Neutrophil dysfunction in a family with a SAPHO syndrome-like phenotype

SAPHO syndrome (synovitis, acne, pustulosis, hyperostosis, osteitis) is an inflammatory disorder of the bone, skin, and joints. We describe a family with multiple affected members who segregate a SAPHO syndrome-like phenotype, and we report the results of neutrophil studies and candidate gene analysis. We obtained written informed consent and a family history and reviewed medical records. We collected DNA and sequenced candidate genes, and we performed functional studies on neutrophils isolated from the proband and her mother. The pedigree segregated chronic osteomyelitis and cutaneous inflammation in a pattern that suggested an autosomal-dominant disorder. No coding sequence mutations were detected in PSTPIP1, PSTPIP2, LPIN2, SH3BP2, or NCF4. Analysis of neutrophil function in the proband, including nitroblue tetrazolium tests, myeloperoxidase assays, neutrophil chemotaxis, and neutrophil chemotaxis assays, revealed no identifiable abnormalities. However, an abnormality in the luminol, but not the isoluminol, respiratory burst assays following stimulation with phorbol myristate acetate (PMA) was detected in neutrophils isolated from the affected proband. Internal oxidant production was also reduced in the proband and her mother when neutrophils were treated with fMLP with or without platelet-activating factor, PMA alone, or tumor necrosis factor alpha alone. This family segregates a disorder characterized by chronic inflammation of the skin and bone. Functional differences in neutrophils exist between affected individuals and controls. The biologic significance of this defect remains unknown. Identification of the gene defect will help identify an immunologic pathway that, when dysregulated, causes inflammation of the skin and bone.     

Prevention of cytokine-induced changes in leukocyte adhesion receptors by nonsteroidal antiinflammatory drugs from the oxicam family

Objective. To explore the effect of the nonsteroidal antiinflammatory drugs (NSAIDs) piroxicam and meloxicam on quantitative and qualitative changes in leukocyte adhesion receptors induced by cytokines and other activation stimuli. Methods. The expression of CD11b and L-selectin during neutrophil activation with tumor necrosis factor α (TNFα), granulocyte-macrophage colony-stimulating factor (GM-CSF), FMLP, phorbol myristate acetate (PMA), and calcium ionophore A23187 was assessed by flow cytometry. Enzyme-linked immunosorbent assays were used to quantitate soluble L-selectin shed after neutrophil stimulation. Enzyme release was measured to determine neutrophil degranulation by proinflammatory stimuli. Changes in affinity state of β1 and β2 integrins after neutrophil and T lymphocyte stimulation were assessed, by flow cytometry, using the monoclonal antibodies (MAb) HUTS-21 (anti-β1) and CBRM1/5 (anti-CD11b), which recognize activation-dependent epitopes on these two integrins. Results. Pretreatment of neutrophils with either NSAID prevented the changes in L-selectin and CD11b expression induced by TNFα, GM-CSF, and FMLP, but not those induced by PMA or A23187. Furthermore, piroxicam significantly decreased the amount of L-selectin shed by cytokine-treated neutrophils, whereas it did not exert this effect on PMA- or A23187-treated neutrophils. Piroxicam also decreased the release of gelatinase and lysozyme induced by TNFα, but not by PMA. Interestingly, piroxicam prevented the conformational changes that β2 integrins underwent upon activation of neutrophils: the appearance of the activation epitope of CD11b, detected by the CBRM1/5 MAb, was blocked by piroxicam in TNFα-treated neutrophils. Moreover, in chemokine-treated T lymphocytes, the expression of activation epitopes on β1 integrins was also diminished by piroxicam. In contrast, this NSAID did not affect the β1 integrin conformational changes induced by PMA or Mn⁺⁺. Conclusion. Our results indicate that members of the oxicam family are able to interfere with events of neutrophil function, such as their degranulation and cytokine-mediated activation changes in adhesion molecules, both in neutrophils and in lymphocytes. Such effects may significantly contribute to the antiinflammatory activity of these drugs.     

Neuropeptides and inflammation. A somatostatin analog as a selective antagonist of neutrophil activation by substance P

Objective. Substance P and somatostatin are neuropeptides found in peripheral sensory nerves. In vitro, these have opposing effects on inflammatory cells. We compared the effects of these peptides on the activation of neutrophils. Methods. Neutrophils were isolated from healthy volunteers, and chemotaxis, superoxide anion generation, aggregation, and changes in cytosolic calcium and GTPase activity were measured in the presence of substance P, somatostatin, and the chemoattractant FMLP. Results. Substance P was an effective chemoattractant, 20% as potent as FMLP at equimolar concentrations. Substance P also stimulated GTPase activity in neutrophil plasma membranes. Somatostatin did not activate neutrophils; however, it effectively inhibited neutrophil chemotaxis and GTPase activity provoked substance P, but not by FMLP. Conclusions. These studies demonstrate that substance P can effectively stimulate chemotaxis, possible via effects on a GTP-binding protein distinct from that triggered by FMLP, and that somatostatin is a selective antagonist of substance P. The biochemical specificities of these peptides on cells may modulate neurogenic inflammation at the local level.     

In vivo neutrophil and lymphocyte function studies in a patient with leukocyte adhesion deficiency type II

We investigated in vivo neutrophil and lymphocyte function in a patient who lacks Sialyl-Lewis-X, a ligand for the selectin family of leukocyte adhesion molecules (leukocyte adhesion deficiency II, LAD II). As assessed by skin chamber and skin window techniques, in vivo chemotaxis of neutrophils was markedly impaired (less than 6% of normal values). A marginal pool was present as determined by an increase in circulating neutrophils after epinephrine injection and calculated recovery of infused radiolabeled autologous neutrophils. Kinetic studies showed a reduced half-life of 3.2 hours (normal 7 hours) and markedly increased turnover rate (cells/kg/d) of approximately eight times the normal value. A normal antibody response to the T-cell-dependent antigen bacteriophage phi X174 showed that T/B-cell interaction is not affected in LAD II. These findings provide direct evidence that the selectin family and its ligands play an important role in neutrophil function.     

Secretory leukocyte protease inhibitor inhibits neutrophil apoptosis

Background and objective: The secretory leukocyte protease inhibitor (SLPI) is a major anti‐elastase barrier at the epithelial surfaces of upper respiratory tract. In addition to its anti‐protease activity, SLPI has been shown to express anti‐bacterial, anti‐viral and anti‐inflammatory properties. Methods: We measured SLPI concentration in nasal lavage fluid of healthy volunteers after challenge with endotoxin (LPS) and evaluated SLPI effects in vitro on neutrophil chemotaxis, adhesion, cytokine (IL‐8) release and apoptosis. Results: SLPI concentration in nasal lavage (n = 9) 2, 6 and 24 h after the challenge with LPS (25 µg) increased from 32% to 238% compared with baseline (226 ± 71 ng/mL). In vitro, SLPI (20–80 µg/mL) induced neutrophil chemotaxis (sixfold, P < 0.001) and decreased neutrophil apoptosis by 73% (P = 0.006), relative to controls. However, SLPI had no affect on IL‐8 release or neutrophil adhesion to fibronectin. SLPI‐positive immunoreactivity was co‐localized with neutrophils in lung specimens from patients with COPD. Conclusions: Our findings indicate upregulation of SLPI in response to LPS in nasal secretions and show anti‐apoptotic effects of SLPI in primary human neutrophils suggesting a new role of SLPI during neutrophilic inflammation.     

Tumor suppressor PTEN is a physiologic suppressor of chemoattractant-mediated neutrophil functions

The recruitment and activation of neutrophils at infected tissues is essential for host defense against invading microorganisms. However, excessive neutrophil recruitment or activation can also damage the surrounding tissues and cause unwanted inflammation. Hence, the responsiveness of neutrophils needs to be tightly regulated. In this study, we have investigated the functional role of tumor suppressor PTEN in neutrophils by using a mouse line in which PTEN is disrupted only in myeloid-derived cells. Chemoattractant-stimulated PTEN(-/-) neutrophils displayed significantly higher Akt phosphorylation and actin polymerization. A larger fraction of these neutrophils displayed membrane ruffles in response to chemoattractant stimulation. In addition, chemoattractant-induced transwell migration and superoxide production were also augmented. Single-cell chemotaxis assays showed that PTEN(-/-) neutrophils have a small (yet statistically significant) defect in directionality. However, these neutrophils also showed an increase in cell speed. As a result, overall chemotaxis, which depends on speed and directionality, was not affected. Consistent with the increased responsiveness of PTEN(-/-) neutrophils, the in vivo recruitment of these cells to the inflamed peritoneal cavity was significantly enhanced. Thus, as a physiologic-negative regulator, PTEN should be a promising therapeutic target for modulating neutrophil functions in various infectious and inflammatory diseases.     

Alterations in rat peripheral blood neutrophil function as a consequence of colitis

Altered peripheral neutrophil function is a feature of IBD that may contribute to the chronicity and extragastrointestinal manifestations of this disease, but clinical evidence for such alterations is confounded by variations in patient characteristics, disease onset, and use of therapeutics that can influence neutrophil function. The use of a rat model of colitis has permitted us to characterize, in a controlled manner, the causal relationship between colitis and altered peripheral neutrophil function. At various times after induction of colitis with trinitrobenzene sulfonic acid (TNBS), peripheral neutrophils were isolated and assays of phagocytosis, chemotaxis, leukotriene B₄ (LTB₄) synthesis, and superoxide production were performed using a variety of stimuli. Circulating neutrophil numbers increased about fourfold within 12 hr of TNBS administration and returned to normal levels over the following two weeks. LTB₄ synthesis in response to calcium ionophore decreased at 12 hr after induction of colitis, then returned to control levels. The chemotactic responses of peripheral neutrophils to LTB₄ and FMLPin vitro and to LTB₄ and IL-8in vivo were profoundly suppressed through the two-week study period. Phagocytosis of nitroblue tetrazolium was significantly enhanced (ca. threefold) at 12 hr after induction of colitis and remained elevated throughout the study period. Superoxide production was also significantly elevated in the early phase of colitis (by ca. fourfold), but was not different from control levels at seven and 14 days. These results demonstrate that colonic inflammation profoundly influences peripheral blood neutrophil function, although the direction and magnitude of the alteration varied among the various functions assessed. The prolonged depression of chemotactic activity may represent a physiological reaction to limit the inflammatory response.     

Participation of Mac-1, LFA-1 and VLA-4 integrins in the in vitro adhesion of sickle cell disease neutrophils to endothelial layers, and reversal of adhesion by simvastatin

Background

Pharmacological approaches to inhibit increased leukocyte adhesive interactions in sickle cell disease may represent important strategies for the prevention of vaso-occlusion in patients with this disorder. We investigated, in vitro, the adhesion molecules involved in endothelial-sickle cell disease neutrophil interactions and the effect of simvastatin on sickle cell disease neutrophil adhesion to tumor necrosis factor-α-activated endothelial monolayers (human umbilical vein endothelial cells), and neutrophil chemotaxis.

Design and Methods

Sickle cell disease patients in steady state and not on hydroxyurea were included in the study. Endothelial cells treated, or not, with tumor necrosis factor-α and simvastatin were used for neutrophil adhesion assays. Neutrophils treated with simvastatin were submitted to interleukin 8-stimulated chemotaxis assays.

Results

Sickle cell disease neutrophils showed greater adhesion to endothelial cells than control neutrophils. Adhesion of control neutrophils to endothelial cells was mediated by Mac-1 under basal conditions and by the Mac-1 and LFA-1 integrins under inflammatory conditions. In contrast, adhesion of sickle cell disease neutrophils to endothelium, under both basal and tumor necrosis factor-α-stimulated conditions, was mediated by Mac-1 and LFA-1 integrins and also by VLA-4. Under stimulated inflammatory conditions, simvastatin significantly reduced sickle cell disease neutrophil adhesion, and this effect was reversed by inhibition of nitric oxide synthase. Furthermore, intercellular adhesion molecule-1 expression was significantly abrogated on tumor necrosis factor-α-stimulated endothelium incubated with simvastatin, and statin treatment inhibited the interleukin-8-stimulated migration of both control and sickle cell disease neutrophils.

Conclusions

The integrins Mac-1, LFA-1 and, interestingly, VLA-4 mediate the adhesion of sickle cell disease leukocytes to activated endothelial cell layers, in vitro. Our data indicate that simvastatin may be able to reduce endothelial activation and consequent leukocyte adhesion in this in vitro model; future experiments and clinical trials may determine whether simvastatin therapy could be employed in patients with sickle cell disease, with beneficial effects on vaso-occlusion.     

Persistent neutrophil dysfunction in an adult. Combined defect in chemotaxis, phagocytosis and intracellular killing

Impaired phagocytosis and killing of bacteria by neutrophils were demonstrated repeatedly in a 65 year old man. He had staphylococcal pyarthrosis and osteomyelitis. Phagocytosis and intracellular killing of Staphylococcus aureus by neutrophils of the patient were impaired, but no significant defect was noted when Candida was used as the test organism. Production of ¹⁴CO₂ from glucose-1-¹⁴C, glucose-6-phosphate dehydrogenase levels, nitroblue tetrazolium reduction test and myeloperoxidase levels were all normal in his neutrophils. The monocyte bactericidal capacity for Staph. aureus was normal. Despite normal migration demonstrated by the skin window technic and by the presence of numerous neutrophils in the joint aspirate, studies of neutrophil chemotaxis revealed an impaired response to chemotaxin, and an inhibitor of chemotaxis of normal neutrophils was also found in his serum. However, his serum generated chemotaxin normally. This neutrophil dysfunction appears to differ from that found in chronic granulomatous disease of childhood and other known defects of neutrophil function.     

Human filarial Wolbachia lipopeptide directly activates human neutrophils in vitro

The host inflammatory response to the Onchocerca volvulus endosymbiont, Wolbachia, is a major contributing factor in the development of chronic pathology in humans (onchocerciasis/river blindness). Recently, the toll‐like pattern recognition receptor motif of the major inflammatory ligands of filarial Wolbachia, membrane‐associated diacylated lipoproteins, was functionally defined in murine models of pathology, including mediation of neutrophil recruitment to the cornea. However, the extent to which human neutrophils can be activated in response to this Wolbachia pattern recognition motif is not known. Therefore, the responses of purified peripheral blood human neutrophils to a synthetic N‐terminal diacylated lipopeptide (WoLP) of filarial Wolbachia peptidoglycan‐associated lipoprotein (PAL) were characterized. WoLP exposure led to a dose‐dependent activation of healthy, human neutrophils that included gross morphological alterations and modulation of surface expressed integrins involved in tethering, rolling and extravasation. WoLP exposure induced chemotaxis but not chemokinesis of neutrophils, and secretion of the major neutrophil chemokine, interleukin 8. WoLP also induced and primed the respiratory burst, and enhanced neutrophil survival by delay of apoptosis. These results indicate that the major inflammatory motif of filarial Wolbachia lipoproteins directly activates human neutrophils in vitro and promotes a molecular pathway by which human neutrophils are recruited to sites of Onchocerca parasitism.     

Increased sensitivity to the C-X-C chemokine CINC/gro in a model of chronic inflammation

OBJECTIVE: The C-C chemokine MCP-1 elicits significant neutrophil emigration in rats with chronic adjuvant-induced inflammation, but not in naive animals. We examined responses to the C-X-C chemokine CINC/gro to determine whether this class of chemokine elicits altered neutrophil responses during chronic inflammation. METHODS: CINC/gro was superfused over mesenteric venules of naive rats or animals with chronic adjuvant-induced vasculitis. Antibodies were used to characterize adhesive mechanisms. RESULTS: CINC/gro elicited leukocyte transendothelial migration in adjuvant-immunized rats at 100-fold lower concentrations than required to elicit transmigration in naive animals. In both groups, neutrophils constituted > 95% of the leukocytes recruited by CINC/gro. Using in vitro chemotaxis assays, neutrophils from control and adjuvant-immunized rats responded equally to CINC/gro, suggesting differences in migration were not related to neutrophil phenotype. Differences in adhesion molecule usage were noted in vivo. In control animals, CD18 antibodies blocked CINC/gro-induced neutrophil adhesion and emigration. In adjuvant-immunized animals, an alpha 4-integrin antibody reduced adhesion and emigration, while a CD18 antibody selectively inhibited emigration. CONCLUSIONS: This study demonstrates increased sensitivity to a C-X-C chemokine in a model of chronic inflammation, implicates the alpha 4-integrin in neutrophil adhesion, and demonstrates that CD18 mediates leukocyte transendothelial migration independent from firm adhesion.     

Lipopolysaccharide: a p38 MAPK-dependent disrupter of neutrophil chemotaxis

In sepsis, and in models of sepsis including endotoxemia, impaired neutrophil recruitment and chemotaxis have been reported. The inability of the endotoxemic neutrophil to chemotax could be attributed to the fact that intracellular signaling via LPS overrides signals from endogenous chemokines or, alternatively, that sequestration of neutrophils into lungs prevents access to peripheral tissues. Using both in vitro and in vivo chemotaxis assays the authors established that neutrophils from healthy mice chemotaxed in vivo toward MIP-2, whereas endotoxemic neutrophils did not. Since LPS activates leukocytes via the p38 MAPK pathway, SKF86002, a p38 MAPK inhibitor, was given to endotoxemic animals. SKF86002 significantly reversed the LPS-induced impairment in emigration of endotoxic neutrophils in response to MIP-2. Neutrophil chemotaxis in vitro was also impaired by LPS, via a p38 MAPK-dependent pathway, and this impairment could be reversed via p38 MAPK inhibition. Although neutrophil numbers dropped in the circulation and trapped in lungs during endotoxemia, SKF86002 did not reverse these parameters, demonstrating that p38 MAPK inhibition did not release trapped neutrophils from the lungs. In conclusion, the data suggest that the impaired emigration and chemotaxis of neutrophils at peripheral sites during endotoxemia may be partially due to a p38 MAPK-mediated inhibition of neutrophil responses to endogenous chemokines.     

Acute skin exposure to ultraviolet light triggers neutrophil-mediated kidney inflammation

Photosensitivity to ultraviolet (UV) light affects up to ∼80% of lupus patients. Sunlight exposure can exacerbate local as well as systemic manifestations of lupus, including nephritis, by mechanisms that are poorly understood. Here, we report that acute skin exposure to UV light triggers a neutrophil-dependent injury response in the kidney characterized by upregulated expression of endothelial adhesion molecules as well as inflammatory and injury markers associated with transient proteinuria. We showed that UV light stimulates neutrophil migration not only to the skin but also to the kidney in an IL-17A–dependent manner. Using a photoactivatable lineage tracing approach, we observed that a subset of neutrophils found in the kidney had transited through UV light–exposed skin, suggesting reverse transmigration. Besides being required for the renal induction of genes encoding mediators of inflammation (vcam-1, s100A9, and Il-1b) and injury (lipocalin-2 and kim-1), neutrophils significantly contributed to the kidney type I interferon signature triggered by UV light. Together, these findings demonstrate that neutrophils mediate subclinical renal inflammation and injury following skin exposure to UV light. Of interest, patients with lupus have subpopulations of blood neutrophils and low-density granulocytes with similar phenotypes to reverse transmigrating neutrophils observed in the mice post-UV exposure, suggesting that these cells could have transmigrated from inflamed tissue, such as the skin.

Sensitivity to ultraviolet (UV) sunlight rays is a well-recognized feature of systemic lupus erythematosus (SLE) (1). Skin exposure to UV light triggers both local and systemic inflammation and has been associated with systemic disease flares, including lupus nephritis (LN), in SLE patients (2–4). How photosensitivity in the skin leads to systemic manifestations remains poorly understood. Shared gene signatures in the skin and kidney of SLE patients (5) suggest common pathogenesis. We recently observed that acute skin exposure to UV light triggers both a local and a systemic type I interferon (IFN-I) response (6), indicating inflammatory responses to UV light are not limited to the skin. How exactly skin exposure to UV light impacts the kidney is not known.We and others have shown that UV light induces rapid neutrophil infiltration into the skin (6, 7). Although neutrophils are major contributors to inflammation at local injury sites, it has recently been recognized that neutrophils can also home to organs distant from the primary site of inflammation (8–10), where they contribute to lung tissue injury via production of reactive oxygen species (ROS) (11) or activate the adaptive immune system in the lymph nodes and the bone marrow (9, 12). In SLE, neutrophils are thought to play an important role in both local and systemic disease. Neutrophils are present in the skin lesions of SLE patients (13, 14) and in the kidney tissue of patients with LN (15, 16). High expression of a neutrophil gene signature is a strong predictor of active disease, including LN and cutaneous flares (17, 18). Proinflammatory low-density granulocytes (LDGs) are increased particularly in patients with skin disease (19). While these findings implicate neutrophils in local tissue injury in SLE, whether neutrophils provide the pathogenic link between skin inflammation and kidney injury is not understood.To elucidate how skin exposure to UV light impacts the kidney and the role neutrophils play in these processes, we evaluated changes in renal gene expression in concert with neutrophil migration. We found that acute skin exposure to UV light stimulates inflammatory processes in the kidney, including expression of endothelial adhesion molecules, inflammatory mediators, injury markers, and transient proteinuria. Notably, we found that neutrophils migrated to the kidney after UV light exposure in an IL-17A–dependent manner, localizing to the tubulointerstitial (TI) areas where they exhibited proinflammatory phenotypes. Blocking neutrophil trafficking by anti–G-CSF immunoglobulin (IgG) treatment abrogated renal inflammation and prevented up-regulation of tubular injury markers. Together, these findings demonstrate that skin exposure to UV light triggers subclinical renal inflammatory and injury processes mediated by neutrophils.     

Identification of two novel mutations in RASGRP2 affecting platelet CalDAG-GEFI expression and function in patients with bleeding diathesis

The RASGRP2 gene encodes the Ca²⁺ and DAG-regulated guanine nucleotide exchange factor I (CalDAG-GEFI), which plays a key role in integrin activation in platelets and neutrophils. We here report two new RASGRP2 variants associated with platelet dysfunction and bleeding in patients. The homozygous patients had normal platelet and neutrophil counts and morphology. Platelet phenotyping showed: prolonged PFA-100 closure times; normal expression of major glycoprotein receptors; severely reduced platelet aggregation response to ADP and collagen (both patients); aggregation response to PAR1 and arachidonic acid markedly impaired in one patient; PMA-induced aggregation unaffected; platelet secretion, clot retraction, and spreading minimally affected. Genetic analysis identified two new homozygous variants in RASGRP2: c.706C>T (p.Q236X) and c.887G>A (p.C296Y). In both patients, CalDAG-GEFI protein was not detectable in platelet lysates, and platelet αIIbβ3 activation, as assessed by fibrinogen binding, was greatly impaired in response to all agonists except PMA. Patient neutrophils showed normal integrin expression, but impaired Mn²⁺-induced fibrinogen binding. In summary, we have identified two new RASGRP2 mutations that can be added to this rapidly growing form of inherited platelet function disorder.     

The chemotactic function of neutrophils in ulcerative colitis

Neutrophil chemotactic function was investigated in 30 patients with ulcerative colitis (UC) in an active (no. = 15) or an inactive (no. = 15) stage. Peripheral blood neutrophils from active cases had a significantly decreased stimulated migration in modified Boyden chambers. Simultaneously done investigations of neutrophil migration into skin window chambers showed normal results in both active and inactive cases of UC. Thus, the defective chemotaxis during in vitro conditions was not reflected in a quantifiable neutrophil dysfunction in experimental acute inflammation.     

Inhibition of neutrophil oxidative metabolism by trichinellosis patient sera. Parasite origin or host induction?

The presence of sera factors able to inhibit both neutrophil chemotaxis and phagocytosis was observed in all patients studied at two months from infection caused by Trichinella britovi and in most of them after one year. Human neutrophils with eosinophils are able to kill T. spiralis newborn larvae in an ADCC system and their major cytotoxic mechanism is oxidative metabolism products. We evaluated the effect of trichinellosis sera on neutrophil oxidative burst to determine if neutrophils are affected by circulating factors during infection. Cells were incubated with sera from trichinellosis patients. Basal or stimulated Superoxide Anion (SA) production and chemilumines-cence in response to different stimulation (PMA, f-MLP, opsonized yeasts) of neutrophils incubated with trichinellosis sera were evaluated and compared with those of cells incubated with control sera. The results show that basal SA production was inhibited by 66% of sera and stimulated by 11%. On the contrary f-MLP stimulated production was significantly increased by 22% sera, and inhibited by none. Chemiluminescence in response to f-MLP or PMA was inhibited by 46 and 80% of sera, respectively. These results show that trichinellosis sera can modulate not only SA production but also other steps of the oxidative burst, irrespective of the stimulating agent, so suggesting that different neutrophil activation pathways are affected. Increased IL-2 levels observed in most of the sera did not correlate with the inhibiting capacity of sera. The hypothesis of a parasite origin of the inhibiting factors is discussed in the light of host-parasite relationship.,     

Rare human leukocyte antigen genotype in two siblings with type 1 diabetes in a Japanese family clustered with type 1 diabetes

Multiplex families with type 1 diabetes are important for identification of rare variants that cannot be identified in case–control association studies. The very low incidence of type 1 diabetes in the Japanese population, however, makes identification of such families difficult. We identified a Japanese family in which three members developed type 1 diabetes, and studied the genotype of the human leukocyte antigen. All three members with type 1 diabetes had the DRB1*08:02‐DQB1*03:02 haplotype, which is specific to the Asian population and strongly susceptible for type 1 diabetes. In particular, a proband and his sister had the same genotype, DRB1*08:02‐DQB1*03:02/DRB1*08:02‐DQB1*03:02, which is extremely rare even in the Japanese population. Both parents also had DRB1*08:02‐DQB1*03:02, but in combination with different human leukocyte antigen haplotypes. Weakly susceptible DRB1*13:02‐DQB1*06:04 was present in the affected mother, and resistant DRB1*15:01‐DQB1*06:02 in the unaffected father. These data suggest DRB1*08:02‐DQB1*03:02 to be a contributing factor for familial clustering of type 1 diabetes in this family.     

Whole-exome sequencing identifies the first French MODY 6 family with a new mutation in the NEUROD1 gene

AimThe aim of the present study was to identify the affected gene in a French family with maturity-onset diabetes of the young (MODY) using whole-exome sequencing (WES).MethodsWES was performed in one patient with MODY, and candidate variants were confirmed in members of the immediate family by Sanger sequencing.ResultsIn the proband, a new heterozygous missense mutation (c.340A>C) was identified in the NEUROD1 gene by WES analysis and confirmed by Sanger sequencing. Additional Sanger sequencing of the proband's sister and mother revealed the same heterozygous mutation. The proband and his sister displayed typical clinical characteristics of MODY, while their mother had the same typical MODY features except for later onset. When clinical and biological profiles were established for all three patients, the severity of diabetes-related complications varied substantially from one family member to another.ConclusionA novel missense mutation found in NEUROD1 was associated with MODY 6 features in a single French family.     

Cell-surface heparan sulfate proteoglycan-mediated regulation of human neutrophil migration by the serpin antithrombin III

The serpin antithrombin III (AT III) is reported to have hemostasis-regulating and anti-inflammatory properties. To determine its ability to influence thrombin-independent leukocyte responses, the direct effects of the AT III concentrate Kybernin P and a monoclonal antibody-purified AT III on neutrophil migration were studied. Chemotactic activity of human neutrophils isolated from the blood of healthy donors was determined in modified Boyden microchemotaxis chambers, and binding studies were performed according to standard experimental protocols. Preincubation in vitro of neutrophils with Kybernin P or immune-adsorbed AT III significantly deactivated migration toward fMet-Leu-Phe, or interleukin-8 (IL-8), in a concentration-dependent manner. In the absence of additional attractants, neutrophils exhibited a migratory response toward gradients of AT III preparations. True chemotaxis was confirmed in checkerboard assays. Analyses revealed that the AT III heparin-binding site interacts with neutrophil membrane-associated heparan sulfate proteoglycan receptors. Mechanisms of intracellular signaling differed; the deactivation of IL-8-induced chemotaxis resulted from tyrphostin-sensitive interactions of AT III-signaling with the IL-8 signal transduction pathway, whereas AT III-induced chemotaxis involved protein kinase C and phosphodiesterases. Signaling similarities between AT III and the proteoglycan syndecan-4 may suggest the binding of AT III to this novel type of membrane receptor. Under physiological conditions, AT III may prevent neutrophils from premature activation. Moreover, the systemic administration of AT III concentrate could have beneficial effects in combating systemic inflammation.     

Neutrophil elastase augments acute edematous injury in isolated rat lungs perfused with neutrophil cytoplasts

To assess the contribution of neutrophil elastase to neutrophil-mediated acute edematous lung injury, isolated rat lungs were perfused with human neutrophil cytoplasts that produce O2 metabolites normally but contain little of the neutrophil elastase usually found in neutrophils. We found that addition of neutrophil cytoplasts and phorbol myristate acetate (PMA) caused less edematous injury in isolated lungs than addition of neutrophils and PMA. However, addition of neutrophil elastase along with neutrophil cytoplasts and PMA increased the amounts of lung injury found in lungs perfused with neutrophil cytoplasts and PMA to levels that equaled the amount of lung injury found after addition of normal neutrophils and PMA. By comparison, addition of purified neutrophil elastase, PMA alone, or human neutrophils alone did not cause lung injury. The results indicate that neutrophil elastase is a necessary component for the maximal development of acute edematous injury in isolated lungs perfused with neutrophils and PMA.