Very Late Antigen-4 Expression Research Articles

Albumin-Binding Lutetium-177-Labeled LLP2A Derivatives as Theranostics for Melanoma.

Very late antigen-4 (VLA-4) is a transmembrane integrin protein that is highly expressed in aggressive forms of metastatic melanoma. A small-molecule peptidomimetic, LLP2A, was found to have a low pM affinity binding to VLA-4. Because LLP2A itself does not inhibit cancer cell proliferation and survival, it is an ideal candidate for the imaging and delivery of therapeutic payloads. An analog of [177Lu]Lu-labeled-LLP2A was previously investigated as a therapeutic agent in melanoma tumor-bearing mice, resulting in only a modest improvement in tumor growth inhibition, likely due to rapid clearance of the agent from the tumor. To improve the pharmacokinetic profile, DOTAGA-PEG4-LLP2A with a 4-(p-iodophenyl)butyric acid (pIBA) albumin binding moiety was synthesized. We demonstrate the feasibility of this albumin binding strategy by comparing in vitro cell binding assays and in vivo biodistribution performance of [177Lu]Lu-DOTAGA-PEG4-LLP2A ([177Lu]Lu-1) to the albumin binding [177Lu]Lu-DOTAGA-pIBA-PEG4-LLP2A ([177Lu]Lu-2). In vitro cell binding assay results for [177Lu]Lu-1 and [177Lu]Lu-2 showed Kd values of 0.40 ± 0.07 and 1.75 ± 0.40 nM, with similar Bmax values of 200 ± 6 and 315 ± 15 fmol/mg, respectively. In vivo biodistribution data for both tracers exhibited specific uptake in the tumor, spleen, thymus, and bone due to endogenous expression of VLA-4. Compound [177Lu]Lu-2 exhibited a much longer blood circulation time compared to [177Lu]Lu-1. The tumor uptake for [177Lu]Lu-1 was highest at 1 h (∼15%ID/g) and that for [177Lu]Lu-2 was highest at 4 h (∼23%ID/g). Significant clearance of [177Lu]Lu-1 from the tumor occurs at 24 h (<5%ID/g) while[177Lu]Lu-2 is retained for greater than 96 h (∼10%ID/g). An efficacy study showed that melanoma tumor-bearing mice receiving compound [177Lu]Lu-2 given in two fractions (2 × 14.8 MBq, 14 days apart) had a greater median survival time than mice administered a single 29.6 MBq dose of compound [177Lu]Lu-1, while a single 29.6 MBq dose of [177Lu]Lu-2 imparted hematopoietic toxicity. The in vitro and in vivo data show addition of pIBA to [177Lu]Lu-DOTAGA-PEG4-LLP2A slows blood clearance for a higher tumor uptake, and there is potential of [177Lu]Lu-2 as a theranostic in fractionated administered doses.

Targeted imaging of very late antigen-4 for noninvasive assessment of lung inflammation-fibrosis axis

BackgroundThe lack of noninvasive methods for assessment of dysregulated inflammation as a major driver of fibrosis (i.e., inflammation-fibrosis axis) has been a major challenge to precision management of fibrotic lung diseases. Here, we determined the potential of very late antigen-4 (VLA-4)-targeted positron emission tomography (PET) to detect inflammation in a mouse model of bleomycin-induced fibrotic lung injury.MethodSingle time-point and longitudinal VLA-4-targeted PET was performed using a high-affinity peptidomimetic radiotracer, 64Cu-LLP2A, at weeks 1, 2, and 4 after bleomycin-induced (2.5 units/kg) lung injury in C57BL/6J mice. The severity of fibrosis was determined by measuring the hydroxyproline content of the lungs and expression of markers of extracellular matrix remodeling. Flow cytometry and histology was performed to determine VLA-4 expression across different leukocyte subsets and their spatial distribution.ResultsLung uptake of 64Cu-LLP2A was significantly elevated throughout different stages of the progression of bleomycin-induced injury. High lung uptake of 64Cu-LLP2A at week-1 post-bleomycin was a predictor of poor survival over the 4-week follow up, supporting the prognostic potential of 64Cu-LLP2A PET during the early stage of the disease. Additionally, the progressive increase in 64Cu-LLP2A uptake from week-1 to week-4 post-bleomycin correlated with the ultimate extent of lung fibrosis and ECM remodeling. Flow cytometry revealed that LLP2A binding was restricted to leukocytes. A combination of increased expression of VLA-4 by alveolar macrophages and accumulation of VLA-4-expressing interstitial and monocyte-derived macrophages as well as dendritic cells was noted in bleomycin-injured, compared to control, lungs. Histology confirmed the increased expression of VLA-4 in bleomycin-injured lungs, particularly in inflamed and fibrotic regions.ConclusionsVLA-4-targeted PET allows for assessment of the inflammation-fibrosis axis and prediction of disease progression in a murine model. The potential of 64Cu-LLP2A PET for assessment of the inflammation-fibrosis axis in human fibrotic lung diseases needs to be further investigated.

Novel Strategy to Regulate PD-L1 Expression and Enhance Anti-Tumor Effect

Abstract The programmed death 1/PD ligand 1(PD-1/PD-L1 axis is a major player in the tumor immune evasion strategy. We show that the immunomodulatory small tellurium molecules, AS101 [ammonium trichloro(dioxoethylene-o, o′)tellurate] and SAS [octa-O-bis-(R,R)-tartarate ditellurane], suppress PD-L1 expression on a variety of human and mouse malignant cells by inhibition of the α4β1 VLA-4 (very late antigen-4) integrin activity. As a consequence, pAkt and its downstream effector pNFκB are inhibited. VLA-4/pAkt/pNFκB signaling blockade was found crucial for decreasing PD-L1 expression. Moreover, the tellurium compounds had physiological effects. SAS augmented killing of human and mouse malignant cells by stimulated syngeneic splenocytes or CD8+ T cells. Furthermore, SAS prevented the development of chemoresistance in malignant cells. IL-10 production by tumor cells, the inhibition of which by AS101 we showed suppressed chemoresistance, is shown here to be closely associated with inhibition of VLA-4 and PD-L1 expression suggesting that AS101 and SAS compounds may, at least partially, induce chemosensitivity by virtue of inhibition of the VLA-4/IL-10/PD-L1 pathway. AS101 or SAS treatment of B16 melanoma-bearing mice decreased tumor cell PD-L1 expression, led to increased CD8+ T-cell infiltration into the tumors and tumor shrinkage. Combined treatment with αPD-1 antibody and either of the tellurium compounds significantly increased antitumor effects. These results collectively suggest that VLA-4 integrin signaling serves as a critical component of cancer evasion and is a potential target for cancer treatment. Furthermore, AS101 or SAS may be suitable for effective combinatorial cancer immunotherapy with αPD-1.

The main e-cigarette component vegetable glycerin enhances neutrophil migration and fibrosis in endotoxin-induced lung injury via p38 MAPK activation

We investigated the effects of vegetable glycerin (VG), a main e-cigarette constituent, on endotoxin-induced acute lung injury (ALI). Mice received intratracheal administration of 30% VG in phosphate buffered saline (PBS) vehicle or only PBS (control) for 4 days. On Day 5, mice received an intratracheal instillation of lipopolysaccharide (LPS) (LPS group and VG + LPS group) or PBS (VG group and control group). Lung histopathology, expression of chemokine receptors, and regulatory signaling were analyzed 24 h after the Day 5 treatment. VG significantly increased ALI-associated histopathological and fibrotic changes in both the VG group and LPS-induced ALI mice (VG + LPS group). Immunohistochemistry (IHC) and western blot analyses revealed that VG administration resulted in upregulation of neutrophil markers [lymphocyte antigen 6 complex locus G6D (Ly6G) and myeloperoxidase (MPO)] as well as upregulation of the expression of transforming growth factor-β (TGF-β), a central mediator of fibrogenesis, in the lungs of both VG and VG + LPS groups. VG enhanced the expression of adhesion molecules [very late antigen 4 (VLA-4) and vascular cell adhesion molecule 1 (VCAM-1)] and increased activation of p38 mitogen-activated protein kinase (p38 MAPK) to prompt neutrophil recruitment in the lungs of mice with ALI. Intraperitoneal administration of a p38 inhibitor attenuated these histopathological changes significantly as well as VG-induced upregulation in expression of Ly6G, MPO, VLA-4, VCAM-1, TGF-β, and collagen-1 in mice with ALI. In conclusion, VG enhances neutrophil chemotaxis and fibrosis and it amplifies the inflammatory response associated with LPS-induced ALI in the lungs via enhancement of p38 MAPK activity.

First-in-Humans Evaluation of Safety and Dosimetry of 64Cu-LLP2A for PET Imaging.

There remains an unmet need for molecularly targeted imaging agents for multiple myeloma (MM). The integrin very late antigen 4 (VLA4), is differentially expressed in malignant MM cells and in pathogenic inflammatory microenvironmental cells. [64Cu]Cu-CB-TE1A1P-LLP2A (64Cu-LLP2A) is a VLA4-targeted, high-affinity radiopharmaceutical with promising utility for managing patients diagnosed with MM. Here, we evaluated the safety and human radiation dosimetry of 64Cu-LLP2A for potential use in MM patients. Methods: A single-dose [natCu]Cu-LLP2A (Cu-LLP2A) tolerability and toxicity study was performed on CD-1 (Hsd:ICR) male and female mice. 64Cu-LLP2A was synthesized in accordance with good-manufacturing-practice-compliant procedures. Three MM patients and six healthy participants underwent 64Cu-LLP2A-PET/CT or PET/MRI at up to 3 time points to help determine tracer biodistribution, pharmacokinetics, and radiation dosimetry. Time-activity curves were plotted for each participant. Mean organ-absorbed doses and effective doses were calculated using the OLINDA software. Tracer bioactivity was evaluated via cell-binding assays, and metabolites from human blood samples were analyzed with analytic radio-high-performance liquid chromatography. When feasible, VLA4 expression was evaluated in the biopsy tissues using 14-color flow cytometry. Results: A 150-fold mass excess of the desired imaging dose was tolerated well in male and female CD-1 mice (no observed adverse effect level). Time-activity curves from human imaging data showed rapid tracer clearance from blood via the kidneys and bladder. The effective dose of 64Cu-LLP2A in humans was 0.036 ± 0.006 mSv/MBq, and the spleen had the highest organ uptake, 0.142 ± 0.034 mSv/MBq. Among all tissues, the red marrow demonstrated the highest residence time. Image quality analysis supports an early imaging time (4-5 h after injection of the radiotracer) as optimal. Cell studies showed statistically significant blocking for the tracer produced for all human studies (82.42% ± 13.47%). Blood metabolism studies confirmed a stable product peak (>90%) up to 1 h after injection of the radiopharmaceutical. No clinical or laboratory adverse events related to 64Cu-LLP2A were observed in the human participants. Conclusion: 64Cu-LLP2A exhibited a favorable dosimetry and safety profile for use in humans.

SVCAM1 in the Hippocampus Contributes to Postoperative Cognitive Dysfunction in Mice by Inducing Microglial Activation Through the VLA-4 Receptor.

Postoperative cognitive dysfunction (POCD) is a severe postsurgical complication, but its underlying mechanisms remain unclear. Neuroinflammation mediated by microglial activation plays a major role in POCD pathophysiology. Upregulation of vascular cell adhesion molecule 1 (VCAM1) on brain endothelial cells is closely correlated with microglial activation in the mouse hippocampus. However, the role of VCAM1 upregulation in microglial activation remains unknown. Soluble VCAM1 (sVCAM1) activates the very late antigen-4 (VLA-4) receptor under inflammatory conditions. Therefore, we hypothesized that sVCAM1 which is shed from VCAM1 contributes to POCD by triggering hippocampal microglial activation through the VLA-4 receptor. We found that VCAM1 and sVCAM1 expression in the mouse hippocampus was upregulated after surgery, and the upregulation was accompanied by hippocampal microglial activation. sVCAM1 levels in mouse and human serum were increased after surgery. Anti-VCAM1 treatment inhibited microglial activation, proinflammatory cytokine production, VLA-4 expression and P38 mitogen-associated protein kinase (MAPK) pathway activation and attenuated hippocampal-dependent cognitive dysfunction. In vitro, recombinant sVCAM1 promoted M1 polarization in BV2 cells, increased VLA-4 expression and activated the P38 MAPK pathway. These effects were reversed by VLA-4 receptor blockade. Anti-VLA-4 treatment ameliorated hippocampal-dependent cognitive dysfunction after surgery by inhibiting microglial activation, proinflammatory cytokine production and P38 pathway activation. In conclusion, increased sVCAM1 in the hippocampus is involved in microglial activation and cognitive dysfunction induced by surgery. Inhibiting the sVCAM1-VLA-4 interaction in microglia may be a therapeutic strategy for POCD.

Molecular Imaging of Very Late Antigen-4 in Acute Lung Injury.

Inflammation plays a central role in the pathogenesis of acute lung injury (ALI) during both the acute pneumonitis stage and progression into the chronic fibroproliferative phase, leading to pulmonary fibrosis. Currently, there is an unmet clinical and research need for noninvasive ways to monitor lung inflammation through targeting of immunoregulatory pathways contributing to ALI pathogenesis. In this study, we evaluated the role of targeted imaging of very late antigen-4 (VLA-4), as a key integrin mediating the adhesion and recruitment of immune cells to inflamed tissues, in quantifying lung inflammation in a mouse model of lipopolysaccharide-induced ALI. Methods: ALI was induced by a single intratracheal administration of lipopolysaccharide (10, 20, or 40 μg per mouse) in C57BL/6J mice. Control mice were intratracheally instilled with sterile phosphate-buffered saline. VLA-4-targeted PET/CT was performed 24 h after intravenous injection of a 64Cu-labeled high-affinity peptidomimetic ligand referred to as 64Cu-LLP2A, which is conjugated with the chelator (1,4,8,11-tetraazacyclotetradecane-1-(methane phosphonic acid)-8-(methane carboxylic acid) and a polyethylene glycol 4 linker, at day 2 after the induction of ALI. Ex vivo biodistribution of 64Cu-LLP2A was determined by γ-counting of harvested organs. The severity of lung inflammation was assessed histologically and by measuring the expression of inflammatory markers in the lung tissue lysates using reverse transcription quantitative polymerase chain reaction. Results: Intratracheal lipopolysaccharide instillation led to an acute inflammatory response in the lungs, characterized by increased expression of multiple inflammatory markers and infiltration of myeloid cells, along with a significant and specific increase in 64Cu-LLP2A uptake, predominantly in a peribronchial distribution. There was a strong correlation between the lipopolysaccharide dose and 64Cu-LLP2A uptake, as quantified by in vivo PET (R = 0.69, P < 0.01). Expression levels of both subunits of VLA-4, that is, integrins α4 and β1, significantly correlated with the expression of multiple inflammatory markers, including tumor necrosis factor-α, interleukin-1β, and nitric oxide synthase-2, highlighting the potential of VLA-4 as a surrogate marker of acute lung inflammation. Notably, in vivo 64Cu-LLP2A uptake significantly correlated with the expression of multiple inflammatory markers and VLA-4. Conclusion: Our study demonstrates the feasibility of molecular imaging of VLA-4, as a mechanistically relevant target in ALI, and the accuracy of VLA-4-targeted PET in quantification of ongoing lung inflammation in a murine model.

Nintedanib Reduces Neutrophil Chemotaxis via Activating GRK2 in Bleomycin-Induced Pulmonary Fibrosis.

Neutrophils are involved in the alveolitis of idiopathic pulmonary fibrosis (IPF). However, their pathogenic mechanisms are still poorly understood. Nintedanib has antifibrotic and anti-inflammatory activity in IPF. This study aimed to investigate the regulatory mechanism of nintedanib on neutrophil chemotaxis in bleomycin (BLM)-induced pulmonary fibrosis. Nintedanib was administered via oral gavage to male C57BL/6 mice 24 h after a bleomycin intratracheal injection (1.5 U/kg). Lung histopathological findings, the expression of cytokines, and the regulatory signaling pathways of neutrophil chemotaxis were analyzed. The effect of nintedanib was also investigated in a mouse model with adoptive neutrophil transfer in vivo. Nintedanib significantly decreased the histopathological changes and neutrophil recruitment in BLM-induced pulmonary fibrosis. Nintedanib mediated a downregulation of chemokine (C-X-C motif) receptor 2 (CXCR2) and very late antigen 4 (VLA-4) expression, as well as an upregulation of G protein-coupled receptor kinase 2 (GRK2) activity in peripheral blood neutrophils in BLM-induced pulmonary fibrosis. Nintedanib also decreased the activation of endothelial cells by the decreased expression of vascular cell adhesion molecule 1 (VCAM-1). The effect of nintedanib on regulating neutrophil chemotaxis was also confirmed by a mouse model with adoptive neutrophil transfer in vivo. In conclusion, nintedanib reduces neutrophil chemotaxis and endothelial cell activation to regulate the severity of BLM-induced pulmonary fibrosis. These effects are associated with an enhancement of GRK2 activity and a reduction in CXCR2 and VLA-4 expression on neutrophils and a decrease in VCAM-1 expression on endothelial cells.

Polymorphonuclear Neutrophils Play a Decisive Role for Brain Injury and Neurological Recovery Poststroke.

Polymorphonuclear Neutrophils Play a Decisive Role for Brain Injury and Neurological Recovery Poststroke.

VLA4 expression by B cells enables B cell-restricted antigen presentation to support CD4 T cell driven central nervous system autoimmunity

Abstract Clonal expansion along with immunoglobulin (Ig) class-switching and secretion result from B cell antigen presentation to cognate CD4 T cells. The presence of oligoclonal Ig in the cerebrospinal fluid of most patients with multiple sclerosis raises the important question of where cognate B:T cell interactions occurs during disease. Previously we showed that increasing the frequency of MOG-specific B cells is sufficient to drive EAE in mice expressing MHCII solely by B cells. We have now established a system in which EAE reliably occurs upon expression of MHCII using Tamoxifen-induced temporal regulation in MOG-specific B cells. While B cells express MHCII in the central nervous system (CNS) in these models, whether or not B cells acquire and present antigen in the CNS compartment is unknown. Large clusters of B cells rapidly formed in the subarachnoid space of the spinal cord during EAE in our model, suggesting homing to the CNS is important for antigen presentation. Genetic regulation of B cell MOG-specificity and expression of MHCII and Very Late Antigen-4 (VLA-4) was used to determine the dependence of passive EAE susceptibility on B cell access to the CNS. Mice in which VLA-4 expression was abrogated simultaneously with MHCII expression by MOG-specific B cells were subjected to passive EAE. Disease exclusively mediated by B cell antigen presentation was dependent on VLA-4 expression by B cells. MOG-specific CD4 T cell hybridomas did not detect endogenous MOG presented by B cells harvested from the periphery of mice prior to the onset of disease. Taken together, our findings indicate that B cells require access to CNS myelin targets in order to propagate antigen-specific CD4 T cell-dependent neuro-inflammation.

Expression of Sirpα Tailless Mutant in Mice Impairs Naïve CD4+ T Cell Adhesion to Immobilized ICAM‐1 and TCR Induced Proliferation.

Signal Regulatory Protein alpha (SIRPα) interaction with its ligand CD47 plays an important role in immune cell homeostasis. CD47 is expressed by essentially all cell types, and acts as a “don't eat me signal” and a marker of self. We previously demonstrated that CD47 on both endothelium and T cells plays an important role in T cell adhesion and diapedesis in vivo and in vitro, and that CD47−/− CD4+ T cells had impaired antigen mediated proliferation in a model of Experimental Autoimmune Encephalomyelitis (EAE). In contrast to CD47, SIRPα has been reported as selectively expressed in the Central Nervous System (CNS), in most myeloid cells and at very low levels‐to‐absent in lymphoid cells in humans. To gain insight into the function of SIRPα in leukocyte recruitment, Sirpα truncated cytoplasmic, non‐signaling mutant (Sirpatm1Nog) transgenic mice and WT mice were studied in TNFα‐induced dermal air pouch model of inflammation. Unexpectedly, CD4+ T cells in Sirpatm1Nog mice, compared to WT mice, had significantly decreased recruitment into the air pouch (WT, 11.9 ± 2.9 ×103 vs Sirpatm1Nog, 7.8 ± 2.6 ×103, p&lt;0.05), whereas no differences in CD8+ T cell or myeloid cell recruitment were observed. Interestingly, flow cytometric analysis using P84 monoclonal antibody showed that a low but significant fraction of CD4+ T cells in both WT and Sirpatm1Nog mice expressed SIRPα. Expression of Very Late Antigen‐4 (VLA‐4) and Lymphocyte Function Associated Antigen‐1 (LFA‐1) integrins, which are ligands of Vascular Cell Adhesion Molecule‐1 (VCAM‐1) and Intercellular Adhesion Molecule‐1 (ICAM‐1), respectively, were similar in T cells from WT and Sirpatm1Nog mice. Additional studies showed naïve CD4+ isolated from Sirpatm1Nog, compared to WT mice, had significantly blunted proliferation 48 hrs after T cell receptor crosslinking (TCR‐XL), and that CD4 T cells had impaired SDF‐1α induced arrest on immobilized ICAM‐1 under shear flow conditions. In summary, these studies demonstrate that SIRPα plays an unexpected and important role in T cell recruitment in vivo and in vitro and in TCR‐XL induced proliferation in vitro. These data suggest that loss of SIRPα signaling in SIRPα+ T cells leads to impaired LFA‐1 activation.Support or Funding InformationR01 HL125780 and T32HL007627.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Preliminary evaluation of 18F-labeled LLP2A-trifluoroborate conjugates as VLA-4 (α4β1 integrin) specific radiotracers for PET imaging of melanoma

IntroductionThe transmembrane α4β1 integrin receptor, or very-late antigen 4 (VLA-4), is associated with tumor metastasis and angiogenesis, the development of chemotherapeutic drug resistance, and is overexpressed in multiple myelomas, osteosarcomas, lymphomas, leukemias, and melanomas. The peptidomimetic, LLP2A, is a high-affinity ligand with specificity for the extracellular portion of VLA-4 and several conjugates have been evaluated in vivo by NIR-fluorescence, 111In-SPECT and 68Ga- and 64Cu-PET imaging, but to date, not with 18F-PET. MethodsUsing two highly stable organotrifluoroborate prosthetic groups: ammoniumdimethyl-trifluoroborate (AMBF3) and a new N-pyridinyl-para-trifluoroborate (N-Pyr-p-BF3), both capable of facile aqueous 18F-labeling by isotope exchange (IEX), we present the first PET imaging evaluations of two [18F]R-BF3−–PEG2-LLP2A tracers using VLA-4 overexpressing B16-F10 murine melanoma tumor mouse models. ResultsHere, we demonstrate successful one-step 18F-labeling of both conjugates with wet NCA [18F]F− in radiochemical yields of up to 11.6% within 75 min at molar activities of 40–100 GBq/μmol. Average tumor uptake values based on ex vivo biodistribution values were 4.4%ID/g (11) and 2.8%ID/g (12) using 18F-labeled LLP2A-conjugates with the two prosthetic groups: N-Pyr-p-BF3 (5) and alkyl-N,N-dimethylammonio-BF3 (AMBF3) (7), respectively, and was found to be target-specific as evidenced by in vivo blocking controls. Dynamic PET scanning and biodistribution studies revealed slow clearance of the [18F]R-BF3−–PEG2-LLP2A tracers from the tumors, and also substantial uptake in the intestines, gall bladder, liver and bladder. Observed bone uptake was blockable, consistent with known VLA-4 expression in hematopoietic stem cells found in bone marrow. ConclusionsThese studies show that these [18F]R-BF3−–PEG2-LLP2A conjugates (11 and 12) are promising VLA-4 targeting radiotracers, yet, further optimization will be required to reduce uptake in the gastro-intestinal tract.

In vitro VLA-4 blockade results in an impaired NK cell-mediated immune surveillance against melanoma

Natalizumab (NTZ) is a monoclonal antibody targeting the α4β1 integrin (CD49d/CD29), very late antigen-4 (VLA-4), which is approved for treatment of relapsing-remitting multiple sclerosis (RR-MS). A possible association between NTZ treatment and a higher risk of melanoma is under debate. Natural Killer (NK) cells, which express VLA-4, represent an innate barrier limiting spreading of melanoma under steady state conditions. Indeed, because of their expression of activating receptors, they are very efficient in recognizing and killing melanoma cells without the need of a previous priming. For this reason, we aimed at assessing whether NK-cell functions might be impaired by sustained exposure to NTZ. To investigate this possibility we isolated NK cells from healthy donors and tested their cytotoxic and migratory functions against primary melanoma cells derived from subcutaneous and lymph node metastases. Flow cytometry analysis demonstrated expression of CD49d on both freshly isolated NK cells and activated NK cells. Moreover, VLA-4 and its receptor, vascular cell adhesion protein-1 (VCAM-1) were similarly expressed on freshly isolated NK cells. However, upon a short exposure to NTZ, expression of VLA-4 on NK cells decreased. Analysis of NK receptor expression upon exposure of NK cells from three healthy donors to NTZ indicated that DNAM-1 and NKp46 are apparently decreased, while NKG2A is increased. The degranulation of NK cells towards melanoma cells, which express both VLA-4 and VCAM-1, was not affected when NTZ was added to the co-culture or when both NK cells and melanoma cells were each pre-exposed to NTZ for over 12h. In contrast, degranulation was significantly inhibited after 48h of pre-incubation indicating that NTZ can influence NK-cell degranulation towards melanoma cells only after a prolonged exposure. Using a migration chamber assay, we observed that the migration of NK cells towards melanoma cells was dependent upon the concentration of melanoma cells in the lower chamber, and that it was significantly reduced in presence of NTZ. Our results show that upon exposure to NTZ both cytolytic activity and migration toward melanoma cells were affected, suggesting that binding of NTZ to NK cells affects pathways involved in these NK-cell functions. We analyzed the expression of CD49d on NK cells from MS patients treated with NTZ and observed that it decreases with time of treatment.These data suggest that blockade of VLA-4 on NK-cell surface alters some key functions involved in the immune surveillance toward melanoma by NK cells and may provide a mechanistic explanation for the reported occurrence of melanoma in MS patients treated with NTZ.

Nicotine Mediates CD161a+ Renal Macrophage Infiltration and Premature Hypertension in the Spontaneously Hypertensive Rat.

Renal inflammation contributes to the pathophysiology of hypertension. CD161a+ immune cells are dominant in the (SHR) spontaneously hypertensive rat and expand in response to nicotinic cholinergic activation. We aimed to phenotype CD161a+ immune cells in prehypertensive SHR after cholinergic activation with nicotine and determine if these cells are involved in renal inflammation and the development of hypertension. Studies used young SHR and WKY (Wistar-Kyoto) rats. Splenocytes and bone marrow cells were exposed to nicotine ex vivo, and nicotine was infused in vivo. Blood pressures, kidney, serum, and urine were obtained. Flow cytometry, Luminex/ELISA, immunohistochemistry, confocal microscopy, and Western blot were used. Nicotinic cholinergic activation induced proliferation of CD161a+/CD68+ macrophages in SHR-derived splenocytes, their renal infiltration, and premature hypertension in SHR. These changes were associated with increased renal expression of MCP-1 (monocyte chemoattractant protein-1) and VLA-4 (very-late antigen-4). LLT1 (lectin-like transcript 1), the ligand for CD161a, was overexpressed in SHR kidney, whereas vascular cellular and intracellular adhesion molecules were similar to those in WKY. Inflammatory cytokines were elevated in SHR kidney and urine after nicotine infusion. Nicotine-mediated renal macrophage infiltration/inflammation was enhanced in denervated kidneys, not explained by angiotensin II levels or expression of angiotensin type-1/2 receptors. Moreover, expression of the anti-inflammatory α7-nAChR (α7-nicotinic acetylcholine receptor) was similar in young SHR and WKY rats. A novel, inherited nicotinic cholinergic inflammatory effect exists in young SHR, measured by expansion of CD161a+/CD68+ macrophages. This leads to renal inflammation and premature hypertension, which may be partially explained by increased renal expression of LLT-1, MCP-1, and VLA-4.

VLA-4 and CXCR4 expression levels show contrasting prognostic impact (favorable and unfavorable, respectively) in acute myeloid leukemia

Very late antigen-4 (VLA-4) and CXC chemokine receptor 4 (CXCR4) perform critical roles in the adhesion of hematopoietic and leukemic stem cells to marrow stromal cells. This mechanism is associated with chemoresistance in patients with acute myeloid leukemia (AML). Here, we measured VLA-4 and CXCR4 expressions in leukemic myeloblasts to determine their prognostic implications. Using multicolor flow cytometry, positive VLA-4 and CXCR4 expressions were measured in leukemic myeloblasts in bone marrow aspirates that were obtained from newly diagnosed adult AML patients (n = 98). VLA-4 expression was higher in patients at favorable or intermediate cytogenetic risk than in patients at poor risk (p < 0.001 and p = 0.002, respectively), but CXCR4 expression was not significantly different. Among the 72 non-promyelocytic leukemia patients analyzed who received cytarabine + anthracycline-based induction chemotherapy, high VLA-4 expression was independently associated with a high probability of complete remission (p = 0.019) and superior relapse-free survival (RFS) (p < 0.001). However, high CXCR4 expression independently increased the probability of relapse (p = 0.002) and was associated with a shorter RFS (p = 0.006). When categorizing patients into three groups according to VLA-4 and CXCR4 expression levels, the group of high VLA-4 and low CXCR4 showed longer RFS (p = 0.001) and overall survival (OS) (p = 0.011) than the group of low VLA-4 or high CXCR4.

Adhesion of monocytes to periodontal fibroblasts requires activation of NOD1/2- and TLR4-mediated LFA-1 and VLA-4

ObjectiveThe aim of this study was to investigate the roles of nucleotide-binding oligomerization domain-containing protein 1/2 (NOD1/2) and Toll-like receptor 4 (TLR4) in mediating the adhesion of monocytes to periodontal fibroblasts through leucocyte function-associated antigen-1 (LFA-1) and very late antigen-4 (VLA-4). DesignThe expression of NOD1, NOD2, and TLR4 was detected in the gingival tissue of patients with chronic periodontitis by immunohistochemistry. Then the adhesion of cells of human monocytic cell line U937 to human gingival fibroblasts (hGFs) and human periodontal ligament cells (hPDLCs) was investigated after U937 cells were treated with the agonists of NOD1, NOD2, and TLR4 for 24h, or transfected with small interfering RNAs (siRNAs) targeting NOD1, NOD2, and TLR4 for 48h. Meanwhile, the expression of LFA-1 and VLA-4 was examined in U937 cells through real-time polymerase chain reaction (PCR), Western blot, and flow cytometry. To confirm the roles of LFA-1 and VLA-4 involved in the process of adhesion, the adhesion blockade assay was performed using the corresponding blocking antibodies against these adhesion molecules. ResultsThe immunostaining results showed that NOD1, NOD2, and TLR4 were highly expressed in the gingival tissue of patients with periodontitis, especially in the monocyte-infiltrated area. The activation of these receptors by agonists upregulated the expression of LFA-1 and VLA-4 in U937 cells, and it increased the affinity of U937 cells to hGFs or hPDLCs. On the other hand, knockdown of these receptors by specific siRNAs resulted in the opposite results. In addition, blocking either LFA-1 or VLA-4 in U937 cells significantly attenuated the agonist-triggered adhesion of U937 to periodontal fibroblasts (P<0.001). ConclusionsThese results suggested that NOD1/2 and TLR4 mediated monocyte–periodontal fibroblast adhesion via the modulation of LFA-1 and VLA-4.

Innate IFN-γ promotes development of experimental autoimmune encephalomyelitis: a role for NK cells and M1 macrophages.

The role of IFN-γ in the pathogenesis of autoimmune diseases is controversial. Although Th1 cells can induce experimental autoimmune encephalomyelitis (EAE), IFN-γ can suppress Th17 cells that are pathogenic in EAE. Here we show that NK cells provide an early source of IFN-γ during development of EAE. Depletion of NK cells or neutralization of IFN-γ delayed the onset of EAE and was associated with reduced infiltration of IL-17(+) and GM-CSF(+) T cells into the CNS. In the passive transfer model, immune cells from myelin oligodendrocyte glycoprotein (MOG)-immunized IFN-γ(-/-) mice failed to induce EAE, despite producing IL-17 and GM-CSF. The macrophages expressed markers of M2 activation and the T cells had low very late antigen-4 (VLA-4) expression and failed to infiltrate the CNS. Addition of recombinant IFN-γ to immune cells from the IFN-γ(-/-) mice activated M1 macrophages and restored VLA-4 expression, migratory, and encephalitogenic activity of T cells. Furthermore, treatment of recipient mice with anti-VLA-4 neutralizing antibody abrogated EAE induced by transfer of T cells from WT mice. Our findings demonstrate IFN-γ-producing T cells are not required for development of EAE, but NK cell-derived IFN-γ has a key role in promoting M1 macrophage expansion and VLA-4-mediated migration of encephalitogenic T cells into the CNS.

Aberrant regulation of the integrin very late antigen-4 in systemic lupus erythematosus

Integrin very late antigen-4 (VLA4) is induced during inflammation and can regulate monocyte migration. It has been implicated in atherogenesis, a significant concern in systemic lupus erythematosus (SLE). The aim of this study was to define VLA4 expression in SLE monocytes. Flow cytometry, reverse transcription polymerase chain reaction, Western blotting, and immunohistochemistry staining with confocal microscopy were used to evaluate VLA4 expression in SLE patients and controls. We found elevated expression of VLA4 in SLE patients with significantly increased VLA4 staining intracellularly compared to control. Exposure of control monocytes to SLE sera or immune complexes led to increased intracellular expression, and immune complexes were capable of driving redistribution of surface VLA4 to the cytoplasm. Therefore, VLA4 was found to be subject to complex regulation with SLE sera driving both RNA expression and redistribution of protein. Stimulation of SLE monocytes with a VLA4 ligand induced significant TNFα expression, confirming a functional effect. This behavior may contribute to increased atherosclerosis and monocyte infiltrates in end organs.

The Role of ROS in Ionizing Radiation-Induced VLA-4 Mediated Adhesion of RAW264.7 Cells to VCAM-1 Under Flow Conditions

Alteration of adhesion molecule expression on endothelial cells has a direct connection with ionizing radiation-induced atherosclerosis, which is an adverse effect observed after radiotherapy. However, minimal attention has been given to monocytes/macrophages role in atherosclerosis development, which are exposed to the radiation at the same time. Under flow conditions using a parallel plate flow chamber to mimic physiological shear stress, we demonstrate here that the avidity between very late antigen-4 (VLA-4) of RAW264.7 cells and its ligand vascular cell adhesion molecule-1 (VCAM-1), was increased after low dose (0.5 Gy) irradiation, but was reduced after higher dose (5 Gy) treatment of ionizing radiation despite the fact that the surface expression of VLA-4 was up-regulated at 5 Gy of ionizing radiation. Treating the cells with free radical scavenger N-acetylcysteine had no effect on VLA-4 expression, but did reduce the avidity between RAW264.7 cells and VCAM-1 to a similar level, independent of ionizing radiation dose. The effect of H(2)O(2) treatment (from 1-100 μM) on RAW264.7 cell adhesion to VCAM-1 generated a similar bell-shaped graph as ionizing radiation. These results suggest that ionizing radiation regulates adhesive interactions between VLA-4 and VCAM-1, and that reactive oxygen species might function as a regulator, for this increased adhesiveness but with altered expression of integrin not play a major role.

Prognostic Significance of VLA4 and CD44 Expression by Flow Cytometry in Multiple Myeloma (MM)

AbstractAbstract 4975 Background:Our group has developed a peptide referred to as HYD1 which binds VLA-4 integrin/CD44 containing complex and induces necrotic cell death in myeloma cells (Nair et al Mol Cancer Ther 2009, Emmons et al, Mol Cancer Ther, 2011). Furthermore, we previously demonstrated in a small subset of MM patients that HYD1 was more active in specimens obtained from relapsed/refractory patients, a finding that correlated with VLA-4 expression. The frequency of expression and prognostic significance of two key surface markers VLA4 (very late antigen 4) and CD44 in patients with MM remains poorly defined. Methods:We retrospectively reviewed records of patients with MM who had a complete flow cytometry panel (which includes assessment for CD44 and VLA4) at Moffitt Cancer Center between 1/1/2004 and 12/31/2009. CD44 and VLA4 were gated by CD138 expression and we categorized expression as negative, dim, moderate or bright. We collected demographic information, disease related characteristics (including ISS stage, cytogenetics, and baseline laboratory testing) and treatment and outcomes related data (prior therapies, response to first line therapy, follow up and vital status). Responses were per IMWG criteria and for the purpose of this study, a partial remission or better qualified as a response to therapy. High risk cytogenetics (HRC) was defined by the presence of one of the following; 17p deletion, t(4;14), t(14;16) and 13q deletion by metaphase cytogenetics. The study was approved by the IRB at the University of South Florida. Results:A four color flow cytometry panel was available for review on 101 myeloma patients including 57 males, median age at diagnosis was 60 (range 39–83) years. The percentage of ISS stage I, II and III at diagnosis was 36. 4%, 34. 8% and 28. 8% respectively and 28. 7% of the patients had HRC. At the time of the flow panel, 32 patients (31. 7%) were untreated and overall patients had a median of 3 prior therapies (range 0–11). 52 patients (51. 5%), 37 (36. 6%) and 12 (11. 9%) had no, dim or moderate CD44 expression respectively while 26 (26. 3%), 68 (68. 7%) and 5 (5. 1%) had no, dim or moderate VLA4 expression respectively. Subsequently both were sub-grouped according to presence (dim or greater) or absence of expression. A correlation between the expression of CD44 and VLA4 was noted (Fisher’s exact p < 0. 001), 45 patients express both markers, 24 express neither markers, 28 express VLA4 but not CD44 while only 4 express CD44 and not VLA4 (p<0. 001). However neither CD44 nor VLA4 expression correlated with prior therapy (untreated versus previously treated), ISS stage, or poor risk cytogenetics (not shown). 76 patients (75. 2%) responded to first line therapy of which 50 expressed VLA4 (66%) while 25 patients did not respond to first line therapy, of which 23 expressed VLA4 (92%) suggesting VLA4 expression is a negative prognostic factor for response to therapy (p=0. 011). Alternatively CD44 did not correlate with response to first line therapy (not shown). The median OS for patients with VLA4 expression was 60. 5 months (95% CI 44. 9–74. 6) while it was 106. 8 months (95% CI 56. 3-Not reached) for patients who did not express VLA4 (log rank p=0. 023). For CD44 expressing patients, the median OS was 65. 8 months (95% CI 49. 3–86. 8) versus 98. 1 months (95% CI 48. 2–106. 8) for patients without CD44 expression (log rank p= 0. 817). On multivariable analysis, age at diagnosis (p=0. 02) and DS stage (p=0. 003) were the only predictors of poor survival while VLA4 expression (p=0. 09) had a trend towards poor survival. Conclusions:While total CD44 expression was not prognostic in this study, the expression of VLA4 was associated with a decreased response to therapy and decreased survival in multiple myeloma patients and validates this marker as a therapeutic target in myeloma. Although total CD44 expression did not correlate with prognosis in this study, expression of specific CD44 splice variants could be prognostic and this does not dismiss CD44 as a target for the novel therapeutic HYD-1 peptide. These studies are currently being pursued by our laboratory. Disclosures:Hazlehurst:Modulation Therapeutics Inc: Patents & Royalties, President and Co-founder Other. Emmons:Modulation Therapeutic Inc: Patents & Royalties.