PMC

Regulation of TSH-βv by LPS, LPS/IFN-γ (IFNG), and IL-4/IL-10. Differentiated THP-1 macrophage cultures were treated with LPS, LPS/IFNγ, and IL-4/IL-10 for 24 hours and gene expression examined for TSH-βv (A), MCSF (B), CCL-2 (C), Wnt5A (D), MARC1 (E), and Wnt10-A (F). These data revealed that both M1 stimulation with LPS/IFNγ and M2 stimulation with IL-4/IL-10 induced TSHβv gene expression by up to 30-fold (A). *, P < .05; **, P < .001. cont, control.

Macrophage gene expression. Real-time PCR analysis revealed that differentiated THP-1 cells show enhanced expression of MCSF (A), CCL-22 (B), MARC1 (C), CCL-2 (D), and WNT-5A (E) gene expression, confirming their macrophage phenotype. Conventional RT-PCR demonstrated enhanced expression of the correct-size transcripts for the same macrophage genes after differentiation from the original cell cultures (F, G). *, P < .05.

The hTSH-β sequence identified. A schematic outlining the native and novel exon sequence of the hTSH-β vs the hTSH-βv with the corresponding amino acid sequence for exon 3 and the nine amino acids from the intronic region (italicized) as reported elsewhere (). The sequences identified in the THP-1 macrophages (A), human PBMCs (B), and human bone marrow (C) are bold and underlined.

Thyroid hormone regulation of mouse TSH-βv in vivo. A, Bone marrow cells from normal WT mice treated with a T₄ hormone pellet for 21 days showed greatly increased TSH-βv gene expression. B, Pituitary tissue from WT mice treated with T₄ pellets for 21 days showed suppression of both full-length TSH-β and TSH-βv in contrast to the bone marrow cell data. *, P < .05; **, P < .001.

Identification of a novel hTSH-βv in human macrophages. RT-PCR analyses of human pituitary extract illustrated the presence of both full-length hTSH-β and the novel hTSH-βv (A and B). The THP-1 cells (C and D), human bone marrow (E), PBMCs (F), and human macrophages (G) showed only the hTSHβv. RT-PCR amplification of the TSH-α gene was positive only in the pituitary sample (H) and not in THP-1 cells (I). *, P < .05.

T₃ hormone regulation of mouse TSH-βv in vitro. Raw cells (264.7 mouse macrophage cell) were stained for F480, a macrophage marker (A), TSH-β (B), and the results overlaid (C) to see the coexpression of both markers. Control staining of CHO cells is shown in the insert to panel C. Raw cells were then treated with T₃ hormone for 1 hour and PCR analyzed. The T₃ hormone increased TSH-βv gene expression in a dose-dependent manner (D). **, P < .001, also enhanced inducible nitric oxide synthase- and arginase-2 (E, F).

TSH-βv protein expression. THP-1 macrophages were differentiated and cultured for 4 days with PMA (100 ng/mL) and their nuclei labeled with 4′,6′-diamidino-2-phenylindole. Cells were counterstained for CD14 expression (A) and TSH-β (B), and the merged pictures are shown in (C) (×630). FACS analysis of these THP-1 cells (D) showed 47% of double-staining cells. E, Control CHO cells stained with 4′,6′-diamidino-2-phenylindole, anti-CD14, and anti-TSH-β within the same experiment. F, A Western blot analysis (at 40 μg protein) with anti-TSH-β, which confirmed the presence of appropriately sized TSH-βv expression in the THP-1 macrophage whole-cell lysate.

Macrophage phenotypes. A, Human THP-1 monocytes were a suspension of cells that were then differentiated into macrophages with PMA (100 ng/mL) (×20). B, Differentiated cells were rested for 48 hours and consisted of two populations: one population was of a fried egg shape and the second population of a spindle-like shape. C, Both populations of cells stained for surface and intracellular CD14 (Toll-like receptor) expression and 4′,6′-diamidino-2-phenylindole (DAPI) stain was used to illustrate the nuclei. D, Human PBMCs were allowed to differentiate with 100 ng/mL MCSF. E, Differentiated human macrophages. F, These differentiated cells from panel E were also CD14 positive. G, FACS analysis showed that 46.1% of the differentiated THP-1 cells were CD14 positive.

T₃ hormone regulation of human TSH-βv in vitro. A, THP-1 cells were treated with T₃ hormone for 1 hour in serum-free macrophage media (Gibco) and PCR analyzed for hTSH-βv. T₃ hormone increased TSH-βv gene expression in a dose-dependent manner as well as CCL-22 and MCSF gene expression, suggesting that T₃ hormone increased both M1- and M2-polarized states (see also ). *, P < .05. B, THP-1 macrophages also showed enhanced TSH-βv protein expression when treated with increasing concentrations of T₃ hormone as shown by in-cell Western blot assay. C, When THP-1 macrophages (MØs) were cocultured with HEK293 cells transfected with the TSHR (HEK293 +TSHR), a cAMP response was elicited that was approximately 5-fold greater than when THP-1 macrophages were cocultured with HEK293 cells without the TSHR. The TSHR-related cAMP generation in response to MØs in coculture was greater than that generated by 1 mU/mL of TSH. Note that MØs with HEK293 cells alone induced a small cAMP response likely secondary to local chemokine release. Data were analyzed by a one-way ANOVA shown as mean ± SEM of two experiments carried out in duplicates. *, P < .001.