Invariant natural killer T cells recognize glycolipids from pathogenic Gram-positive bacteria.

Natural killer T cells (NKT cells) recognize glycolipid antigens presented by CD1d. These cells express an evolutionarily conserved, invariant T cell antigen receptor (TCR), but the forces that drive TCR conservation have remained uncertain. Here we show that NKT cells recognized diacylglycerol-containing glycolipids from Streptococcus pneumoniae, the leading cause of community-acquired pneumonia, and group B Streptococcus, which causes neonatal sepsis and meningitis. Furthermore, CD1d-dependent responses by NKT cells were required for activation and host protection. The glycolipid response was dependent on vaccenic acid, which is present in low concentrations in mammalian cells. Our results show how microbial lipids position the sugar for recognition by the invariant TCR and, most notably, extend the range of microbes recognized by this conserved TCR to several clinically important bacteria.

Dendritic cell-based immunization ameliorates pulmonary infection with highly virulent Cryptococcus gattii.

Cryptococcosis due to a highly virulent fungus, Cryptococcus gattii, emerged as an infectious disease on Vancouver Island in Canada and surrounding areas in 1999, causing deaths among immunocompetent individuals. Previous studies indicated that C. gattii strain R265 isolated from the Canadian outbreak had immune avoidance or immune suppression capabilities. However, protective immunity against C. gattii has not been identified. In this study, we used a gain-of-function approach to investigate the protective immunity against C. gattii infection using a dendritic cell (DC)-based vaccine. Bone marrow-derived dendritic cells (BMDCs) efficiently engulfed acapsular C. gattii (Δcap60 strain), which resulted in their expression of costimulatory molecules and inflammatory cytokines. This was not observed for BMDCs that were cultured with encapsulated strains. When Δcap60 strain-pulsed BMDCs were transferred to mice prior to intratracheal R265 infection, significant amelioration of pathology, fungal burden, and the survival rate resulted compared with those in controls. Multinucleated giant cells (MGCs) that engulfed fungal cells were significantly increased in the lungs of immunized mice. Interleukin 17A (IL-17A)-, gamma interferon (IFN-γ)-, and tumor necrosis factor alpha (TNF-α)-producing lymphocytes were significantly increased in the spleens and lungs of immunized mice. The protective effect of this DC vaccine was significantly reduced in IFN-γ knockout mice. These results demonstrated that an increase in cytokine-producing lymphocytes and the development of MGCs that engulfed fungal cells were associated with the protection against pulmonary infection with highly virulent C. gattii and suggested that IFN-γ may have been an important mediator for this vaccine-induced protection.

The first reported case of central venous catheter-related fungemia caused by Cryptococcus liquefaciens.

We describe a case of central venous catheter-related fungemia caused by Cryptococcus liquefaciens, a non-neoformans and non-gattii Cryptococcus, in a non-HIV patient. A 71-year-old man with diffuse large B-cell lymphoma receiving antineoplastic chemotherapy was febrile approximately 30 weeks after central venous port insertion, and C. liquefaciens was isolated from all three performed blood cultures as well as a central venous catheter tip culture. In vitro antifungal susceptibility tests showed that this yeast isolate was susceptible to low concentrations of amphotericin B, fluconazole, itraconazole and voriconazole yet was resistant to 5-fluorocytosine (MIC: >64 µg/ml), unlike Cryptococcus neoformans. Treatment of the patient with oral and intravenous voriconazole was effective and consistent with the susceptibility tests. Although non-neoformans and non-gattii Cryptococcus spp. are considered non-pathogenic environmental yeast, they may rarely be the causative agents of serious infections in humans, as in the present case.

Exacerbation of invasive Candida albicans infection by commensal bacteria or a glycolipid through IFN-γ produced in part by iNKT cells.

BACKGROUND: The commensal yeast Candida albicans is a major cause of invasive fungal infections. Despite treatment with antifungal agents, the mortality rate attributed to these types of infection is high. Although numerous cases have been reported regarding a poor outcome for patients with bacterial and C. albicans coinfection, the mechanisms by which the coinfecting bacteria exacerbate the C. albicans infection remain elusive. METHODS AND RESULTS: We evaluated how glycolipid-mediated activation of invariant natural killer T (iNKT) cells affects the clearance of C. albicans. Surprisingly, C. albicans-infected, glycolipid-treated mice exhibited significantly lower survival rates, increased fungal burden, and higher interleukin (IL)-6 production in the kidneys compared with control mice. Glycolipid-induced exacerbation of C. albicans infection was not observed in interferon-gamma knockout (IFN-γKO) mice. In the C. albicans-infected, glycolipid-treated mice, the number of neutrophils in the blood and bone marrow dramatically decreased in an IFN-γ-dependent manner. Furthermore, mice that were coinfected with C. albicans and nonfermentative gram-negative commensal bacteria exhibited increased fungal burden and inflammatory cytokine production in the kidneys that were dependent on IFN-γ and iNKT cells. CONCLUSIONS: Our results indicate that coinfecting commensal bacteria exacerbate C. albicans infection through IFN-γ produced, in part, by iNKT cells.

The mannan of Candida albicans lacking ß-1,2-linked oligomannosides increases the production of inflammatory cytokines by dendritic cells.

Mannans are mannose polymers attached to cell wall proteins in all Candida species, including the pathogenic fungus Candida albicans. Mannans are sensed by pattern recognition receptors expressed on innate immune cells. However, the detailed structural patterns affecting immune sensing are not fully understood because mannans have a complex structure that includes α- and ß-mannosyl linkages. In this study, we focused on the ß-1,2-mannosides of N-linked mannan in C. albicans because this moiety is not present in the non-pathogenic yeast Saccharomyces cerevisiae. To investigate the impact of ß-1,2-mannosides on immune sensing, we constructed a C. albicans ∆mnn4/∆bmt1 double deletant. Thin-layer chromatography and nuclear magnetic resonance analyses revealed that the deletant lacked ß-1,2-mannosides in N-linked mannan. Mannans lacking the ß-1,2-mannosides induced the production of higher levels of inflammatory cytokines, including IL-6, IL-12p40 and TNF-α, in mice dendritic cells compared to wild-type mannan. Our data show that ß-1,2-mannosides in N-linked mannan reduce the production of inflammatory cytokines by dendritic cells.

Administration of human immunoglobulin suppresses development of murine systemic vasculitis induced with Candida albicans water-soluble fraction: an animal model of Kawasaki disease.

We investigated the inhibitory effect of human immunoglobulin (h-Ig) on the development of coronary arteritis in a murine model of vasculitis induced with a Candida albicans water-soluble fraction (CAWS). CAWS was intraperitoneally injected to C57BL/6 mice for 5 days. Then h-Ig was administered according to various schedules. The animals were sacrificed in week 5, and the status of vasculitis in the coronary arteries and the aortic root was investigated histologically. The groups in which h-Ig was administered for 5 days from day 3 and from day 5 of the experiment showed a significant reduction in the incidence of panvasculitis. In addition, the scope and severity of the inflammation of the aortic root and the coronary arteries were reduced in both groups. In the group administered h-Ig for 5 days from day 1 and the group administered a high dose of h-Ig once on day 1 or day 3, no suppression of development of vasculitis was observed. The h-Ig acted by suppressing the generation and progression of vasculitis in this CAWS-induced murine vasculitis model.

Nanocrystal quantum dot-conjugated anti-myeloperoxidase antibody as the detector of activated neutrophils.

Fluorescent nanocrystal quantum dots (QDs) have been applied to a wide range of biological studies by taking advantage of their fluorescence properties. Here we show that QDs conjugated with antibody against neutrophil peroxidase, myeloperoxidase (MPO). We designed a novel method to conjugate QDs to antibody without losing any antibody function including their antigen recognizing and Fc-receptor binding activities. When we applied anti-MPO antibody (Ab) with conventional organic probes in the case of immunostaining of living cells, the antibodies lost their fluorescence because of MPO enzymic activity to produce reactive oxygen species. Our QD-conjugated anti-MPO (alpha-MPO-QDs) can detect MPO on the surface of activated neutrophils. In addition, anti-MPO-QDs did not react to the inactivated neutrophils. In conclusion, we demonstrated that antibody visualized the expression of MPO on the neutrophil surface after stimulation with proinflammatory cytokines. Taken together, these techniques have the possibility that QDs can reveal the activation of neutrophils by immunostaining and flow cytometric analysis as a powerful tool for diagnosis of the neutrophil activation in vitro.

Lethal and severe coronary arteritis in DBA/2 mice induced by fungal pathogen, CAWS, Candida albicans water-soluble fraction.

CAWS is a microbial pathogen-associated molecular patterns (PAMPs) produced by Candida albicans. CAWS is a mannoprotein-beta-glucan complex and secreted into the culture supernatant. CAWS has various biological effects, causing acute shock and disrupting vascular permeability. Intraperitoneal administration of CAWS induces coronary arteritis in various strains of inbred mice. The CAWS-induced coronary arteritis is strain-dependent and most severe in DBA/2 mice with a significant number of these animals expiring with cardiomegaly during the observation period. In vivo and in vitro, splenocytes of DBA/2 mice produced various cytokines, such as IL-6, TNF-alpha, and IFN-gamma in response to CAWS. GM-CSF was also produced in response to CAWS. The production of cytokines was significantly enhanced in the presence of recombinant GM-CSF. In contrast, anti-GM-CSF significantly reduced the production of TNF-alpha and IFN-gamma. Augmented production of cytokines in response to CAWS would be a key to the severity of coronary arteritis.

Contribution of the myeloperoxidase-dependent oxidative system to host defence against Cryptococcus neoformans.

The in vivo contribution of reactive oxygen species produced by neutrophils against Cryptococcus infection is not widely recognized. Myeloperoxidase (MPO) is a neutrophil-specific enzyme that catalyses the production of hypohalous acids such as HOCl from H2O2. This study investigated the role of MPO in immunological defence against Cryptococcus neoformans in an MPO-deficient (MPO-/-) mouse model. The survival of MPO-/- mice infected either intranasally or intravenously with C. neoformans was lower than that of identically challenged wild-type mice. The MPO-/- mice that received intranasal injection of C. neoformans had significantly larger lung fungal burdens than wild-type mice. On day 7, MPO-/- mice had a significantly higher lung concentration of interleukin (IL)-4 and lower concentrations of IL-2, IL-12p70 and interferon (IFN)-gamma than wild-type mice, suggesting a weak Th1 response in the MPO-/- mice to C. neoformans. Pathologically, the MPO-/- mice with intranasal infection showed more severe pneumonia than wild-type mice, which was associated with an increase in the levels of IL-1alpha/beta in the lungs. In addition, in MPO-/- mice, the pulmonary infection disseminated to the brain with occasional meningitis. The keratinocyte-derived cytokine (KC) level in the brain of infected MPO-/- mice was higher than that of control mice. Both intranasal and intravenous infections resulted in a higher number of fungi in the spleen of MPO-/- mice compared to wild-type, suggesting decreased resistance to C. neoformans not only in the lungs but also in the spleen in the absence of MPO. Taken together, these data suggest a major role of MPO in the response to cryptococcal infection.

Myeloperoxidase has directly-opposed effects on nitration reaction--study on myeloperoxidase-deficient patient and myeloperoxidase-knockout mice.

Myeloperoxidase (MPO) catalyzes a nitration reaction to form nitrotyrosine in the presence of high nitrite, the metabolite of NO. Human leukocyte was shown to cause phenolic nitration using released MPO as a catalyst in the presence of nitrite. It opposes our previous finding that inhibition of MPO was essential for phenol nitration in human leukocyte study. To clarify the role of MPO, we utilized MPO-deficient human leukocytes and MPO-knockout mice. Even in the absence of exogenously added nitrite, high nitration product was observed in MPO-deficient leukocytes. In liver subjected to ischemia/reperfusion injury, a significantly higher amount of nitrotyrosine was produced in MPO-knockout mice than in normal mice. These results clearly demonstrate that MPO inhibits the accumulation of nitration products in vivo. Further experiments showed that MPO could degrade nitrotyrosine in the presence of glutathione. Thus, MPO-induced degradation of nitration products may cause the underestimation of the nitration product generated in vivo. We conclude that MPO may act predominantly to scavenge nitrotyrosine under physiological nitrite condition, and protect against injurious effect of nitrotyrosine.

A limited role of iNKT cells in controlling systemic Candida albicans infections.

Candida albicans is a major cause of invasive fungal infections. Mortality attributable to candidemia is very high, even when patients are treated with adequate antifungal agents. Therefore, it is important to investigate the mechanisms of immune response to C. albicans. Invariant natural killer T (iNKT) cells, innate lymphocytes that express an invariant T cell receptor α chain, participate in the response to various microbes, including two fungal pathogens, Aspergillus fumigatus and Cryptococcus neoformans. However, it is unknown whether iNKT cells play a role in the immune response to C. albicans. In this study, we have investigated the role of iNKT cells in the host defense against systemic C. albicans infection in mice. We compared the survival and fungal clearance between control mice and Jα18KO mice, which specifically lack iNKT cells, after intravenous C. albicans infection. There was no difference in the survival and fungal burden in the kidneys of the control and Jα18KO mice. Furthermore, production of inflammatory cytokines in several organs during C. albicans infection did not significantly differ between these two groups. These results suggest that iNKT cells play a minor role in controlling systemic C. albicans infections in mice.

An outbreak of histoplasmosis among healthy young Japanese women after traveling to Southeast Asia.

Histoplasmosis, caused by Histoplasma capsulatum, is an endemic mycosis in many countries of the world except for Japan. Outbreaks of histoplasmosis among Japanese people are very rare and are mainly imported by travelers. We report an outbreak of histoplasmosis among healthy Japanese people who traveled to a resort area in Southeast Asia. Three young Japanese women traveled to Langkawi island, Malaysia and stayed on the island for five days without visiting caves, a known reservoir of H. capsulatum. All three individuals developed flu-like symptoms with multiple nodule shadows on chest X rays or chest CT scans at around ten days after their return to Japan. Serum samples obtained from the three subjects were positive for anti-Histoplasma antibody and specific PCR for H. capsulatum on lung biopsy specimens and the serum from one patient was positive. The clinical course of all three patients improved without the use of anti-fungal agents and no recurrence has been confirmed. Clinical attendants should consider histoplasmosis when they see patients with flu-like symptoms with abnormal chest X-rays after visiting H. capsulatum endemic areas, especially Southeast Asia.

A pilot-controlled study of myeloperoxidase-specific anti-neutrophil cytoplasmic autoantibody (MPO-ANCA) in the coronary circulation.

Hereby we report our observations derived from a pilot-study of 39 subjects (30 patients with coronary artery disease [CAD] and 9 non-CAD controls). In this work, we aimed to evaluate MPO-ANCA titer in the human coronary circulation for the first time; and examine its possible association with CAD and some cytokines/inflammatory markers. We found higher mean coronary MPO-ANCA titer in CAD subjects than in non-CAD controls; beside significant positive correlations between MPO-ANCA titers and both C-reactive protein and interleukin-6 levels. Thus, we might suggest the possible involvement of MPO-ANCA in coronary atherogenesis indirectly through modulating some pro-inflammatory cytokines/markers; that a large-scale study of MPO-ANCA in CAD patients may be warranted in the future.

Suppressive role of leukocyte cell-derived chemotaxin 2 in mouse anti-type II collagen antibody-induced arthritis.

OBJECTIVE: We previously reported that the Val58Ile polymorphism of the leukocyte cell-derived chemotaxin 2 gene (LECT2) is associated with the severity of rheumatoid arthritis (RA). To define the role of LECT2 in inflammatory arthritides, we investigated the development of collagen antibody-induced arthritis (CAIA) in LECT2-deficient (LECT2(-/-)) mice. METHODS: CAIA was induced in mice by administering anti-type II collagen antibodies followed by lipopolysaccharide. Daily assessment of hind paw swelling was used to monitor the development of arthritis. The histopathologic features and expression of inflammatory cytokines were also analyzed. We confirmed the role of LECT2 by introducing a LECT2 expression vector into LECT2(-/-) mice, using a hydrodynamic gene transfer method. RESULTS: Arthritis in LECT2(-/-) mice was significantly exacerbated compared with that in wild-type (WT) controls. Histopathologic assessment of the tarsal joints showed that inflammation and erosion of cartilage and bone in LECT2(-/-) mice were more severe than that in controls. Interleukin-1beta (IL-1beta), IL-6, and certain chemokines were present at significantly higher levels in the arthritic hind paws of LECT2(-/-) mice. In contrast, the amount of LECT2 in the serum and locally in the hind paws was higher in arthritic WT mice. Finally, hydrodynamic gene transfer experiments revealed that the severity of arthritis was reduced by the systemic expression of exogenous mouse LECT2 protein in LECT2(-/-) mice. CONCLUSION: These results strongly suggest that LECT2 directly suppresses the development of CAIA. Manipulation of LECT2 might provide a rationale for novel therapeutic approaches to the treatment of inflammatory arthritides such as RA.

Up-regulation of adhesion molecule expression in glomerular endothelial cells by anti-myeloperoxidase antibody.

BACKGROUND: Anti-neutrophil cytoplasmic antibody directed against myeloperoxidase (MPO-ANCA) has been implicated in pauci-immune crescentic glomerulonephritis. It stimulates primed neutrophils to adhere to glomerular endothelial cells (GECs), thereby releasing reactive oxygen and other toxic substances and ultimately damaging the GECs. Though, a pathogenic role for MPO-ANCA is not fully understood, we hypothesized that MPO-ANCA modulates GEC functions by the increases in expression of adhesion molecules. METHODS: A polyclonal rabbit anti-recombinant mouse MPO antibody (anti-rmMPO IgG) was evaluated in mouse GEC (mGEC) for its effect on adhesion molecule expression. The primary culture of mGEC was incubated with anti-rmMPO IgG or isotype control and the expression of intercellular adhesion molecules-1 (ICAM-1) was evaluated by real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis and ICAM-1 cell ELISA. RESULTS: The real-time RT-PCR analysis showed that a treatment with 100 microg/ml anti-rmMPO IgG increased the expression of mRNAs for ICAM-1, vascular cell adhesion molecule-1 and E-selectin by approximately 12.5, 7.5 and 10.5-fold, respectively. ICAM-1 cell ELISA also substantiated increased expression of ICAM-1. This enhancement of ICAM-1 expression was mediated by the antigen specificity of anti-rmMPO IgG. In addition, there were several proteins in mGEC specifically immunoprecipitated with anti-rmMPO IgG. CONCLUSIONS: These results showed that anti-MPO antibody activates not only neutrophils, but also GEC, indicating that anti-rmMPO IgG-induced direct activation of GEC contributes to neutrophil adhesion to GEC, thereby increasing glomerular neutrophil infiltration in initiation and progression of pauci-immune glomerulonephritis.

Neutrophil activation and arteritis induced by C. albicans water-soluble mannoprotein-beta-glucan complex (CAWS).

We have established a mouse model which shows the symptoms of coronary arteritis after consecutive injections of CAWS, which is released from Candida albicans. In this study, we examined neutrophil activation in the initial period after CAWS injection intraperitoneally. During 10 min to 16 h after the injection, blood profiles and neutrophil functions were determined. At the same time, levels of inflammatory cytokines and chemokines in plasma were measured. Furthermore, level of ICAM-1 as a marker of lesion in arterial endothelial cells was measured. Counts of the peripheral leukocytes increased immediately after CAWS injection, especially involving neutrophil. In vitro sensitivity of neutrophils to stimuli was enhanced. Moreover, proinflammatory cytokines (IL-1beta, IL-12 and IL-6) increased in plasma initially followed by an increase in IL-10, G-CSF, MIP-2 and soluble ICAM-1. Locally, ICAM-1 message in arterial walls was significantly increased 16 h after CAWS injection. A decrease in C3 levels was observed in plasma, suggesting complement activation and consumption. In summary, neutrophil activation occurred after CAWS injection, followed by complement activation, and production of proinflammatory cytokines chemokines and G-CSF which may be involved in development of coronary arteritis.

Trafficking of QD-conjugated MPO-ANCA in murine systemic vasculitis and glomerulonephritis model mice.

In systemic vasculitis, the serum level of myeloperoxidase (MPO)-specific anti-neutrophil cytoplasmic autoantibodies (MPO-ANCA) is significantly elevated with the progression of disease. We have established a model of murine systemic vasculitis by administration of MPO-ANCA and fungal mannoprotein to C57BL/6 mice. We examined the role of MPO and MPO-ANCA in the pathogenesis of glomerulonephritis and systemic vasculitis in this model using quantum dots (QDs). We demonstrated that QD-conjugated MPO-ANCA (ANCA-QD) visualized the translocation of MPO on the neutrophil membrane surface after stimulation with proinflammatory cytokines. We also observed that MPO translocation on neutrophils in both patients with rapid progressive glomerulonephritis and these model mice without any stimulation, suggesting that MPO translocation is certain to contribute to the development of glomerular lesion. In addition, blood flow on the kidney surface vessel was significantly decelerated in both SCG/Kj mice and this model, suggesting that ANCA induces the damage of blood vessel. These results indicate that MPO-ANCA and surface-translocated MPO on the activated neutrophils coordinately plays essential roles in the initial steps of the glomerulonephritis.

A novel mouse model for MPO-ANCA-associated glomerulonephritis.

We established a novel model mouse for myeloperoxidase anti-neutrophil cytoplasmic antibody (MPO-ANCA)-associated glomerulonephritis with crescentic formation, which was induced by administering bovine serum albumin (BSA). Neutrophil infiltration into the renal glomeruli began at 8 weeks and crescent formation was observed from 10 weeks after the first BSA injection. Platelet and neutrophil counts significantly increased, and proteinuria was observed from 5 weeks. MPO-ANCA increased slightly at 4 and markedly at 9 weeks, and the TNF-alpha level increased at 11 weeks. Glomerular neutrophil infiltration was correlated with MPO-ANCA levels. In addition, proteinuria also significantly correlated with MPO-ANCA levels. Finally, renal crescent formation was associated with an increase of MPO-ANCA levels and neutrophil infiltration into glomeruli. The glomerular immune deposition of IgG and C3 was observed. These findings indicate that BSA induces neutrophil activation of peripheral blood followed by the elevation of MPO-ANCA, resulting in the development of crescentic glomerulonephritis in mice.

Beta-mannosyl linkages negatively regulate anaphylaxis and vasculitis in mice, induced by CAWS, fungal PAMPS composed of mannoprotein-beta-glucan complex secreted by Candida albicans.

Candida albicans water soluble fraction (CAWS) is a water-soluble extracellular mannoprotein-beta-glucan complex obtained from the culture supernatant of Candida albicans, which grows in a chemically defined medium. CAWS induced toxic reactions, such as acute anaphylactoid reaction, by intravenous administration and coronary arteritis by intraperitoneal administration. To clarify the structure responsible for these toxic reactions, C. albicans was cultured in pH- and temperature-controlled conditions and prepared with CAWS with or without the beta-1,2-linked mannosyl segment (BM). The structure of CAWS was assessed by immunochemical and spectroscopic methodologies, and we found that CAWS prepared under the natural culture conditions contained only small amounts of BM and CAWS prepared at neutral conditions at 27 degrees C contained a significantly higher percentage of BM. Both the acute lethal toxicity and coronary arteritis induction was significantly more severe in the absence of BM. Activation of a complement pathway, the lectin pathway, by CAWS was significantly stronger in the absence of BM. These facts strongly suggest that BM linkages in CAWS negatively modulate acute and chronic toxicity of CAWS, and may be strongly related to the lectin pathway of the complement activation.

Genetic dissection of vasculitis, myeloperoxidase-specific antineutrophil cytoplasmic autoantibody production, and related traits in spontaneous crescentic glomerulonephritis-forming/Kinjoh mice.

The spontaneous crescentic glomerulonephritis-forming/Kinjoh (SCG/Kj) mouse is a model of human crescentic glomerulonephritis and vasculitis associated with the production of the myeloperoxidase (MPO)-specific antineutrophil cytoplasmic autoantibody (MPO-ANCA). Although the disease is mediated initially by mutation of the Fas gene (lpr), SCG/Kj mice also have non-Fas predisposing genetic factors. To define these factors, genome-wide quantitative trait locus (QTL) mapping was performed on female (B(6)x SCG/Kj) F(2) intercross mice. Fourteen non-Fas QTLs were identified. QTLs of glomerulonephritis were located on chromosomes 1, 10, 13, 16, and 17, vasculitis on chromosomes 1 and 17, splenomegaly on chromosome 1, hypergammaglobulinemia on chromosomes 1, 2, 4, 6, 7, 11, 13, and 17, antinuclear Ab on chromosomes 1, 8, 10, and 12, and MPO-ANCA production on chromosomes 1 and 10. Significant QTLs derived from SCG/Kj on chromosomes 1, 2, 7, and 13 were designated Scg-1 to Scg-5, respectively, and those derived from B(6) on chromosomes 4, 6, 17, and 10 were designated Sxb-1 to Sxb-4, respectively. Two loci linked to MPO-ANCA production on chromosomes 1 and 10 were designated Man-1 and Man-2 (for MPO-ANCA), respectively. Although both Scg-1 and Scg-2 were on chromosome 1 and shared several functions, it was of interest that aberrant MPO-ANCA production was exclusively controlled by Man-1, the centromeric half region of the Scg-2 chromosomal segment. We also examined the epistatic effects between the lpr mutation and non-Fas susceptibility genes. QTLs are discussed in relation to previously described loci, with emphasis on their candidate genes.