FcR gamma-chain is essential for both surface expression and function of human Fc gamma RI (CD64) in vivo

Most Ig receptors exist as hetero-oligomeric complexes with separate ligand binding (alpha) and signal transducing (beta, gamma, or zeta) subunits. For Fc gamma RIIIa and Fc epsilon RI, association with the FcR gamma-chain is essential for surface expression. However, the human high affinity IgG receptor, hFc gamma RI, was found to be surface- expressed by itself in transient transfection models. We have now analyzed the integrity of hFc gamma RI expression in more detail in stable transfectants. In vitro we noted that, in the absence of FcR gamma-chain, surface expression of hFc gamma RI rapidly declined to background levels, in both IIA1.6 B cells and NIH3T3 fibroblasts. The effect of FcR gamma-chain on hFc gamma RI surface expression in vivo was evaluated by using two newly generated transgenic mouse lines, selectively expressing hFc gamma RI on myeloid cells. These transgenic mice were crossed with FcR gamma-chain-deficient mice. Analysis of blood monocytes and peritoneal macrophages showed that surface expression of hFc gamma RI was reduced by approximately 80%. The remaining approximately 20% of receptors were still capable of binding IgG-opsonized RBC, suggesting FcR gamma-chain not to be critical for hFc gamma RI ligand-binding capacity. Importantly, however, hFc gamma RI signaling capacity was lost in FcR gamma-chain-deficient cells. No phagocytosis could be observed using either ligand sensitized (EA- IgG2a) or CD64-targeted erythrocytes (using a bispecific antibody) in both hFc gamma RI transgenic lines. This documents the FcR gamma-chain to be indispensable for both surface membrane expression and function of human Fc gamma RI in vivo.     

Therapeutic action and underlying mechanisms of a combination of two pentacyclic triterpenes, α- and β-amyrin,in a mouse model of colitis

Background and purpose:

α- and β-amyrin are pentacyclic triterpenes found in plants and are known to exhibit pronounced anti-inflammatory effects. Here, we evaluated the effects of a 1:1 mixture of α- and β-amyrin (α,β-amyrin) on an experimental model of colitis in mice.

Experimental approach:

Colitis was induced in Swiss male mice by trinitrobenzene sulphonic acid (TNBS) and followed up to 72 h; animals were treated systemically with α,β-amyrin, dexamethasone or vehicle. Macro- and microscopic damage, myeloperoxidase activity and cytokine levels were assessed in colons. Histological sections were immunostained for cyclooxygenase-2 (COX-2), vascular endothelial growth factor, phospho-p65 nuclear factor-κB (NF-κB) and phospho-cyclic AMP response element-binding protein (CREB)

Key results:

TNBS-induced colitis was associated with tissue damage, neutrophil infiltration and time-dependent increase of inflammatory mediators. Treatment with α,β-amyrin (3 mg·kg⁻¹, i.p.) or dexamethasone (1 mg·kg⁻¹, s.c.) consistently improved tissue damage scores and abolished polymorphonuclear cell infiltration. α,β-Amyrin, like dexamethasone, significantly diminished interleukin (IL)-1β levels and partially restored IL-10 levels in colon tissues 72 h after colitis induction, but only α,β-amyrin reduced vascular endothelial growth factor expression by immunohistochemistry. The colonic expression of COX-2 at 24 h and that of phospho-NF-κB and phospho-CREB (peaking at 6 h) after colitis induction were consistently inhibited by both α,β-amyrin and dexamethasone.

Conclusions and implications:

Systemic administration of α,β-amyrin exerted a marked and rapid inhibition of TNBS-induced colitis, related to the local suppression of inflammatory cytokines and COX-2 levels, possibly via inhibition of NF-κB and CREB-signalling pathways. Taken together, our data suggest a potential use of α,β-amyrin to control inflammatory responses in bowel disease.     

Monocyte-mediated antibody-dependent cell-mediated cytotoxicity: the role of the metabolic burst

Human monocytes respond to opsonized microorganisms with a "metabolic burst" composed of an increase in oxygen consumption, an increase in hexose monophosphate shunt (HMPS) activity, and the generation of reactive oxygen species (ROS). We investigated the role of the metabolic burst in antibody-dependent cell-mediated cytotoxicity (ADCC) by human monocytes toward anti-D coated erythrocyte target cells because recent studies suggested a role for oxygen-dependent bactericidal mechanisms in ADCC. In normal monocytes, we found that ADCC was nearly halved under hypoxic conditions. Several agents known to impair activation of the burst, such as vincristine, cation chelators, and a sulfhydryl reagent, all decreased cytotoxicity if added before initiation of contact between target and effector cells. Cytotoxicity was inhibited by 2-deoxyglucose but not fluoride, suggesting a nonglycolytic role for glucose in ADCC, perhaps in the HMPS pathway. Although these data suggested a role for the metabolic burst in ADCC, scavengers of ROS did not impair cytotoxicity, and monocytes from chronic granulomatous disease (CGD) patients who had a defective metabolic burst had normal levels of ADCC. We conclude that ADCC toward anti-D coated erythrocyte target cells was the result of at least two independent but closely related cytotoxic pathways. Although one of these pathways appeared to involve the metabolic burst, the potentially cytotoxic reactive oxygen species did not appear to play a role in this system.