C-terminal deletion mutants of gp96 are unable to chaperone TLRs and integrins.

<p><i>A</i>, gp96 null preB cells were transduced with retroviral expression vectors for empty vector (EV), full-length gp96 (FL) or gp96 deletion mutants (N355, N603), followed by Western blot with gp96N Ab or β–actin as a loading control. <i>B</i>, Various transfectants in (A) were analyzed by flow cytometry for cell surface expression of gp96 clientele TLR2 and α4 integrin (open histogram). Shaded histogram depicts isotype control.</p

Bone marrow basophils are marked by strong unfolded protein response.

<p><i>A</i>, Western blot for grp78 and CRT of sorted populations. <i>B</i>, RT-PCR of XBP-1 and its spliced form XBP-1s.</p

Basophil does not express full-length gp96.

<p><i>A</i>, Intracellular stain of <i>WT</i> and <i>KO</i> B cells with isotype control (shaded histogram) or antibody against gp96N and gp96C (open histogram). <i>B</i>, Basophil (population I) does not express gp96C (open histogram). Shaded histograms are staining result with isotype control antibody. <i>C</i>, Western blot for gp96N and gp96C of sorted populations. β–actin was blotted to indicate equal loading of cell lysates.</p

gp96MD expression is restricted to mouse basophils.

<p><i>A</i>, Murine mast cells express intact gp96 without evidence for gp96MD. Left panel showed flow profile of bone marrow-derived mast cells. Right panel demonstrates gp96 immunoblot. <i>B</i>, Purified human basophils (left panel) only express full-length gp96 (right panel). <i>C</i>, Human KU812 basophil cell line does not cleave gp96 with and without ER stress induced by tunicamycin (Tu) or thapsigargin (Thap), demonstrated by gp96 immunoblot. <i>D</i>, Western blots for gp96 in the whole cell lysate of KU812 cells with and without ectopic expression of mMCP11.</p

Expression of gp96MD is mediated by proteolysis.

<p><i>A</i>, BMDBs were treated with AEBSF for 6 hours followed by intracellular stain for gp96 using either C-terminal or N-terminal specific antibody. <i>B</i>, BMDBs were treated with AEBSF for 6 hours followed by subjecting total cell lysate to electrophoresis and immunoblot for gp96. <i>C</i>, Kinetic emergence of intracellular gp96 C-terminal antibody reactivity after treatment of B220⁻IgG⁺ population with AEBSF for the indicated time. Number in the quadrant indicates mean fluorescence intensity of gp96 stain. For comparison, peak intensity of the stain at 6 hours were indicated with a vertical line.</p

Expression of gp96MD is not due to a basophil-specific alternative splicing.

<p>Exon (Ex)-specific PCR analysis of gp96 cDNA from sorted populations of bone marrow cells. (Ex2-3, Ex3-5, Ex3-8, et al. indicates the amplified fragments between different exons; NS: non-specific band). Population I: B220⁻IgG^high, Population II: B220⁺IgG^high, Population III: B220⁺IgG⁻.</p

gp96 deletion does not compromise the global secretory machinery of basophils.

<p><i>A</i>, gp96 immunoblot to demonstrate the efficient knockdown of gp96 by shRNA. <i>B</i>, Efficient cytokine and chemokine production by both <i>WT</i> and gp96 knockdown basophils in response to ionomycin. <i>C</i>, Immunoblot of gp96 after treatment with ionomycin.</p

Basophil develops in the absence of gp96.

<p><i>A</i>, Presence of B220⁻IgG^high basophil population (boxed) in the bone marrow of gp96 <i>KO</i> mice. <i>B</i>, Intracellular stain demonstrated an efficient deletion of gp96 in bone marrow cells of <i>Hsp90b1^floxRosa26^ERcre</i> (<i>KO</i>) mice by TAM. <i>C</i>, Both <i>WT</i> and gp96 <i>KO</i> B220⁻IgG^high basophils express FcεRI (open histogram indicates gp96 or FcεRI; shaded histogram, isotype control).</p

Bone marrow B220⁻IgG^high cells are basophils.

<p><i>A</i>, Phenotype of B220⁻IgG^high cells. Numbers represent percentages of gated population or corresponding quadrants. <i>B</i>, Phenotypic analysis of FACS sorted BM cells based on cell surface markers of B220 and IgG. (C) RT-PCR analysis of IL-4 and Igα mRNA.</p

Sex Differences in Monocyte Activation in Systemic Lupus Erythematosus (SLE)

<div><p>Introduction</p><p>TLR7/8 and TLR9 signaling pathways have been extensively studied in systemic lupus erythematosus (SLE) as possible mediators of disease. Monocytes are a major source of pro-inflammatory cytokines and are understudied in SLE. In the current project, we investigated sex differences in monocyte activation and its implications in SLE disease pathogenesis.</p><p>Methods</p><p>Human blood samples from 27 healthy male controls, 32 healthy female controls, and 25 female patients with SLE matched for age and race were studied. Monocyte activation was tested by flow cytometry and ELISA, including subset proportions, CD14, CD80 and CD86 expression, the percentage of IL-6-producing monocytes, plasma levels of sCD14 and IL-6, and urine levels of creatinine.</p><p>Results</p><p>Monocytes were significantly more activated in women compared to men and in patients with SLE compared to controls <i>in vivo</i>. We observed increased proportions of non-classic monocytes, decreased proportions of classic monocytes, elevated levels of plasma sCD14 as well as reduced surface expression of CD14 on monocytes comparing women to men and lupus patients to controls. Plasma levels of IL-6 were positively related to sCD14 and serum creatinine.</p><p>Conclusion</p><p>Monocyte activation and TLR4 responsiveness are altered in women compared to men and in patients with SLE compared to controls. These sex differences may allow persistent systemic inflammation and resultant enhanced SLE susceptibility.</p></div