Enhanced osteoclast development in collagen-induced arthritis in interferon-γ receptor knock-out mice as related to increased splenic CD11b+ myelopoiesis

Collagen-induced arthritis (CIA) in mice is accompanied by splenomegaly due to the selective expansion of immature CD11b⁺ myeloblasts. Both disease manifestations are more pronounced in interferon-γ receptor knock-out (IFN-γR KO) mice. We have taken advantage of this difference to test the hypothesis that the expanding CD11b⁺ splenic cell population constitutes a source from which osteoclast precursors are recruited to the joint synovia. We found larger numbers of osteoclasts and more severe bone destruction in joints of IFN-γR KO mice than in joints of wild-type mice. Osteoclast-like multinucleated cells appeared in splenocyte cultures established in the presence of macrophage colony-stimulating factor (M-CSF) and stimulated with the osteoclast-differentiating factor receptor activator of NF-κB ligand (RANKL) or with tumour necrosis factor-α (TNF-α). Significantly larger numbers of such cells could be generated from splenocytes of IFN-γR KO mice than from those of wild-type mice. This was not accompanied, as might have been expected, by increased concentrations of the intracellular adaptor protein TRAF6, known to be involved in signalling of RANKL- and TNF-α-induced osteoclast formation. Splenocyte cultures of IFN-γR KO mice also produced more TNF-α and more RANKL than those of wild-type mice. Finally, splenocytes isolated from immunised IFN-γR KO mice contained comparatively low levels of pro-interleukin-1β (pro-IL-1β) and pro-caspase-1, indicating more extensive conversion of pro-IL-1β into secreted active IL-1β. These observations provide evidence that all conditions are fulfilled for the expanding CD11b⁺ splenocytes to act as a source of osteoclasts and to be indirectly responsible for bone destruction in CIA. They also provide a plausible explanation for the higher susceptibility of IFN-γR KO mice to CIA.     

Effector mechanisms of interleukin-17 in collagen-induced arthritis in the absence of interferon-γ and counteraction by interferon-γ

Introduction  

Interleukin (IL)-17 is a pro-inflammatory cytokine in rheumatoid arthritis (RA) and collagen-induced arthritis (CIA). Since interferon (IFN)-γ inhibits Th17 cell development, IFN-γ receptor knockout (IFN-γR KO) mice develop CIA more readily. We took advantage of this model to analyse the mechanisms of action of IL-17 in arthritis. The role of IFN-γ on the effector mechanisms of IL-17 in an in vitro system was also investigated.     

AMD3100, a potent and specific antagonist of the stromal cell-derived factor-1 chemokine receptor CXCR4, inhibits autoimmune joint inflammation in IFN-gamma receptor-deficient mice

Autoimmune collagen-induced arthritis (CIA) in IFN-gammaR-deficient DBA/1 mice was shown to be reduced in severity by treatment with the bicyclam derivative AMD3100, a specific antagonist of the interaction between the chemokine stromal cell-derived factor-1 (SDF-1) and its receptor CXCR4. The beneficial effect of the CXCR4 antagonist was demonstrable when treatment was initiated between the time of immunization and appearance of the first symptoms. Treatment also reduced the delayed-type hypersensitivity response to the autoantigen, collagen type II. These observations are indicative of an action on a late event in the pathogenesis, such as chemokine-mediated attraction of leukocytes toward joint tissues. The notion of SDF-1 involvement was further supported by the observation that exogenous SDF-1 injected in periarthritic tissue elicited an inflammatory response that could be inhibited by AMD3100. The majority of leukocytes harvested from inflamed joints of mice with CIA were found to be Mac-1(+) and CXCR4(+), and AMD3100 was demonstrated to interfere specifically with chemotaxis and Ca(2+) mobilization induced in vitro by SDF-1 on Mac-1(+)/CXCR4(+) splenocytes. We conclude that SDF-1 plays a central role in the pathogenesis of murine CIA, by attracting Mac-1(+)/CXCR4(+) cells to the inflamed joints.     

Insights into the mechanism of enhanced mobilization of hematopoietic progenitor cells and release of CXCL12 by a combination of AMD3100 and aminoglycoside-polyarginine conjugates

Mobilization of hematopoietic stem and progenitor cells (HSPCs) from the bone marrow to the peripheral blood is utilized in clinical HSPC transplantation protocols. Retention of HSPCs in the bone marrow is determined by relationships between the chemokine chemokine (C-X-C motif) ligand 12 (CXCL12) and its major receptor C-X-C chemokine receptor type 4 (CXCR4), and disruption of this retention by CXCR4 antagonists such as AMD3100 induces rapid HSPC mobilization. Here, we report that aminoglycoside-polyarginine conjugates (APACs) and N-α-acetyl-nona-D-arginine (r9) induce mobilization of white blood cells and, preferentially, immature hematopoietic progenitor cells (HPCs) in mice, similarly to AMD3100. Remarkably, administration of AMD3100 with each one of the APACs or r9 caused additional HPC mobilization. The mobilizing activity of APACs and r9 was accompanied by a significant elevation in plasma CXCL12 levels. To further understand how APACs, r9 and their combinations with AMD3100 compete with CXCL12 binding to CXCR4, as well with antibody against CXCR4 for CXCR4 binding, we have undertaken an approach combining experimental validation and docking to determine plausible binding modes for these ligands. On the basis of our biological and docking findings, and recently published NMR data, we suggest that combination of pairs of compounds such as APACs (or r9) with AMD3100 induces more efficient disruption of the CXCL12-CXCR4 interaction than AMD3100 alone, resulting in enhanced HPC mobilization.     

肠道病毒71型感染小鼠心肌损害与CXCL12/CXCR4通路激活的关系

重症手足口病患儿中心肌损害常见,肠道病毒71型(Enterovirus 71,EV71)是引起手足口病的主要病原体之一,EV71感染小鼠可以出现心肌炎的病理改变,但EV71引起心肌损害的机制尚不明确。为了阐明EV71引起心肌损害的机制,本实验观察了EV71感染小鼠心肌损害与CXC趋化因子配体12(CXC chemokine ligand 12,CXCL12)/CXC趋化因子受体4(CXC chemokine receptor 4,CXCR4)通路激活的关系。BALB/c乳鼠随机分为对照组、EV71组、EV71+AMD3100组、AMD3100组,对照组、AMD3100组给予生理盐水腹腔注射,EV71组、EV71+AMD3100组给予EV71病毒液腹腔注射;而后EV71+AMD3100组、AMD3100组给予CXCR4抑制剂AMD3100腹腔注射、连续7d。比较四组间血清中CXCL12、磷酸肌酸激酶同工酶(CreatineKinase-MB,CK-MB)、乳酸脱氢酶(Lactate dehydrogenase,LDH)含量、心肌病理改变及细胞凋亡率、心肌中CXCR4、含半胱氨酸的天冬氨酸蛋白水解酶-3(caspase-3)表达及肿瘤坏死因子-α(Tumor necrosis factor,TNF-α)、白介素-6(Interleukin-6,IL-6)含量的差异。与对照组比较,EV71组血清中CXCL12、CK-MB、LDH的含量明显增加,心肌出现了典型的心肌炎病理改变且细胞凋亡率、CXCR4及caspase-3表达水平、TNF-α及IL-6含量明显增加;与EV71组比较,EV71+AMD3100组血清中CXCL12、CK-MB、LDH的含量明显降低,心肌的病理改变改善且细胞凋亡率、CXCR4及caspase-3表达水平、TNF-α及IL-6含量明显降低。以上结果表明EV71感染小鼠心肌中CXCL12/CXCR4通路过度激活,该通路的激活介导了心肌细胞凋亡及炎症反应。本研究创新点为阐明了CXCL12/CXCR4通路在EV71病毒感染引起心肌损害中的作用,CXCL12/CXCR4通路激活能够激活EV71感染小鼠心肌的炎症反应及细胞凋亡,这为今后研究手足口病发病过程中心肌损害的机制提供了依据。     

Retracted: Role of CXCL12–CXCR4 axis in ovarian cancer metastasis and CXCL12–CXCR4 blockade with AMD3100 suppresses tumor cell migration and invasion in vitro

Ovarian cancer (OC) is a lethal gynecologic tumor, which brings its mortality to the head. CXCL12 and its receptor chemokine receptor 4 ( CXCR4) have been found to be highly expressed in OC and contribute to the disease progression by affecting tumor cell proliferation and invasion. Here, in this study, we aim to explore whether the blockade of CXCL12–CXCR4 axis with AMD3100 (a selective CXCR4 antagonist) has effects on the progression of OC. On the basis of the gene expression omnibus database of OC gene expression chips, the OC differentially expressed genes were screened by microarray analysis. OC (nonmetastatic and metastatic) and normal ovarian tissues were collected to determine the expressions of CXCL12 and CXCR4. A series of AMD3100, shRNA against CXCR4, and pCNS-CXCR4 were introduced to treat CAOV3 cells with the highest CXCR4 was assessed. Cell viability, apoptosis, migration, and invasion were all evaluated. The microarray analysis screened out the differential expression of CXCL12–CXCR4 in OC. CXCL12 and CXCR4 expressions were increased in OC tissues, particularly in the metastatic OC tissues. Downregulation of CXCR4 by AMD3100 or shRNA was observed to have a critical role in inhibiting cell proliferation, migration, and invasion of the CAOV3 OC cell line while promoting cell apoptosis. Overexpressed CXCR4 brought significantly promoting effects on the proliferation and invasiveness of OC cells. These results reinforce that the blockade of CXCL12–CXCR4 axis with AMD3100 inhibits the growth of OC cells. The antitumor role of the inhibition of CXCL12–CXCR4 axis offers a preclinical validation of CXCL12–CXCR4 axis as a therapeutic target in OC.     

Interferon Gamma Suppresses Collagen-Induced Arthritis by Regulation of Th17 through the Induction of Indoleamine-2,3-Deoxygenase

C57BL/6 mice are known to be resistant to the development of collagen-induced arthritis (CIA). However, they show a severe arthritic phenotype when the Ifng gene is deleted. Although it has been proposed that IFN-γ suppresses inflammation in CIA via suppressing Th17 which is involved in the pathogenesis of CIA, the exact molecular mechanism of the Th17 regulation by IFN-γ is poorly understood. This study was conducted to 1) clarify that arthritogenic condition of IFN-γ knockout (KO) mice is dependent on the disinhibition of Th17 and 2) demonstrate that IFN-γ-induced indoleamine2,3dioxgenase (IDO) is engaged in the regulation of Th17. The results showed that the IFN-γ KO mice displayed increased levels of IL-17 producing T cells and the exacerbation of arthritis. Also, production of IL-17 by the splenocytes of the IFN-γ KO mice was increased when cultured with type II collagen. When Il17 was deleted from the IFN-γ KO mice, only mild arthritis developed without any progression of the arthritis score. The proportion of CD44^highCD62L^low memory-like T cells were elevated in the spleen, draining lymph node and mesenteric lymph node of IFN-γ KO CIA mice. Meanwhile, CD44^lowCD62L^high naïve T cells were increased in IFN-γ and IL-17 double KO CIA mice. When Th17 polarized CD4+ T cells of IFN-γ KO mice were co-cultured with their own antigen presenting cells (APCs), a greater increase in IL-17 production was observed than in co-culture of the cells from wild type mice. In contrast, when APCs from IFN-γ KO mice were pretreated with IFN-γ, there was a significant reduction in IL-17 in the co-culture system. Of note, pretreatment of 1-methyl-DL- tryptophan, a specific inhibitor of IDO, abolished the inhibitory effects of IFN-γ. Given that IFN-γ is a potent inducer of IDO in APCs, these results suggest that IDO is involved in the regulation of IL-17 by IFN-γ.     

Imaging CXCL12-CXCR4 Signaling in Ovarian Cancer Therapy

Chemokine CXCL12 and receptor CXCR4 have emerged as promising therapeutic targets for ovarian cancer, a disease that continues to have a dismal prognosis. CXCL12-CXCR4 signaling drives proliferation, survival, and invasion of ovarian cancer cells, leading to tumor growth and metastasis. Pleiotropic effects of CXCR4 in multiple key steps in ovarian cancer suggest that blocking this pathway will improve outcomes for patients with this disease. To quantify CXCL12-CXCR4 signaling in cell-based assays and living mouse models of ovarian cancer, we developed a click beetle red luciferase complementation reporter that detects activation of CXCR4 based on recruitment of the cytosolic adapter protein β-arrestin 2. Both in two-dimensional and three-dimensional cell cultures, we established that bioluminescence from this reporter measures CXCL12-dependent activation of CXCR4 and inhibition of this pathway with AMD3100, a clinically-approved small molecule that blocks CXCL12-CXCR4 binding. We used this imaging system to quantify CXCL12-CXCR4 signaling in a mouse model of metastatic ovarian cancer and showed that treatment with AMD3100 interrupted this pathway in vivo. Combination therapy with AMD3100 and cisplatin significantly decreased tumor burden in mice, although differences in overall survival were not significantly greater than treatment with either agent as monotherapy. These studies establish a molecular imaging reporter system for analyzing CXCL12-CXCR4 signaling in ovarian cancer, which can be used to investigate biology and therapeutic targeting of this pathway in cell-based assays and living mice.     

Early Repeated Administration of CXCR4 Antagonist AMD3100 Dose-Dependently Improves Neuropathic Pain in Rats After L5 Spinal Nerve Ligation

AMD3100 is a specific C-X-C chemokine receptor type 4 (CXCR4) antagonist which blocks the interaction between CXCR4 and CXCL12. Multiple lines of evidence suggest that AMD3100 has analgesic effects on many pathological pain states, including peripheral neuropathic pain. However, little is known about the underlying mechanisms. In the current study, we investigated the effect of different doses of AMD3100 on neuropathic pain in rats after L5 spinal nerve ligation. We used naloxone methiodide (NLXM) to further determine whether AMD3100-mediated analgesic effect was opioid-dependent. Behavioral study showed that early repeated administration of AMD3100 (2 and 5 mg/kg, i.p.) dose-dependently alleviates peripheral neuropathic pain. Flow cytometry, immunofluorescence and NLXM experiments showed that AMD3100 alleviates neuropathic pain partially by augmenting leukocyte-derived endogenous opioid secretion. Furthermore, we found that pro-inflammatory cytokines were down-regulated by AMD3100 using Enzyme-linked Immunosorbent Assay. Our data indicate that AMD3100 dose-dependently alleviates neuropathic pain partially by augmenting leukocyte-derived endogenous opioid secretion. This finding suggests that AMD3100 may be a viable pharmacotherapeutic strategy for the treatment of neuropathic pain.     

AMD3100 improves ovariectomy-induced osteoporosis in mice by facilitating mobilization of hematopoietic stem/progenitor cells

Inhibition of an increase of osteoclasts has become the most important treatment for osteoporosis. The CXCR4 antagonist, AMD3100, plays an important role in the mobilization of osteoclast precursors within bone marrow (BM). However, the actual therapeutic impact of AMD3100 in osteoporosis has not yet been ascertained. Here we demonstrate the therapeutic effect of AMD3100 in the treatment of ovariectomy-induced osteoporosis in mice. We found that treatment with AMD3100 resulted in direct induction of release of SDF-1 from BM to blood and mobilization of hematopoietic stem/progenitor cells (HSPCs) in an osteoporosis model. AMD3100 prevented bone density loss after ovariectomy by mobilization of HSPCs, suggesting a therapeutic strategy to reduce the number of osteoclasts on bone surfaces. These findings support the hypothesis that treatment with AMD3100 can result in efficient mobilization of HSPCs into blood through direct blockade of the SDF-1/CXCR4 interaction in BM and can be considered as a potential new therapeutic intervention for osteoporosis. [BMB Reports 2014; 47(8): 439-444]     

Defective CD4<Superscript>+</Superscript>CD25<Superscript>+</Superscript>regulatory T cell functioning in collagen-induced arthritis: an important factor in pathogenesis,counter-regulated by endogenous IFN-γ

Mice with a deficiency in IFN-γ or IFN-γ receptor (IFN-γR) are more susceptible to collagen-induced arthritis (CIA), an experimental autoimmune disease that relies on the use of complete Freund's adjuvant (CFA). Here we report that the heightened susceptibility of IFN-γR knock-out (KO) mice is associated with a functional impairment of CD4⁺CD25⁺ T_reg cells. Treatment of wild-type mice with depleting anti-CD25 antibody after CFA-assisted immunisation with collagen type II (CII) significantly accelerated the onset of arthritis and increased the severity of CIA. This is an indication of a role of T_reg cells in the effector phase of CIA. IFN-γR deficiency did not affect the number of CD4⁺CD25⁺ T cells in the central and peripheral lymphoid tissues. In addition, CD4⁺CD25⁺ T cells isolated from naive IFN-γR KO mice had a normal potential to suppress T cell proliferation in vitro. However, after immunisation with CII in CFA, the suppressive activity of CD4⁺CD25⁺ T cells became significantly more impaired in IFN-γR-deficient mice. Moreover, expression of the mRNA for Foxp3, a highly specific marker for T_reg cells, was lower. We further demonstrated that the effect of endogenous IFN-γ, which accounts for more suppressive activity in wild-type mice, concerns both T_reg cells and accessory cells. Our results demonstrate that the decrease in T_reg cell activity in CIA is counter-regulated by endogenous IFN-γ.     

Enhanced osteoclast development in collagen-induced arthritis in interferon-gamma receptor knock-out mice as related to increased splenic CD11b+ myelopoiesis

Collagen-induced arthritis (CIA) in mice is accompanied by splenomegaly due to the selective expansion of immature CD11b+ myeloblasts. Both disease manifestations are more pronounced in interferon-gamma receptor knock-out (IFN-gammaR KO) mice. We have taken advantage of this difference to test the hypothesis that the expanding CD11b+ splenic cell population constitutes a source from which osteoclast precursors are recruited to the joint synovia. We found larger numbers of osteoclasts and more severe bone destruction in joints of IFN-gammaR KO mice than in joints of wild-type mice. Osteoclast-like multinucleated cells appeared in splenocyte cultures established in the presence of macrophage colony-stimulating factor (M-CSF) and stimulated with the osteoclast-differentiating factor receptor activator of NF-kappaB ligand (RANKL) or with tumour necrosis factor-alpha (TNF-alpha). Significantly larger numbers of such cells could be generated from splenocytes of IFN-gammaR KO mice than from those of wild-type mice. This was not accompanied, as might have been expected, by increased concentrations of the intracellular adaptor protein TRAF6, known to be involved in signalling of RANKL- and TNF-alpha-induced osteoclast formation. Splenocyte cultures of IFN-gammaR KO mice also produced more TNF-alpha and more RANKL than those of wild-type mice. Finally, splenocytes isolated from immunised IFN-gammaR KO mice contained comparatively low levels of pro-interleukin-1beta (pro-IL-1beta) and pro-caspase-1, indicating more extensive conversion of pro-IL-1beta into secreted active IL-1beta. These observations provide evidence that all conditions are fulfilled for the expanding CD11b+ splenocytes to act as a source of osteoclasts and to be indirectly responsible for bone destruction in CIA. They also provide a plausible explanation for the higher susceptibility of IFN-gammaR KO mice to CIA.     

Long-term high-fat diet induces pancreatic injuries via pancreatic microcirculatory disturbances and oxidative stress in rats with hyperlipidemia

Stromal cell-derived factor 1 (CXCL12) is an angiogenic chemokine that is believed to act solely via its cognate receptor CXCR4. Evidence is now provided for the existence of a different CXCL12 binding and signaling receptor on endothelial cells. Bovine aortic endothelial cells (BAECs) strongly expressed CXCR4 and exhibited high binding capacity for fluorescently labeled CXCL12. However, CXCL12 binding was not correlated with the CXCR4 expression level and was virtually unaffected by the specific CXCR4 antagonists AMD3100 or T22. Similar observations were made in endothelial cells of mouse and human origin. Also, AMD3100 failed to block CXCL12 internalization and CXCL12-induced intracellular signal transduction via extracellular signal-regulated kinases 1/2 in BAECs. In contrast, CXCL12 binding and signaling were almost completely inhibited by the CXCR4 antagonist in T-lymphoid SupT1 cells. Together, our data point to the existence of an additional receptor through which CXCL12 exerts its biological effects in endothelial cells.     

CXCL12/CXCR4 Axis Improves Migration of Neuroblasts Along Corpus Callosum by Stimulating MMP-2 Secretion After Traumatic Brain Injury in Rats

To investigate the effect of CXCL12 on migration of neural precursor cells after traumatic brain injury (TBI). We randomly divided 48 rats into four groups: (1) the sham group, rats were performed craniotomy only, (2) the control group, saline were injected into the ipsilateral cortex after TBI, (3) the CXCL12 group, CXCL12 were injected into the ipsilateral cortex after TBI, and (4) the CXCL12 + AMD3100 group, CXCL12 and AMD3100 were mixed together and injected into the ipsilateral cortex after TBI. At 7 days after TBI, the brain tissues were subjected to immunofluorescent double-labeled staining with the antibodies of CXCR4/DCX, MMP-2/DCX, MMP-2/GFAP, MMP-2/NeuN. Western blot assay was used to measure the protein levels of MMP-2. Compared with the control group, the number of CXCR4/DCX and MMP-2 positive cells around the injured corpus callosum area were significantly increased in the CXCL12 treatment group. The area occupied by these cells expanded and the shape changed from chain distribution to radial. CXCL12 + AMD3100 treatment significantly decreased the number and distribution area of CXCR4/DCX and MMP-2 positive cells compared with the CXCL12 treatment and control group. The DCX positive cells could not form chain or radial distribution. The protein expressions of MMP-2 had the similar change trends as the results of immunofluorescent staining. MMP-2 could be secreted by DCX, GFAP and NeuN positive cells. CXCL12/CXCR4 axis can improve the migration of the neuroblasts along the corpus callosum by stimulating the MMP-2 secretion of different types of cells.     

Inhibition of human immunodeficiency virus replication by a dual CCR5/CXCR4 antagonist

Here we report that the N-pyridinylmethyl cyclam analog AMD3451 has antiviral activity against a wide variety of R5, R5/X4, and X4 strains of human immunodeficiency virus type 1 (HIV-1) and HIV-2 (50% inhibitory concentration [IC(50)] ranging from 1.2 to 26.5 microM) in various T-cell lines, CCR5- or CXCR4-transfected cells, peripheral blood mononuclear cells (PBMCs), and monocytes/macrophages. AMD3451 also inhibited R5, R5/X4, and X4 HIV-1 primary clinical isolates in PBMCs (IC(50), 1.8 to 7.3 microM). A PCR-based viral entry assay revealed that AMD3451 blocks R5 and X4 HIV-1 infection at the virus entry stage. AMD3451 dose-dependently inhibited the intracellular Ca(2+) signaling induced by the CXCR4 ligand CXCL12 in T-lymphocytic cells and in CXCR4-transfected cells, as well as the Ca(2+) flux induced by the CCR5 ligands CCL5, CCL3, and CCL4 in CCR5-transfected cells. The compound did not interfere with chemokine-induced Ca(2+) signaling through CCR1, CCR2, CCR3, CCR4, CCR6, CCR9, or CXCR3 and did not induce intracellular Ca(2+) signaling by itself at concentrations up to 400 microM. In freshly isolated monocytes, AMD3451 inhibited the Ca(2+) flux induced by CXCL12 and CCL4 but not that induced by CCL2, CCL3, CCL5, and CCL7. The CXCL12- and CCL3-induced chemotaxis was also dose-dependently inhibited by AMD3451. Furthermore, AMD3451 inhibited CXCL12- and CCL3L1-induced endocytosis in CXCR4- and CCR5-transfected cells. AMD3451, in contrast to the specific CXCR4 antagonist AMD3100, did not inhibit but enhanced the binding of several anti-CXCR4 monoclonal antibodies (such as clone 12G5) at the cell surface, pointing to a different interaction with CXCR4. AMD3451 is the first low-molecular-weight anti-HIV agent with selective HIV coreceptor, CCR5 and CXCR4, interaction.     

The peptidomimetic CXCR4 antagonist TC14012 recruits beta-arrestin to CXCR7: roles of receptor domains

CXCR7 is an atypical chemokine receptor that signals through β-arrestin in response to agonists without detectable activation of heterotrimeric G-proteins. Its cognate chemokine ligand CXCL12 also binds CXCR4, a chemokine receptor of considerable clinical interest. Here we report that TC14012, a peptidomimetic inverse agonist of CXCR4, is an agonist on CXCR7. The potency of β-arrestin recruitment to CXCR7 by TC14012 is much higher than that of the previously reported CXCR4 antagonist AMD3100 and differs only by one log from that of the natural ligand CXCL12 (EC(50) 350 nM for TC14012, as compared with 30 nM for CXCL12 and 140 μM for AMD3100). Moreover, like CXCL12, TC14012 leads to Erk 1/2 activation in U373 glioma cells that express only CXCR7, but not CXCR4. Given that with TC14012 and AMD3100 two structurally unrelated CXCR4 antagonists turn out to be agonists on CXCR7, this likely reflects differences in the activation mechanism of the arrestin pathway by both receptors. To identify the receptor domain responsible for these opposed effects, we investigated CXCR4 and CXCR7 C terminus-swapping chimeras. Using quantitative bioluminescence resonance energy transfer, we find that the CXCR7 receptor core formed by the seven-transmembrane domains and the connecting loops determines the agonistic activity of both TC14012 and AMD3100. Moreover, we find that the CXCR7 chimera bearing the CXCR4 C-terminal constitutively associates with arrestin in the absence of ligands. Our data suggest that the CXCR4 and CXCR7 cores share ligand-binding surfaces for the binding of the synthetic ligands, indicating that CXCR4 inhibitors should be tested also on CXCR7.     

Oral administration of milk fermented with Lactobacillus delbrueckii ssp. bulgaricus OLL1073R-1 to DBA/1 mice inhibits secretion of proinflammatory cytokines

We have previously shown that oral administration of skimmed milk(SM) fermented with Lactobacillus delbrueckii ssp. bulgaricus OLL1073R-1 (OLL1073R-1/SM) to DBA/1 mice inhibited the development of collagen-induced arthritis (CIA). In this study, our aim was to examine possible mechanisms of inhibiting the development of CIA. We studied the effect of OLL1073R-1/SM on cytokine secretion from cells of popliteal lymph nodes (lymph node cells; LNC) of mice. The results showed that feeding OLL1073R-1/SM inhibited secretion of proinflammatory cytokines such as interferon γ (IFN-γ), interleukin 6 (IL-6), tumor necrosis factor α (TNF-α) and the chemokine, monocyte chemoattractant protein 1 (MCP-1). The most prominent effect was inhibition of TNF-α. Secretion of IL-2 and IL-4 were not influenced. Feeding OLL1073R-1/SM inhibited secretion of proinflammatory cytokines produced by accessory cells, but not T cells. We conclude that CIA may be prevented via down regulation of secretion of proinflammatory cytokines such as IL-6, TNF-α and IFN-γ, and of the chemokine of MCP-1. This revised version was published online in August 2006 with corrections to the Cover Date.     

AMD3100 is a potent antagonist at CXCR4(R334X) , a hyperfunctional mutant chemokine receptor and cause of WHIM syndrome

WHIM is an acronym for a rare immunodeficiency syndrome (OMIM #193670) caused by autosomal dominant mutations truncating the C-terminus of the chemokine receptor CXC chemokine receptor 4 (CXCR4). WHIM mutations may potentiate CXCR4 signalling, suggesting that the United States Food and Drug Administration (FDA)-approved CXCR4 antagonist AnorMED3100 (AMD3100) (also known as Plerixafor) may be beneficial in WHIM syndrome. We have tested this at the preclinical level by comparing Chinese hamster ovary (CHO) and K562 cell lines matched for expression of recombinant wild-type CXCR4 (CXCR4(WT)) and the most common WHIM variant of CXCR4 (CXCR4(R334X)), as well as leucocytes from a WHIM patient with the CXCR4(R334X) mutation versus healthy controls. We found that CXCR4(R334X) mediated modestly increased signalling (~2-fold) in all functional assays tested, but strongly resisted ligand-dependent down-regulation. AMD3100 was equipotent and equieffective as an antagonist at CXCR4(R334X) and CXCR4(WT) . Together, our data provide further evidence that CXCR4(R334X) is a gain-of-function mutation, and support clinical evaluation of AMD3100 as mechanism-based treatment in patients with WHIM syndrome.