IgG.

Antibodies, Antineutrophil Cytoplasmic↗
PubMed2004

Activation of Syk in neutrophils by antineutrophil cytoplasm antibodies occurs via Fcgamma receptors and CD18.

Antineutrophil cytoplasm antibodies (ANCA) activate TNF-alpha-primed neutrophils to undergo a respiratory burst. The intracellular signals that mediate activation have not been studied extensively but could increase the understanding of the pathogenesis small vessel vasculitis. It was demonstrated that ANCA-IgG induced phosphorylation of the tyrosine kinase Syk in TNF-alpha-primed neutrophils from healthy donors. Syk was not phosphorylated in response to ANCA F(ab')(2). Furthermore, Syk phosphorylation was attenuated by blockade of both low-affinity Fcgamma receptors and CD18. Similarly, low-affinity Fcgamma receptor blockade reduced ANCA-induced superoxide production. In patient-derived neutrophils, the high-affinity Fcgamma receptor FcgammaRI was also demonstrated to be involved in ANCA-induced superoxide production. However, Syk phosphorylation was not attenuated by blockade of the FcgammaRI, present on neutrophils from vasculitis patients. The tyrosine kinase inhibitor 4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine inhibited the ANCA-induced respiratory burst and Syk phosphorylation, suggesting that Src kinases lie upstream of Syk activation but downstream of ANCA engagement of Fcgamma receptors. Piceatannol, another tyrosine kinase inhibitor, also inhibited ANCA-induced Syk phosphorylation and the ANCA-stimulated respiratory burst, supporting the proposed functional role for Syk in ANCA signaling. ANCA-induced phosphorylation of Cbl and intracellular calcium transients, potential downstream mediators of Syk activation, were also blocked by tyrosine kinase inhibitors. While it has previously been shown that pertussis toxin diminishes the ANCA-induced respiratory burst, indicating heterotrimeric G protein involvement, Syk phosphorylation and calcium transients were unaffected by pertussis toxin. Collectively, these data show that Syk phosphorylation is induced during ANCA-triggered neutrophil activation.

Antibodies, Antineutrophil Cytoplasmic↗
PubMed2003

Activation of the G(i) heterotrimeric G protein by ANCA IgG F(ab')2 fragments is necessary but not sufficient to stimulate the recruitment of those downstream mediators used by intact ANCA IgG.

Anti-neutrophil cytoplasm autoantibodies (ANCA) are implicated in the pathogenesis of systemic vasculitis. Intact ANCA IgG activate superoxide generation in cytokine-primed neutrophils after binding their antigens and co-engaging Fcgamma receptors (FcgammaR). The contribution of antigen binding via ANCA F(ab')(2) fragments to signaling has been unclear. This study shows that both ANCA IgG and F(ab')(2) fragments of ANCA IgG induce significant GTPase activity, which could be blocked with pertussis toxin and anti-G(i) protein antibodies. Pertussis toxin inhibited ANCA IgG-induced superoxide generation but was without effect on superoxide production after conventional FcgammaR ligation. ANCA F(ab')(2) fragments did not induce superoxide generation. ANCA IgG activated PI 3-kinase-generating PIP(3), activated protein kinase B (PKB), and p21(ras); activation of each mediator was inhibited with pertussis toxin, but PI3K and PKB were not activated by ANCA IgG F(ab')(2) fragments. Intact ANCA IgG induced tyrosine phosphorylation, whereas F(ab')(2) fragments did not, and ANCA IgG-mediated superoxide generation was inhibited with genistein. Both genistein and pertussis toxin together completely abrogated the ANCA-induced oxidative burst. Genistein also inhibited ANCA IgG-induced PIP(3) generation and p21(ras) activation. These data implicate a novel ANCA IgG stimulated signaling pathway that involves both F(ab')(2)-mediated antigen binding and Fc-mediated FcgammaR ligation in cooperative interactions between G(i) proteins and tyrosine kinases that facilitates activation of downstream mediators.

Antibodies, Antineutrophil Cytoplasmic↗