CAR-T cell-mediated depletion of immunosuppressive tumor-associated macrophages promotes endogenous antitumor immunity and augments adoptive immunotherapy.

The immunosuppressive tumor microenvironment (TME) represents a major barrier for effective immunotherapy. Tumor-associated macrophages (TAMs) are highly heterogeneous and plastic cell components of the TME which can either promote tumor progression (M2-like) or boost antitumor immunity (M1-like). Here, we demonstrate that a subset of TAMs that express folate receptor ß (FRß) possess an immunosuppressive M2-like profile. In syngeneic tumor mouse models, chimeric antigen receptor (CAR)-T cell-mediated selective elimination of FRß+ TAMs in the TME results in an enrichment of pro-inflammatory monocytes, an influx of endogenous tumor-specific CD8+ T cells, delayed tumor progression, and prolonged survival. Preconditioning of the TME with FRß-specific CAR-T cells also improves the effectiveness of tumor-directed anti-mesothelin CAR-T cells, while simultaneous co-administration of both CAR products does not. These results highlight the pro-tumor role of FRß+ TAMs in the TME and the therapeutic implications of TAM-depleting agents as preparative adjuncts to conventional immunotherapies that directly target tumor antigens.

Folate Receptor ß (FRß) Expression in Tissue-Resident and Tumor-Associated Macrophages Associates with and Depends on the Expression of PU.1.

As macrophages exhibit a huge functional plasticity under homeostasis and pathological conditions, they have become a therapeutic target for chronic inflammatory diseases. Hence, the identification of macrophage subset-specific markers is a requisite for the development of macrophage-directed therapeutic interventions. In this regard, the macrophage-specific Folate Receptor ß (FRß, encoded by the FOLR2 gene) has been already validated as a target for molecular delivery in cancer as well as in macrophage-targeting therapeutic strategies for chronic inflammatory pathologies. We now show that the transcriptome of human macrophages from healthy and inflamed tissues (tumor; rheumatoid arthritis, RA) share a significant over-representation of the "anti-inflammatory gene set", which defines the gene profile of M-CSF-dependent IL-10-producing human macrophages (M-MØ). More specifically, FOLR2 expression has been found to strongly correlate with the expression of M-MØ-specific genes in tissue-resident macrophages, tumor-associated macrophages (TAM) and macrophages from inflamed synovium, and also correlates with the presence of the PU.1 transcription factor. In fact, PU.1-binding elements are found upstream of the first exon of FOLR2 and most M-MØ-specific- and TAM-specific genes. The functional relevance of PU.1 binding was demonstrated through analysis of the proximal regulatory region of the FOLR2 gene, whose activity was dependent on a cluster of PU.1-binding sequences. Further, siRNA-mediated knockdown established the importance of PU.1 for FOLR2 gene expression in myeloid cells. Therefore, we provide evidence that FRß marks tissue-resident macrophages as well as macrophages within inflamed tissues, and its expression is dependent on PU.1.

Serum-soluble folate receptor ß as a biomarker for the activity of rheumatoid arthritis synovitis and the response to anti-TNF agents.

This study aims to develop a sandwich ELISA system for the measurement of soluble folate receptor ß (sFRß) and evaluate whether base line levels of serum sFRß are a biomarker for the activity of RA synovitis and the response to anti-TNF agents. Serum sFRß from normal controls (41 samples), patients with OA (29 samples), and patients with RA (27 samples) and synovial fluid sFRß from patients with RA (17 samples) were measured by sandwich ELISA, using anti-FRαß and anti-FRß antibodies as capture and detection antibodies, respectively. Baseline levels of serum sFRß before therapy were evaluated in relation with DAS28-CRP or CRP and response to anti-TNF agents at 3-month follow-up. sFRß levels in RA synovial fluids were higher than those in RA sera, and sFRß levels in RA sera were higher than those in osteoarthritis and normal control sera. A significant relationship was observed between serum sFRß levels and the DAS28-CRP scores or CRP values. The area under curve (AUC) values for receiver-operating characteristic curves defined using the serum sFRß levels of RA patients before therapy had a higher predictive capacity than DAS28-CRP and CRP for the effective response of anti-TNF agents. The high serum sFRß levels with a cutoff value of 8 ng/mL were 100% specificity for the effective response of anti-TNF agents. The findings support that the serum sFRß levels in patients with RA act as a disease activation biomarker and that high serum sFRß levels act as a predictive biomarker for the response to anti-TNF agents.

Extracellular Purine Metabolism Is the Switchboard of Immunosuppressive Macrophages and a Novel Target to Treat Diseases With Macrophage Imbalances.

If misregulated, macrophage (Mϕ)-T cell interactions can drive chronic inflammation thereby causing diseases, such as rheumatoid arthritis (RA). We report that in a proinflammatory environment, granulocyte-Mϕ (GM-CSF)- and Mϕ colony-stimulating factor (M-CSF)-dependent Mϕs have dichotomous effects on T cell activity. While GM-CSF-dependent Mϕs show a highly stimulatory activity typical for M1 Mϕs, M-CSF-dependent Mϕs, marked by folate receptor ß (FRß), adopt an immunosuppressive M2 phenotype. We find the latter to be caused by the purinergic pathway that directs release of extracellular ATP and its conversion to immunosuppressive adenosine by co-expressed CD39 and CD73. Since we observed a misbalance between immunosuppressive and immunostimulatory Mϕs in human and murine arthritic joints, we devised a new strategy for RA treatment based on targeted delivery of a novel methotrexate (MTX) formulation to the immunosuppressive FRß+CD39+CD73+ Mϕs, which boosts adenosine production and curtails the dominance of proinflammatory Mϕs. In contrast to untargeted MTX, this approach leads to potent alleviation of inflammation in the murine arthritis model. In conclusion, we define the Mϕ extracellular purine metabolism as a novel checkpoint in Mϕ cell fate decision-making and an attractive target to control pathological Mϕs in immune-mediated diseases.

Prophylactic and therapeutic activity of alkaline phosphatase in arthritic rats: single-agent effects of alkaline phosphatase and synergistic effects in combination with methotrexate.

Alkaline phosphatase (AP) is a gate-keeper of innate immune system responses by detoxifying inflammation triggering moieties released from endogenous and external sources. We examined whether AP's broad mechanism of action constitutes a safe therapeutic, either as single agent or combined with methotrexate (MTX), for chronic inflammatory disorders, for example, rheumatoid arthritis (RA). A rat model for RA was used with repeated intra-articular methylated bovine serum albumin (mBSA) injections in 1 knee ("arthritic" knee), with the contralateral knee serving as internal control. AP (200 µg, subcut) was administered before mBSA injections (prophylactic setting) or after arthritis induction (therapeutic setting) or combined with MTX (0.3 mg/kg or 1 mg/kg; intraperitoneally). As end point of treatment outcome, macrophage infiltration in knees, liver, and spleen was assessed by immunohistochemistry (ED1 and ED2 expression), immunofluoresence (macrophage marker folate receptor-ß [FRß]), and [18F]fluoro-polyethylene glycol-folate positron emission tomography (PET) (macrophage imaging) and ex vivo tissue distribution. Single-agent AP treatment and combinations with MTX were well tolerated. Both prophylactic and therapeutic AP markedly reduced synovial macrophage infiltration in arthritic knees (ED1: 3.5- to 4-fold; ED2: 3.5- to 6-fold), comparable with MTX treatment. AP-MTX combinations slightly improved on single agent effects. PET monitoring and ex vivo tissue distribution studies corroborated the impact of AP, MTX, and AP-MTX on reducing synovial macrophage infiltration. Beyond localized articular effects, AP also revealed systemic anti-inflammatory effects by a 2-fold reduction of ED1, ED2, and FRß+ macrophages in liver and spleen of arthritic rats. Collectively, single-agent AP and AP combined with MTX elicited local and systemic anti-arthritic activity in arthritic rats.

Imaging and Methotrexate Response Monitoring of Systemic Inflammation in Arthritic Rats Employing the Macrophage PET Tracer [18F]Fluoro-PEG-Folate.

Background: In rheumatoid arthritis, articular inflammation is a hallmark of disease, while the involvement of extra-articular tissues is less well defined. Here, we examined the feasibility of PET imaging with the macrophage tracer [18F]fluoro-PEG-folate, targeting folate receptor ß (FRß), to monitor systemic inflammatory disease in liver and spleen of arthritic rats before and after methotrexate (MTX) treatment. Methods: [18F]Fluoro-PEG-folate PET scans (60 min) were acquired in saline- and MTX-treated (1 mg/kg, 4x) arthritic rats, followed by tissue resection and radiotracer distribution analysis. Liver and spleen tissues were stained for ED1/ED2-macrophage markers and FRß expression. Results: [18F]Fluoro-PEG-folate PET and ex vivo tissue distribution studies revealed a significant (p < 0.01) 2-fold lower tracer uptake in both liver and spleen of MTX-treated arthritic rats. Consistently, ED1- and ED2-positive macrophages were significantly (p < 0.01) decreased in liver (4-fold) and spleen (3-fold) of MTX-treated compared with saline-treated rats. Additionally, FRß-positive macrophages were also significantly reduced in liver (5-fold, p < 0.005) and spleen (3-fold, p < 0.01) of MTX- versus saline-treated rats. Conclusions: MTX treatment reduced activated macrophages in liver and spleen, as markers for systemic inflammation in these organs. Macrophage PET imaging with [18F]fluoro-PEG-folate holds promise for detection of systemic inflammation in RA as well as therapy (MTX) response monitoring.

Activation of interleukin-6 and -8 expressions by methylmercury in human U937 macrophages involves RelA and p50.

The accumulation of macrophages has been observed around lesions of the brain in patients with Minamata disease. In this condition, mercury has been detected histochemically in macrophages throughout the brain. However, the role of macrophages in the neurotoxicity of methylmercury (MeHg) and the molecular mechanisms of their response to MeHg exposure remain to be elucidated. Here, we investigated how MeHg affects the expression of proinflammatory cytokines such as interleukin (IL)-6 and IL-8 in cultured human U937 macrophages. Compared with controls, IL-6 and IL-8 mRNA expression was maximally induced in U937 macrophages after treatment with 10 µM MeHg for 6 h. The protein secretion of IL-6 and IL-8 was significantly stimulated by MeHg in U937 macrophages. Results from luciferase reporter assay indicated functional activation of nuclear factor kappa B and the involvement of subunit RelA and p50 in MeHg-induced IL-6 and IL-8 activation, which was confirmed by siRNA knockdown experiments. MeHg exposure at 4 µM also significantly induced IL-8 expression in U-87 MG cells at mRNA and protein level, indicating that IL-8 induction might be a general mode of action of MeHg treatment among different cell types. These results indicate a possible involvement of an early inflammatory response, including IL-6 and IL-8 expression in the pathogenesis of MeHg. N-acetyl-l-cysteine suppressed MeHg-induced activation of IL-6 and IL-8 mRNA expression in U937 macrophages, indicating the effectiveness of N-acetyl-l-cysteine as a therapeutic drug in MeHg-induced inflammation. Copyright © 2016 John Wiley & Sons, Ltd.

Production of a High-affinity Monoclonal Antibody Reactive with Folate Receptors Alpha and Beta.

Folate receptors α (FRα) and ß (FRß) are two isoforms of the cell surface glycoprotein that binds folate. The expression of FRα is rare in normal cells and elevated in cancer cells. Thus, FRα-based tumor-targeted therapy has been a focus area of laboratory research and clinical trials. Recently, it was shown that a significant fraction of tumor-associated macrophages expresses FRß and that these cells can enhance tumor growth. Although FRα and FRß share 70% identity in their deduced amino acid sequence, a monoclonal antibody (MAb) reactive with both receptors has not been developed. A MAb that can target both FRα-expressing cancer cells and FRß-expressing tumor-associated macrophages may provide a more potent therapeutic tool for cancer than individual anti-FRα or anti-FRß MAbs. In this study, we developed a MAb that recognizes both FRα and FRß (anti-FRαß). The anti-FRαß specifically stained trophoblasts and macrophages from human placenta, synovial macrophages from rheumatoid arthritis patient, liver macrophages from cynomolgus monkey and common marmoset, and cancer cells and tumor-associated macrophages from ovary and lung carcinomas. Surface plasmon resonance showed that the anti-FRαß bound to soluble forms of the FRα and FRß proteins with high affinity (KD=6.26×10(-9) M and 4.33×10(-9) M, respectively). In vitro functional analysis of the anti-FRαß showed that this MAb mediates complement-dependent cytotoxicity, antibody-dependent cellular cytotoxicity, and antibody-dependent cellular phagocytosis of FRα-expressing and FRß-expressing cell lines. The anti-FRαß MAb is a promising therapeutic candidate for cancers in which macrophages promote tumor progression.

Depletion of folate receptor ß-expressing macrophages alleviates bleomycin-induced experimental skin fibrosis.

OBJECTIVES: Folate receptor ß (FRß)-expressing macrophages have been identified as activated macrophages. Here, we investigated the infiltration of FRß-expressing macrophages in a murine model of bleomycin (BLM)-induced skin fibrosis and assessed the antifibrotic effects of depletion of FRß-expressing macrophages in this model using a recombinant immunotoxin to FRß. METHODS: A recombinant immunotoxin (anti-FRß-PE38) was prepared by conjugating the Fv portion of the anti-mouse FRß heavy chain with truncated Pseudomonas exotoxin A (VH-PE38) and the Fv portion of the anti-mouse FRß light chain. BLM-induced skin fibrosis mice were intravenously treated with either anti-FRß-PE38 or VH-PE38 as a control protein. Skin fibrosis was evaluated by the change of skin thickness and hydroxyproline content on Day 29. The TGFß1 mRNA levels in the treated skin were assessed by quantitative real-time RT-PCR on Day 9. RESULTS: Numbers of FRß-expressing macrophages increased in BLM-injected skin. Anti-FRß-PE38 treatment led to a dramatic reduction in the number of FRß-expressing macrophages. Additionally, skin thickness and hydroxyproline content, were markedly reduced. TGFß1 mRNA levels were also down-regulated after the treatment. TGFß1 expression was enriched in FRß-expressing macrophages compared with FRß-negative macrophages. CONCLUSION: These results indicated that anti-FRß-PE38 treatment efficiently depleted FRß-expressing macrophages and consequently alleviated BLM-induced skin fibrosis.

Macrophage uptake and accumulation of folates are polarization-dependent in vitro and in vivo and are regulated by activin A.

Vitamin B9, commonly known as folate, is an essential cofactor for one-carbon metabolism that enters cells through three major specialized transporter molecules (RFC, FR, and PCFT), which differ in expression pattern, affinity for substrate, and ligand-binding pH dependency. We now report that the expression of the folate transporters differs between macrophage subtypes and explains the higher accumulation of 5-MTHF-the major folate form found in serum-in M2 macrophages in vitro and in vivo. M1 macrophages display a higher expression of RFC, whereas FRß and PCFT are preferentially expressed by anti-inflammatory and homeostatic M2 macrophages. These differences are also seen in macrophages from normal tissues involved in folate transit (placenta, liver, colon) and inflamed tissues (ulcerative colitis, RA), as M2-like macrophages from normal tissues express FRß and PCFT, whereas TNF-α-expressing M1 macrophages from inflamed tissues are RFC+. Besides, we provide evidences that activin A is a critical factor controlling the set of folate transporters in macrophages, as it down-regulates FRß, up-regulates RFC expression, and modulates 5-MTHF uptake. All of these experiments support the notion that folate handling is dependent on the stage of macrophage polarization.

Novel Therapy for Atherosclerosis Using Recombinant Immunotoxin Against Folate Receptor ß-Expressing Macrophages.

BACKGROUND: Folate receptor ß (FRß) is induced during macrophage activation. A recombinant immunotoxin consisting of the truncated Pseudomonas exotoxin A (PE38) conjugated to an anti-FRß antibody (anti-FRß-PE38) has been reported to kill activated macrophages in inflammatory diseases. To elucidate the effect of an immunotoxin targeting FRß on atherosclerosis, we determined the presence of FRß-expressing macrophages in atherosclerotic lesions and administered the FRß immunotoxin in apolipoprotein E-deficient mice. METHODS AND RESULTS: The FRß-expressing macrophages were observed in atherosclerotic lesions of apolipoprotein E-deficient mice. At 15 or 35 weeks of age, the apolipoprotein E-deficient mice were divided into 3 groups and were intravenously administered 0.1 mg/kg of anti-FRß-PE38 (immunotoxin group), 0.1 mg/kg of PE38 (toxin group), or 0.1 mL of saline (control group) every 3 days, for a total of 5 times for each age group. The mice were analyzed at 21 or 41 weeks of age. Treatment with the immunotoxin resulted in 31% and 22% reductions in atherosclerotic lesions of the 21- and 41-week-old mice, respectively (P<0.05). Administration of immunotoxin reduced the numbers of FRß- and tumor necrosis factor-α-expressing macrophages, reduced cell proliferation, and increased the number of apoptotic cells (P<0.05). Real-time polymerase chain reaction demonstrated that the expression of FRß and tumor necrosis factor-α mRNA was significantly decreased in the immunotoxin group (P<0.05). CONCLUSIONS: These results suggest that FRß-expressing macrophages exist in the atherosclerotic lesions of apolipoprotein E-deficient mice and that FRß immunotoxin administration reduces the progression of atherosclerotic lesions in younger and older individuals. The recombinant FRß immunotoxin targeting activated macrophages could provide a novel therapeutic tool for atherosclerosis. (J Am Heart Assoc. 2012;1:e003079 doi: 10.1161/JAHA.112.003079.).

The prolyl hydroxylase PHD3 identifies proinflammatory macrophages and its expression is regulated by activin A.

Modulation of macrophage polarization underlies the onset and resolution of inflammatory processes, with polarization-specific molecules being actively sought as potential diagnostic and therapeutic tools. Based on their cytokine profile upon exposure to pathogenic stimuli, human monocyte-derived macrophages generated in the presence of GM-CSF or M-CSF are considered as proinflammatory (M1) or anti-inflammatory (M2) macrophages, respectively. We report in this study that the prolyl hydroxylase PHD3-encoding EGLN3 gene is specifically expressed by in vitro-generated proinflammatory M1(GM-CSF) human macrophages at the mRNA and protein level. Immunohistochemical analysis revealed the expression of PHD3 in CD163(+) lung macrophages under basal homeostatic conditions, whereas PHD3(+) macrophages were abundantly found in tissues undergoing inflammatory responses (e.g., Crohn's disease and ulcerative colitis) and in tumors. In the case of melanoma, PHD3 expression marked a subset of tumor-associated macrophages that exhibit a weak (e.g., CD163) or absent (e.g., FOLR2) expression of typical M2-polarization markers. EGLN3 gene expression in proinflammatory M1(GM-CSF) macrophages was found to be activin A dependent and could be prevented in the presence of an anti-activin A-blocking Ab or inhibitors of activin receptor-like kinase receptors. Moreover, EGLN3 gene expression was upregulated in response to hypoxia only in M2(M-CSF) macrophages, and the hypoxia-mediated upregulation of EGLN3 expression was significantly impaired by activin A neutralization. These results indicate that EGLN3 gene expression in macrophages is dependent on activin A both under basal and hypoxic conditions and that the expression of the EGLN3-encoded PHD3 prolyl hydroxylase identifies proinflammatory macrophages in vivo and in vitro.

Development of ultra-super sensitive immunohistochemistry and its application to the etiological study of adult T-cell leukemia/lymphoma.

Antigen retrieval (AR) and ultra-super sensitive immunohistochemistry (ultra-IHC) have been established for application to archival human pathology specimens. The original ultra-IHC was the ImmunoMax method or the catalyzed signal amplification system (ImmunoMax/CSA method), comprising the streptavidin-biotin complex (sABC) method and catalyzed reporter deposition (CARD) reaction with visualization of its deposition. By introducing procedures to diminish non-specific staining in the original ultra-IHC method, we developed the modified ImmunoMax/CSA method with AR heating sections in an AR solution (heating-AR). The heating-AR and modified ImmunoMax/CSA method visualized expression of the predominantly simple present form of HTLV-1 proviral DNA pX region p40Tax protein (Tax) in adult T-cell leukemia/lymphoma (ATLL) cells in archival pathology specimens in approximately 75% of cases. The simple present form of Tax detected exhibited a close relation with ATLL cell proliferation. We also established a new simplified CSA (nsCSA) system by replacing the sABC method with the secondary antibody- and horse radish peroxidase-labeled polymer reagent method, introducing the pretreatments blocking non-specific binding of secondary antibody reagent, and diminishing the diffusion of deposition in the CARD reaction. Combined with AR treating sections with proteinase K solution (enzymatic-AR), the nsCSA system visualized granular immunostaining of the complex present form of Tax in a small number of ATLL cells in most cases, presenting the possibility of etiological pathological diagnosis of ATLL and suggesting that the complex present form of Tax-positive ATLL cells were young cells derived from ATLL stem cells. The heating-AR and ultra-IHC detected physiological expression of the p53 protein and its probable phosphorylation by Tax in peripheral blood mononuclear cells of peripheral blood tissue specimens from HTLV-1 carriers, as well as physiological and pathological expression of the molecules involved with G1 phase progression and G1-S phase transition (E2F-1, E2F-4, DP-1, and cyclin E) in ATLL and peripheral T-cell lymphoma cells. The ultra-IHC with AR is useful for etiological pathological diagnosis of ATLL since HTLV-1 pathogenicity depends on that of Tax, and can be a useful tool for studies translating advanced molecular biology and pathology to human pathology.

Efficacy of an immunotoxin to folate receptor beta in the intra-articular treatment of antigen-induced arthritis.

INTRODUCTION: We previously demonstrated that synovial sublining macrophages express folate receptor beta (FRß). The aim of this study was to evaluate the efficacy of intra-articular administration of a recombinant immunotoxin to FRß for treating rat antigen-induced arthritis. METHODS: A monoclonal antibody (mAb) to rat FRß was produced by immunizing mice with B300-19 cells (murine pre-B cells) transfected with the rat FRß gene. Recombinant immunotoxin was prepared by conjugating the Fv portion of the anti-rat FRß mAb heavy chain with a truncated Pseudomonas exotoxin A and the Fv portion of the anti-rat FRß mAb light chain. Antigen-induced arthritis was induced through intra-articular injection of methylated bovine serum albumin (mBSA) after two subcutaneous injections of mBSA and complete Freund's adjuvant. Immunotoxin was intra-articularly injected into the arthritis joint every other day for seven days after arthritis onset. Joint swelling was measured and histological scores of inflammation, synovial thickness, cartilage, and bone destruction were determined. Immunohistochemistry was performed to detect osteoclast and osteoclast precursor FRß-expressing macrophages and cathepsin K-positive cells on day 21. RESULTS: Intra-articular administration of the immunotoxin attenuated joint swelling (61% suppression; P < 0.01 compared to the control on day 21) and improved histological findings, particularly cartilage and bone destruction (scores of rats treated with control versus the immunotoxin: 2.2 versus 0.5; P < 0.01), by reducing the number of FRß-expressing macrophages and cathepsin K-positive cells. CONCLUSIONS: Intra-articular administration of an immunotoxin to FRß is effective for improving rat antigen-induced arthritis.

Clinical significance of folate receptor ß-expressing tumor-associated macrophages in pancreatic cancer.

PURPOSE: To examine the appearance and distribution of folate receptor ß-expressing (FRß+) macrophages in the pancreatic tumor microenvironment and their relationship to metastasis and prognosis in pancreatic cancer patients. METHODS: Tumor samples were obtained from 76 patients with pancreatic cancer who underwent curative resection. None of these patients had received any preoperative chemotherapy or radiotherapy. Both FRß+ and tumor-infiltrating (CD68+) macrophages were examined in each tumor specimen by immunohistochemical and immunofluorescence staining using a newly developed anti-human FRß monoclonal antibody and CD68 antibody. The appearance, distribution, expression of vascular endothelial growth factor (VEGF) on FRß-expressing or CD68+ macrophages, and tumor microvessel density (MVD) were assessed. Log rank test and Cox proportional hazard regression were used to investigate the associations among CD68+ or FRß+ macrophages, clinicopathologic factors, and overall survival. RESULTS: FRß+ macrophages were prominent in the perivascular regions of the tumor-invasive front and a specific subset with VEGF expression in the CD68+ macrophages. A high number of FRß+ macrophages showed a positive association with high MVD, a high incidence of hematogenous metastasis, and a poor prognosis in pancreatic cancer patients. CONCLUSIONS: FRß+ macrophages are a novel subset of tumor-associated macrophages in pancreatic cancer and may play an important role in the tumor microenvironment in association with systemic metastasis through the interaction with tumor cells and vessels. FRß+ macrophages may be promising a targeting therapy for pancreatic cancer.

Immunohistochemistry of programmed cell death in archival human pathology specimens.

Immunohistochemistry (IHC) for detecting key signal molecules involved in programmed cell death (PCD) in archival human pathology specimens is fairly well established. Detection of cleaved caspase-3 in lymphocytes in rheumatoid arthritis (RA) and gastric surface foveolar glandular epithelia but not in synoviocytes in RA, gastric fundic glandular epithelia, or nasal NK/T-cell lymphoma (NKTCL) cells suggests anti-apoptotic mechanisms in cell differentiation and in oncogenesis such as the induction of survivin. Enzymatically pretreated and ultra-super sensitive detection of beclin-1 in synoviocytes in RA and gastric fundic glandular epithelia suggests enhanced autophagy. The deposition of beclin-1 in fibrinoid necrosis in RA and expression of beclin-1 in detached gastric fundic glandular cells suggest that enhanced autophagy undergoes autophagic cell death (ACD). NKTCL exhibited enhanced autophagy through LC3 labeling and showed densely LC3 labeled cell-debris in regions of peculiar necrosis without deposition of beclin-1, indicating massive ACD in NKTCL and the alternative pathway enhancing autophagy following autophagic vesicle nucleation. Autophagy progression was monitored by labeling aggregated mitochondria and cathepsin D. The cell-debris in massive ACD in NKTCL were positive for 8-hydroxydeoxyguanosine, suggesting DNA oxidation occurred in ACD. Immunohistochemical autophagy and PCD analysis in archival human pathology specimens may offer new insights into autophagy in humans.

Phenotypic and functional profiles of CRIg (Z39Ig)-expressing macrophages in the large intestine.

Intestinal macrophages (Mφ) play significant roles in maintaining homeostasis by the efficient elimination of foreign particles in the large intestine. However, functional complement receptors have not been fully identified. In this study, we showed that a complement receptor of the Ig superfamily (CRIg, also known as Z39Ig), a receptor for complement fragments (C3b and iC3b), was expressed on a subset of intestinal M in murine and human large intestine. When abilities of uptake of antigens of murine CRIg(+) Mφ were examined, intestinal CRIg(+) Mφ displayed less endocytic and similar phagocytic abilities compared to resident peritoneal F4/80(+)CRIg(-) Mφ and F4/80(+)CRIg(+) Mφ. Additionally, we found that a significant portion of C3b-dependent phagocytosis by large intestinal M involves CRIg, emphasizing the importance of efficient mechanisms to eliminate foreign particles in the large intestine. On the other hand, intestinal Mφ from 2,4,6-trinitrobenzene sulfonic acid-treated mice had decreased CRIg expression but increased CD11b expression, implying some contribution to the removal of immune complexes. This study will shed new light on opsonization and phagocytosis by large intestinal Mφ.

Enhanced Autophagy and Reduced Expression of Cathepsin D Are Related to Autophagic Cell Death in Epstein-Barr Virus-Associated Nasal Natural Killer/T-Cell Lymphomas: An Immunohistochemical Analysis of Beclin-1, LC3, Mitochondria (AE-1), and Cathepsin D in Nasopharyngeal Lymphomas.

This study investigated autophagy in 37 cases of nasopharyngeal lymphomas including 23 nasal natural killer (NK)/T-cell lymphomas (NKTCL), 3 cytotoxic T-cell lymphomas (cytotoxic-TML) and 9 B-cell lymphomas (BML) by means of antigen-retrieval immunohistochemistry of beclin-1, LC3, mitochondria (AE-1) and cathepsin D. Peculiar necrosis was noted in EBV(+) lymphomas comprising 21 NKTCL, 2 cytotoxic-TML and 1 BML. Lymphomas without peculiar necrosis showed high expression of beclin-1, macrogranular cytoplasmal stain of LC3 with sporadic nuclear stain, a hallmark of autophagic cell death (ACD), some aggregated mitochondria and high expression of cathepsin D, suggesting a state of growth with enhanced autophagy with sporadic ACD. EBV(+) NKTCL with the peculiar necrosis, showed significantly low level of macrogranular staining of LC3, aggregated mitochondria and low expression of cathepsin D in the cellular areas when degenerative lymphoma cells showed decreased beclin-1, significantly advanced LC3-labeled autophagy, residual aggregated mitochondria and significantly reduced expression of cathepsin D, suggesting advanced autophagy with regional ACD. Consequently it was suggested that enhanced autophagy and reduced expression of lysosomal enzymes induced regional ACD under EBV infection in NKTCL.

Possible role for external environmental stimuli in nasopharyngeal NK/T-cell lymphomas in the northeast of China with EBV infection-related autophagic cell death: a pathoepidemiological analysis.

This study examined the relationship between external environmental factors and Epstein-Barr virus (EBV) infection in nasal natural killer (NK)/T-cell lymphomagenesis. Archival paraffin sections from 134 cases of nasopharyngeal lymphomas in the northeast of China were investigated by in situ hybridization of EBV-encoded small RNA-1 (EBER-1) and by immunohistochemistry of the status of programmed cell death (PCD). The cases examined included 74 (55.2%) cases of NK/T-cell lymphomas (NKTCL) in T-cell and NK-cell neoplasms as well as 32 (23.9%) cases of B-cell neoplasms (B-MLs) and 9 (6.7%) cases of carcinomas. These cases indicated a significant dominant occurrence of NKTCL in the nasal cavity and of B-MLs in the pharynx. Many EBV-associated NKTCLs were seen in the nasopharynx, all three cases of EBV-associated B-MLs were in the nasal cavity and all three cases of EBV-associated carcinomas were only seen in the pharynx. The low number of NKTCL cases showing little or no EBV association, together with the existence of EBER-1-free lymphoma cells in EBV-associated NKTCLs, suggested EBV-related lymphoma cell expansion during lymphomagenesis. Peculiar necrosis, frequently observed in NKTCLs, was due to accelerated PCD. This PCD was autophagic cell death as judged by labeling of Beclin-1 and LC3, which possibly occurred due to EBV infection, when apoptosis was suppressed by survivin. Very minute squamous carcinomas, observed in 10 of 23 cases of NKTCLs with residual epithelia that were survivin-positive but not EBV-associated suggested that carcinogenesis occurred before lymphomagenesis. These data suggest that external environmental oncogenic factors initiate nasopharyngeal carcinomas and lymphomas whereas EBV infection promotes them.

Expression of endothelia and lymphocyte adhesion molecules in bronchus-associated lymphoid tissue (BALT) in adult human lung.

BACKGROUND: Bronchus-associated lymphoid tissue (BALT) is the secondary lymphoid tissue in bronchial mucosa and is involved in the development of bronchopulmonary immune responses. Although migration of lymphocytes from blood vessels into secondary lymphoid tissues is critical for the development of appropriate adaptive immunity, the endothelia and lymphocyte adhesion molecules that recruit specific subsets of lymphocytes into human BALT are not known. The aim of this study was to determine which adhesion molecules are expressed on lymphocytes and high endothelial venules (HEVs) in human BALT. METHODS: We immunostained frozen sections of BALT from lobectomy specimens from 17 patients with lung carcinoma with a panel of monoclonal antibodies to endothelia and lymphocyte adhesion molecules. RESULTS: Sections of BALT showed B cell follicles surrounded by T cells. Most BALT CD4+ T cells had a CD45RO+ memory phenotype. Almost all BALT B cells expressed alpha4 integrin and L-selectin. In contrast, 43% of BALT T cells expressed alpha4 integrin and 20% of BALT T cells expressed L-selectin. Almost all BALT lymphocytes expressed LFA-1. HEVs, which support the migration of lymphocytes from the bloodstream into secondary lymphoid tissues, were prominent in BALT. All HEVs expressed peripheral node addressin, most HEVs expressed vascular cell adhesion molecule-1, and no HEVs expressed mucosal addressin cell adhesion molecule-1. CONCLUSION: Human BALT expresses endothelia and lymphocyte adhesion molecules that may be important in recruiting naive and memory/effector lymphocytes to BALT during protective and pathologic bronchopulmonary immune responses.