Improved harvest and fixation methodology for isolated human peripheral blood mononuclear cells in cytokinesis-block micronucleus assay.

PURPOSE: In suspected radiation exposures, cytokinesis-block micronucleus (CBMN) assay is used for biodosimetry by detecting micronuclei (MN) in binucleated (BN) cells in whole blood and isolated peripheral blood mononuclear cell (PBMC) cultures. Standardized harvest protocols for whole blood were published by the International Atomic Energy Agency (IAEA) in 2001 (Technical report no. 405) and 2011 (EPR-Biodosimetry). For isolated PBMC harvest, cytocentrifugation of fresh cells is recommended to preserve cytoplasmic boundaries for MN scoring. However, cytocentrifugation utilizes specialized equipment and long-term cell suspension storage is difficult. In this study, an alternative CBMN harvest protocol is proposed for laboratories interested in culturing PBMCs and storing fixed cells with routine biodosimetry methods. MATERIALS AND METHODS: Peripheral blood from 4 males (24, 34, 41, 51 y.o.) and females (26, 37, 44, 56 y.o.) was irradiated with 0 and 2 Gy X-rays. For cells harvested with IAEA 2001 and 2011 protocols, whole blood was used. For cells harvested with our protocol (CRG), isolated PBMCs were used. CRG protocol was validated in DAPI, acridine orange and Giemsa stain, and in three other laboratories. Cytoplasm status, nuclear division index (NDI) and induced MN frequency (MN frequency at 2 Gy - background MN frequency at 0 Gy) (MN/1000 BN) of Giemsa-stained BN cells were compared in IAEA 2001, IAEA 2011, IAEA 2011 + formaldehyde (FA) and CRG protocols. Effects of low and high humidity spreading were evaluated. RESULTS: >94% of 1000 BN cells were scorable with clear cytoplasmic boundaries in all donors harvested with CRG protocol. FA addition in IAEA 2011 protocol reduced cell rupture in whole blood cultures, but cell rupture was affected by age, sex and humidity. Almost all cells harvested with IAEA 2001 protocol had cytoplasm loss. PBMCs harvested with CRG protocol stained well in DAPI, acridine orange and Giemsa, and showed high scorable BN frequency in all laboratories. A higher NDI and a lower induced MN frequency were seen in 2 Gy isolated PBMC than whole blood cultures. CONCLUSION: This quick CBMN harvest protocol for isolated PBMCs is a viable alternative to cytocentrifugation, as many scorable BN cells were obtained with routine biodosimetry reagents and equipment. IAEA 2011 + FA protocol should be used to improve CBMN harvest in whole blood cultures. Humidity during spreading should be optimized depending on the harvest protocol. NDI and MN frequency should be separately evaluated for whole blood and isolated PBMC cultures.

Mediation of Interleukin-23 and Tumor Necrosis Factor-Driven Reactive Arthritis by Chlamydia-Infected Macrophages in SKG Mice.

OBJECTIVE: ZAP-70W163C BALB/c (SKG) mice develop reactive arthritis (ReA) following infection with Chlamydia muridarum. Since intracellular pathogens enhance their replicative fitness in stressed host cells, we examined how myeloid cells infected with C muridarum drive arthritis. METHODS: SKG, Il17a-deficient SKG, and BALB/c female mice were infected with C muridarum or C muridarum luciferase in the genitals. C muridarum dissemination was assessed by in vivo imaging or genomic DNA amplification. Macrophages were depleted using clodronate liposomes. Anti-tumor necrosis factor (anti-TNF) and anti-interleukin-23p19 (anti-IL-23p19) were administered after infection or arthritis onset. Gene expression of Hspa5, Tgtp1, Il23a, Il17a, Il12b, and Tnf was compared in SKG mice and BALB/c mice. RESULTS: One week following infection with C muridarum, macrophages and neutrophils were observed to have infiltrated the uteri of mice and were also shown to have carried C muridarum DNA to the spleen. C muridarum load was higher in SKG mice than in BALB/c mice. Macrophage depletion was shown to reduce C muridarum load and prevent development of arthritis. Compared with BALB/c mice, expression of Il23a and Il17a was increased in the uterine and splenic neutrophils of SKG mice. The presence of anti-IL-23p19 during infection or Il17a deficiency suppressed arthritis. Tnf was overexpressed in the joints of SKG mice within 1 week postinfection, and persisted beyond the first week. TNF inhibition during infection or at arthritis onset suppressed the development of arthritis. Levels of endoplasmic reticulum stress were constitutively increased in the joints of SKG mice but were induced, in conjunction with immunity-related GTPase, by C muridarum infection in the uterus. CONCLUSION: C muridarum load is higher in SKG mice than in BALB/c mice. Whereas proinflammatory IL-23 produced by neutrophils contributes to the initiation of C muridarum-mediated ReA, macrophage depletion reduces C muridarum dissemination to other tissues, tissue burden, and the development of arthritis. TNF inhibition was also shown to suppress arthritis development. Our data suggest that enhanced bacterial dissemination in macrophages of SKG mice drives the TNF production needed for persistent arthritis.