A novel monocyte differentiation pattern in pristane-induced lupus with diffuse alveolar hemorrhage
- Division of Rheumatology, Allergy, & Clinical Immunology, University of Florida, United States
Figures
Figure 1
CD138+CD11b+ cells in the circulation and peritoneum.
(A) B6 mice were injected with either pristane or mineral oil (MO). CD138 and CD11b surface expression on peripheral blood leukocytes and peritoneal exudate cells (PECs) was analyzed by flow …
Figure 2
Flow cytometry of monocytes surface markers.
Peripheral blood mononuclear cells and peritoneal exudate cells (PECs) from pristane- or mineral oil (MO)-treated mice were stained with fluorescently labeled anti-CD11b, Ly6G, Ly6C, and CD138 …
Figure 3
Ccr2 dependence of monocyte egress from the bone marrow (BM).
B6 mice were treated with either mineral oil or pristane and CD11b+Ly6G− myeloid subsets in the blood and peritoneal exudate cell (PEC) were gated as in Figure 2A. (A) Circulating Ly6Chi (R1), Ly6C−C…
Figure 4
Monocyte/Mϕ subsets in Nr4a1−/− mice.
(A) Gating strategy. Peritoneal exudate cells (PECs) from pristane- or mineral oil (MO)-treated wild-type and Nr4a1−/− mice were stained with anti-CD11b, Ly6G, Ly6C, and CD138 and analyzed by flow …
Figure 5
Circulating monocyte subsets.
(A) Gating strategy. Blood cells from pristane- or mineral oil (MO)-treated B6 mice, Nr4a1−/− mice, and PBS-treated controls were stained with anti-CD11b, Ly6G, Ly6C, and CD138 and analyzed by flow …
Figure 6
Circulating monocyte phenotypes.
Circulating monocytes are gated as in Figure 5 and the staining intensity (MFI) of various markers was ascertained by flow cytometry. (A) CD62L, (B) Nr4a1, (C) Cx3Cr1, (D) LFA-1, and (E) CD11c. *p < …
Figure 7
Function of CD138+ monocytes.
Circulating monocytes from pristane-treated B6 mice were gated as in Figure 5 and the staining intensity (MFI) of TLR7 (A) and TremL4 (B) was ascertained by flow cytometry. (C) R848-stimulated TNFα …
Figure 8
Low TremL4 expression in Nr4a1−/− mice.
(A) Blood cells were stained with anti-CD11b and anti-Treml4 monoclonal antibodies. CD11b+Treml4+ cells as a percentage of total CD11b+ cells were determined by flow cytometry in pristane-treated …
Figure 9
Ly6CloCD138+ monocytes are increased in mice that develop diffuse alveolar hemorrhage (DAH).
(A) Intracellular Nr4a1 staining in the CD11b+Ly6Chi and CD11b+Ly6C−/lo monocytes from untreated B6 and BALB/c mice. (B) CD11b and Ly6C staining of circulating Ly6G- cells in mineral oil …
Figure 10
Nr4a1 regulates TremL4.
(A) R848 activated expression (24 hr) of Nr4a1, TremL4, and CD86 proteins in RAW264.7 cells (flow cytometry; MFI, mean fluorescence intensity). (B) R848-stimulated mRNA expression of Nr4a1, Tnfa, …
Figure 11
Monocyte subsets in pristane- and mineral oil (MO)-treated mice.
(A) Pristane-treated B6 mice; (B) pristane-treated B6 Ccr2−/− mice; (C) pristane-treated B6 Nr4a1−/− mice; (D) MO-treated B6 mice, pristane-treated B6 μMT mice, and pristane-treated BALB/c mice. …
Tables
Table 1
Phenotypes of monocyte subsets and pristane- and mineral oil-treated mice.
| Ly6C | CD138 | CD11b | Nr4a1 | CCR2 | Cx3Cr1 | CD62L | CD11c | LFA1 | Treml4 | TLR7 | TNFa | CD43 | CD115 | |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| R1 (P)* | ++ | (−) | + | (−) | ++ | + | ++ | (−) | + | (−) | + | ++ | ++ | + |
| R2A (P) | + | (−) | + | + | + | + | + | + | ++ | + | + | ++ | + | + |
| R2B (P) | (−) | (−) | + | + | (−) | + | + | + | ++ | + | + | + | + | + |
| R3A (P) | + | ++ | ++ | ++ | + | ++ | (−) | + | ++ | ++ | ++ | ++ | + | ++ |
| R3B (P) | (−) | ++ | + | + | (−) | ++ | (−) | ++ | ++ | ++ | ++ | + | ++ | ++ |
| R1 (MO)* | ++ | (−) | + | (−) | n.d. | n.d. | n.d. | (−) | n.d. | (−) | + | + | + | n.d. |
| R3A (MO) | + | (−) | ++ | ++ | n.d. | n.d. | n.d. | + | n.d. | ++ | + | ++ | + | n.d. |
| R3B (MO) | (−) | (−) | + | + | n.d. | n.d. | n.d. | ++ | n.d. | ++ | + | + | + | n.d. |
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*P, pristane; MO, mineral oil; n.d., not determined.
Table 2
Murine monoclonal antibodies.
| Specificity (clone) | Fluorochrome* | Source |
|---|---|---|
| CD138 (281-2) | PE; APC; APC-Cy7 | BioLegend |
| CD11b (M1/70) | BV421, Pacific Blue | BioLegend |
| Ly6C (HK1.4) | APC-Cy7; Alexa Fluor 488 | BioLegend |
| Ly6G (1A8) | APC-Cy7; PE | BioLegend |
| CD43 | FITC | BioLegend |
| TREML4 | PE | BioLegend |
| CD115 | APC-Cy7 | BioLegend |
| F4/80 | PE, BV421 | BioLegend |
| CD62L | PE | BD Pharmingen |
| CD64 | PE | BD Pharmingen |
| CCR2 | FITC | BioLegend |
| Tim4 | APC, PE | BioLegend |
| CD11c | PE | BD Pharmingen |
| CX3CR1 (SA011F11) | FITC | BioLegend |
| LFA-1 | PE | BioLegend |
| NR4A1 | PE | Miltenyi |
| TLR7 | PE | BioLegend |
| CD86 | APC-Cy7 | BioLegend |
| TNFα | APC, PE | BioLegend |
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*PE, phycoerythrin; APC, allophycocyanin; FITC, fluorescein isothiocyanate; BV, brilliant violet; APC-Cy7, APC/cyanine 7.
