In a 3D model of breast morphogenesis, CEACAM1 (carcinoembryonic antigen-related cell adhesion molecule 1) plays an essential role in lumen formation in a subline of the nonmalignant human breast cell line (MCF10A). We show that mammary carcinoma cells (MCF7), which do not express CEACAM1 or form lumena when grown in Matrigel, are restored to a normal morphogenic program when transfected with CEACAM1-4S, the short cytoplasmic isoform of CEACAM1 that predominates in breast epithelia. During the time course of lumen formation, CEACAM1-4S was found initially between the cells, and in mature acini, it was found exclusively in an apical location, identical to its expression pattern in normal breast. Lumena were formed by apoptosis as opposed to necrosis of the central cells within the alveolar structures, and apoptotic cells within the lumena expressed CEACAM1-4S. Dying cells exhibited classical hallmarks of apoptosis, including nuclear condensation, membrane blebbing, caspase activation, and DNA laddering. Apoptosis was mediated by Bax translocation to the mitochondria and release of cytochrome c into the cytoplasm, and was partially inhibited by culturing cells with caspase inhibitors. The dynamic changes in CEACAM1 expression during morphogenesis, together with studies implicating extracellular matrix and integrin signaling, suggest that a morphogenic program integrates cell–cell and cell–extracellular matrix signaling to produce the lumena in mammary glands. This report reveals a function of CEACAM1-4S relevant to cellular physiology that distinguishes it from its related long cytoplasmic domain isoform.

CAR T cell therapy is a promising clinical approach for reducing tumor progression and prolonging patient survival. However, improvements in both the safety and potency of CAR T cell therapy demand quantitative imaging techniques to determine the distribution of cells following adoptive transfer. The purpose of this study is to optimize zirconium-89-oxine (⁸⁹Zr-oxine) labeling of chimeric antigen receptor (CAR) T cells and evaluate positron emission tomography (PET) as a platform for imaging adoptively transferred CAR T cells. Methods: CAR T cells were labeled with 0 – 1.4 MBq of ⁸⁹Zr-oxine per million cells and assessed for radioactivity retention, viability, and functionality. In vivo trafficking of ⁸⁹Zr-oxine labeled CAR T cells was evaluated in two murine xenograft tumor models: glioblastoma brain tumors with intracranially delivered IL13Rα2-targeted CAR T cells and subcutaneous prostate tumors with intravenously delivered PSCA-targeted CAR T cells. Results: CAR T cells were efficiently labeled (75%) and retained greater than 60% of ⁸⁹Zr over 6 days. In vitro cytokine production, migration, and tumor cytotoxicity as well as in vivo antitumor activity were not significantly reduced when labeled with 70 kBq per million cells. IL13Rα2-CAR T cells delivered intraventricularly were detectable by PET for at least six days throughout the central nervous system and within intracranial tumors. When intravenously administered, PSCA-CAR T cells also showed tumor tropism, with a 9-fold greater tumor-to-muscle ratio in comparison to CAR-negative T cells. Conclusion: ⁸⁹Zr-oxine can be utilized for labeling and imaging CAR T cells while maintaining cell viability and function. Based on these studies, we propose that ⁸⁹Zr-oxine is a clinically translatable platform for real-time assessment of cell therapies.

LL‐37, derived from human cathelicidin, stimulates immune responses in neutrophils. Although FPR2 and P2X7 were proposed as LL‐37 receptors, we have shown that among 21 neutrophil receptors only CXCR2 was down‐regulated by LL‐37. LL‐37 functions similarly to CXCR2‐specific chemokines CXCL1 and CXCL7 in terms of receptor down‐regulation and intracellular calcium mobilization on freshly isolated neutrophils. Neutrophils pretreated with CXCL8, a chemokine that binds both CXCR1/2, completely blocked the calcium mobilization in response to LL‐37, while LL‐37 also partially inhibited ¹²⁵I‐CXCL8 binding to neutrophils. SB225002, a selective CXCR2 antagonist, blocked LL‐37‐induced calcium mobilization and migration of neutrophils. LL‐37 stimulates calcium mobilization in CXCR2‐transfected HEK293 cells, CXCR2⁺ THP‐1 cells and monocytes, but not in CXCR1‐transfected HEK293 cells. WKYMVm peptide (ligand for FPR2) does not block LL‐37‐stimulated calcium flux in either THP‐1 (FPR2⁻) or monocytes (FPR2^high), further confirming the specificity of LL‐37 for CXCR2 and not FPR2. Among all ligands tested (ATP, BzATP, WKYMVm, CXCL1, and LL‐37), only LL‐37 stimulated migration of monocytes (CXCR2⁺ and FPR2⁺) and migration was inhibited by the CXCR2 inhibitor SB225002. Moreover, CXCR2 but not CXCR1 was internalized in LL‐37‐treated neutrophils. Thus, our data provide evidence that LL‐37 may act as a functional ligand for CXCR2 on human neutrophils.

LPS-activated neutrophils secrete IL-1β by activation of TLR-4. Based on studies in macrophages, it is likely that ROS and lysosomal destabilization regulated by Syk activation may also be involved. Since neutrophils have abundant expression of the ITIM-containing co-receptor CEACAM1 and Gram-negative bacteria such as Neisseria utilize CEACAM1 as a receptor that inhibits inflammation, we hypothesized that the overall production of IL-1β in LPS treated neutrophils may be negatively regulated by CEACAM1. We found that LPS treated neutrophils induced phosphorylation of Syk resulting in the formation of a complex including TLR4, p-Syk, and p-CEACAM1, which in turn, recruited the inhibitory phosphatase SHP-1. LPS treatment leads to ROS production, lysosomal damage, caspase-1 activation and IL-1β secretion in neutrophils. The absence of this regulation in Ceacam1^−/− neutrophils led to hyper production of IL-1β in response to LPS. The hyper production of IL-1β was abrogated by in vivo reconstitution of wild type but not ITIM-mutated CEACAM1 bone marrow stem cells. Blocking Syk activation by kinase inhibitors or RNAi reduced Syk phosphorylation, lysosomal destabilization, ROS production, and caspase-1 activation in Ceacam1^−/− neutrophils. We conclude that LPS treatment of neutrophils triggers formation of a complex of TLR4 with pSyk and pCEACAM1, which upon recruitment of SHP-1 to the ITIMs of pCEACAM1, inhibits IL-1β production by the inflammasome. Thus, CEACAM1 fine-tunes IL-1β production in LPS treated neutrophils, explaining why the additional utilization of CEACAM1 as a pathogen receptor would further inhibit inflammation. Pathogens often evade the immune system by directly binding to and inhibiting neutrophils, abundant white cells that accumulate at the site of infection. For example Gram-negative Neisseria pathogens, such as those that cause gonorrhea or meningitis, bind the neutrophil receptor CEACAM1. Gram-negative bacteria express lipopolysaccharide (LPS) that interacts with toll-like receptor-4 (TLR4) on neutrophils. Since CEACAM1 is an inhibitory receptor, we hypothesized that LPS activation of TLR4 would be inhibited. In this paper we show that this is the case and that the mechanism of LPS inhibition involves induction of a complex between the LPS receptor TLR4, CEACAM1 and an activating kinase called Syk. In the presence of CEACAM1, an inhibitory phosphatase (opposes the kinase) is recruited to the complex that prevents the activation of Syk. The net effect is the inhibition of the pathway that normally leads to the production of the pro-inflammatory cytokine IL-1β. We show that this inhibition is lost in CEACAM1 deficient neutrophils leading to hyper production of IL-1β. We think that CEACAM1 fine-tunes the normal inflammatory response at the site of infection preventing hyper-inflammation, but in the case of Gram-negative pathogens that actually bind to neutrophils, inflammation is further blunted, favoring the infectious process.

Fibromyalgia (FMS), a predominantly female (85%) syndrome, affects an estimated 2% of the US population with skeletal muscle ache, fatigue, headache, and sleep disorder. The pathogenesis of FMS is unknown and there is no laboratory test for diagnosis. In this study, plasma levels of 25 cytokines and chemokines in 92 female patients with FMS and 69 family members were measured compared to 77 controls. Trans-endothelial migration of normal leukocytes in response to FMS plasma and the cytokine profile of human myoblasts were analyzed. High levels of MCP-1 ( P < 0.001) and eotaxin ( P < 0.01) were found in patients and family members compared to controls. Patients (56/92) treated with the single agent guaifenesin (>3 months) had higher levels of eotaxin than those not treated ( P < 0.01). Diluted plasma from patients increased the migration of normal eosinophils and monocytes, but not neutrophils, through an endothelial/Matrigel barrier only when mast cells are included in the lower wells ( P < 0.05). Furthermore, myoblasts can secrete MCP-1, eotaxin, and IP-10, while treatment with MCP-1 caused secretion of IL-1β, eotaxin and IP-10. FMS is associated with inflammatory chemokines, that MCP-1 and eotaxin may contribute to the symptoms of FMS, and that similar cytokine profiles found in family members support the idea that FMS has a genetic component. Furthermore, the chemokine profile associated with FMS has direct effects on the migration of eosinophils and monocytes in the presence of mast cells, and skeletal muscle itself may secrete

Neutrophils represent the most common granulocyte subtype present in blood. The short half-life of circulating neutrophils is regulated by spontaneous apoptosis, and tissue infiltrating neutrophils die by apoptosis and secondary necrosis. The mechanism of neutrophil apoptosis has been the subject of many studies; however, the mechanism of neutrophil secondary necrosis is less clear. Human cathelicidin cationic peptide 18, proteolytically processed to its active form, LL-37, is secreted by neutrophils and epithelial cells and shown to have effects in addition to bacterial lysis. We demonstrate here that LL-37 affects neutrophil lifespan by the pathway of secondary necrosis, rapidly converting annexin V-positive (AV+), propidium iodide-negative (PI−; apoptotic) cells into PI+ (necrotic) cells with the release of IL-8, IL-1R antagonist, ATP, and intact granules. The effects of LL-37 on apoptotic neutrophils are neither energy-dependent nor affected by pretreatment with G-CSF, GM-CSF, TNF-α, and LPS and are partially inhibited by human serum. Moreover, LL-37 decreases CXCR2 expression of AV−PI− (live) neutrophils, suggesting an effect on the neutrophil response to its chemotactic factors, including IL-8. Thus, the lifespan and inflammatory functions of neutrophils are directly affected by LL-37.