Neutrophil Cytotoxicity Research Articles

Abstract P5-01-07: Select neutrophils inhibit breast cancer metastases in patients via chemokine-mediated mechanisms

Abstract Background: In both breast cancer patients and healthy women, we have previously demonstrated that select neutrophils found in breast cancer patients as opposed to healthy women are cytotoxic to breast cancer cell lines. (Granot Z et al. Cancer Cell. 2010) This work stemmed from our prior research in murine breast cancer models indicating that primary breast tumors can mobilize select neutrophils, termed Tumor Entrained Neutrophils (“TENS”); these entrained neutrophils have the unique capacity to inhibit metastatic seeding in the lung through cell-kill mechanisms. (Granot Z et al. Cancer Cell. 2010) In this study, we evaluated the relationship between select chemokines and neutrophil cytotoxicity in breast cancer patients versus healthy volunteers. Methods: Neutrophils were purified from the blood of 75 randomly selected newly diagnosed pre-operative breast cancer patients without evidence of metastatic disease, and 47 healthy female volunteers with no history of cancer. Cytotoxicity was evaluated by incubating neutrophils with luciferase labeled MDA-MB-231 cells. Luciferase activity was measured as a reflection of% cytotoxicity. Serum was also isolated from breast cancer patients and healthy volunteers. Based on prior experiments, we used the Millipore® Milliplex Human Cytokine Plex Kit to evaluate Il-1Ra, MCP-1 and TNFa in our serum samples in 50/75 of the cancer patients and 25/47 controls. We used multiple linear regression to develop a model to predict cytotoxicity as a function of the chemokines. Results: In comparison to healthy volunteers whose mean neutrophil cytotoxicity to MDA-MB-231 cells was 6.5%, pre-operative breast cancer patients demonstrated a mean neutrophil cytotoxicity of 12.7%, p<0.0001. We then evaluated the serum chemokine levels of 50 of the breast cancer patients; 31/50 had high neutrophil cytotoxicity (>10%), and 19/50 had low neutrophil cytotoxicity (<10%). We compared the serum chemokine levels in the patients to the levels found in the 25 controls. Using a multiple linear regression model, we found that the levels of these three chemokines are associated with cytotoxicity (R2 = 0.126, p = 0.022). An ANOVA decomposition of the model suggested that Il.1RA was the most predictive (p = 0.018) followed by MCP.1 (p = 0.088) and TNF.alpha (p = 0.245). Conclusion: Our work demonstrates the cytotoxic role of select neutrophils in the peripheral blood of breast cancer patients as contrasted with neutrophils from healthy women. We further demonstrate that select chemokines appear to be correlated with neutrophil cytotoxicity. We are currently evaluating the prognostic and therapeutic roles of cytotoxic neutrophils and their related chemokines in breast cancer patients. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-01-07.

The cytotoxicity of select neutrophils in breast cancer patients and the role of chemokines in neutrophil cytotoxicity.

e22148 Background: Recent research in murine breast cancer models demonstrates that primary breast tumors can mobilize select neutrophils, which have the unique capacity to inhibit metastatic seeding in the lung through cell-kill mechanisms. (Granot Z et al. Cancer Cell. 2010). In both breast cancer patients and healthy women, we investigated whether neutrophils were cytotoxic to breast cancer cell lines. We also assessed whether serum chemokines were associated with neutrophil cytotoxicity. Methods: Neutrophils were purified from the blood of 59 randomly selected newly diagnosed pre-operative breast cancer patients without evidence of metastatic disease, 50 metastatic breast cancer patients, 15 DCIS patients, and 45 healthy female volunteers with no history of cancer. Cytotoxicity was evaluated by incubating neutrophils with luciferase labeled MDA-MB-231 cells. Luciferase activity was measured as a reflection of % cytotoxicity. Serum was also isolated from breast cancer patients and healthy volunteers. Millipore Milliplex Human Cytokine Plex Kit was used to assess the concentration of 42 different chemokines in each of the serum samples. Results: In comparison to healthy volunteers whose mean neutrophil cytotoxicity to MDA-MB-231 cells was 6.5%, pre-operative breast cancer patients demonstrated a mean neutrophil cytotoxicity of 13.4%, p<0.0001, metastatic patients mean neutrophil cytotoxicity was 19.4%, p <0.003 and DCIS patients had a mean neutrophil cytotoxicity of 11.7%. p <0.01. By Kruskal-Wallist test, the relative concentrations of three chemokines, Il.1a, MCP.1 and TNF-a, were found to have a statistically significant association with neutrophil cytotoxicity (p-value: 0.005, 0.002, 0.0005 respectively). Conclusions: Our work demonstrates the cytotoxic role of select neutrophils in the peripheral blood of breast cancer patients as contrasted with neutrophils from healthy women. We further demonstrate that select chemokines appear to be correlated with neutrophil cytotoxicity, in varying concentrations. Further studies are needed to evaluate the prognostic and therapeutic role of cytotoxic neutrophils as well as the role of chemokines in neutrophil cytotoxicity.

Early Treatment of High Risk Chronic Lymphocytic Leukemia with Alemtuzumab, Rituximab, and PGG Beta Glucan: A Phase I Clinical Trial

AbstractAbstract 1792High risk disease can be identified in patients with early stage chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL) using biological prognostic markers. We have shown that early therapy of high risk CLL patients with alemtuzumab (ALM) and rituximab (RTX) is effective and could possibly delay first standard treatment (Cancer 2008;113:2110-8). Efficacy of unconjugated monoclonal antibody (mAb) therapy in these patients could be improved by enhancing mAb mediated cellular cytotoxicity. Preclinical studies show that yeast cell wall derived beta glucan, which increases complement receptor 3 (CR3) binding to the complement fragment iC3b on target cells, could increase mAb mediated cellular cytotoxicity. Both ALM and RTX activate complement resulting in deposition of iC3b on the cell membrane. In CLL cells that are not lysed by complement activation, these iC3b molecules are targets for the effector cells mediating cellular cytotoxicity. We hypothesized that PGG beta glucan (Imprime PPG®, Biothera, Eagan MN) an intravenous formulation of a 1,3/1,6 glucose polymer prepared from a strain of Saccharomyces cerevisiae, would improve the efficacy of therapy with ALM and RTX in patients with CLL by increasing CR3 binding to iC3b and thus enhancing macrophage, neutrophil, and NK cell mediated cytotoxicity. We report the results of a Phase I study of the combination of ALM, RTX and PPG beta glucan in patients with CLL. Methods:The primary aim of this IRB approved study (NCT01269385) was to determine the maximum tolerated dose (MTD) of PGG beta glucan that could be safely combined with ALM and RTX. The MTD was defined as the PGG beta glucan dose level below that which induced dose limiting toxicity in at least one third of patients, or the highest dose level tested if all levels were tolerated. Eligibility for the trial required a diagnosis of CLL by standard (IWCLL-NCI 2008) criteria, no prior treatment for CLL, high risk CLL based on molecular markers, absence of standard indications for initiation of therapy for CLL, and adequate performance status and organ function. High risk CLL was defined as at least one of the following: 17p13-; 11q22-; either unmutated (<2%) IGHV or use of VH3–21 as well as CD38+ and/or ZAP70+. Patients received standard premedication for mAb, antimicrobial and allopurinol prophylaxis and weekly PCR testing for CMV reactivation with treatment of viremia. The duration of treatment was 33 days. PGG beta glucan was administered IV on days 1, 5, 10, 17, 24, and 31 and the first dose was premedicated with hydrocortisone 100mg IV, oral acetaminophen 1000 mg and diphenhydramine 50 mg. The starting dose level of PGG beta glucan was 1 mg/kg, 2nd dose level was 2 mg/kg and the 3rd dose level 4 mg/kg. Subcutaneous ALM therapy started on day 3 with daily dose escalation (3 – 10 – 30 mg) and then 30 mg Mon-Wed-Friday for 4 weeks. Weekly RTX started on day 10 at 375 mg/m2 IV × 4 doses. Results:Thirteen patients were enrolled from February 2011 to April 2012. The 11 evaluable patients had a median age of 61 years (range 47 – 77), 73% were male, 3 had early stage disease (Rai 0) and 8 had intermediate stage disease (Rai I n = 7, Rai II n = 1). High-risk parameters were 17p- in 4 patients, 11q22- in 3 patients, and unmutated IGHV and expression of ZAP70 and/or CD38 in 4 patients. There were no dose limiting toxicities. One patient had grade 4 febrile neutropenia, with no grade 3–4 anemias or thrombocytopenias, and there were no grade 3–4 non-hematological toxicities. All patients responded to therapy with 7 CR, 1 CCR, 1 nPR, and 2 PR (IWCLL-NCI 2008 criteria). Median follow up was 6.9 months (2.3 – 13.2) and one patient progressed at 9.7 months. No patients have required treatment for progressive disease and there have been no patient deaths. Two patients were not evaluable: One developed neutropenia and therapy was not held per protocol, and the other developed a grade 2 skin reaction to ALM and treatment was stopped. Discussion:The combination of PGG beta glucan with ALM and RTX is well tolerated at a PGG beta glucan dose of 4 mg/kg. All patients responded to therapy with 64% achieving a CR. These data support continuation of this study in a phase II component. Acknowledgment:This study was funded by the University of Iowa/Mayo Clinic Lymphoma SPORE (CA097274) and Biothera. Disclosures:Zent:Biothera: Research Funding; Genzyme: Research Funding; Genentech: Research Funding; Novartis: Research Funding; GlaxoSmithKline: Research Funding. Off Label Use: Phase I study using PGG beta glucan in CLL.

Model of specific diagnostics of ovarian cancer stages

Aim. To develop the model of specific diagnostics of III-IV stages of ovarian cancer. Methods. 404 patients with I-IV stages of primary ovarian cancer according to International Federation of Gynecology and Obstetrics classification were examined. Ultrasonography of genitalia and internal organs, chest X-ray in order to detect possible remote metastases, cytological, histological and immunohistological examination of tumor mass, as well as «lipid peroxidation - antioxidants» system condition of tumor tissue and patients’ serum were examined. Results. Elevated level of a lipid peroxidation product, malondialdehyde, was found in tumor tissue of patients with ovarian cancer, with levels of malondialdehyde growing from clinical stage I to clinical stage III of the disease. Leukocyte counts of peripheral blood statistically significantly decreased at stages I-III and elevated at stage IV. However, aerobic and anaerobic cytotoxicity of neutrophils decreases. An algorithm of supplementary diagnostic tools use to define the stage of ovarian cancer was developed, as well as differentiation coefficient calculation formula. Conclusion. Defining the serum levels of malondialdehyde, catalase and glutathione reductase activity allows to calculate the coefficient for differentiation between stages III and IV of ovarian cancer, and detection of cation proteins level, myeloperoxidase activity and phagocytic index allows to calculate the coefficient for differentiation between stages II and III of ovarian cancer.

P1-01-03: The Cytotoxicity of Select Neutrophils in Cancer Patients and the Role of Chemokines in Inducing Neutrophil Cytotoxicity.

Abstract Background: Using murine mammary tumor models, recent research conducted by our laboratory at the Sloan-Kettering Institute indicates that select neutrophils are mobilized by a primary breast tumor and uniquely have the capacity to inhibit metastatic seeding in the lung (Granot Z et al. unpublished). We sought to evaluate the cytotoxic role of select neutrophils in the peripheral blood of breast cancer patients as contrasted with those from women without breast cancer and women with DCIS. In addition, we sought to determine whether the addition of select chemokines to neutrophils from a DCIS patient could induce cytotoxicity. Methods: Neutrophils were purified from the blood of 21 newly diagnosed pre-operative breast cancer patients without evidence of metastatic disease, 9 healthy female volunteers with no history of any cancer, and 3 patients with newly diagnosed DCIS. Cytotoxicity was evaluated by incubating isolated neutrophils with luciferase labeled MDA-MB-231 cells. Luciferase activity, as a reflection of% cell kill, was measured using a Bio-Tek microplate luminescence reader. Neutrophils from a DCIS patient with low cytotoxicity were then co-cultured with various CC chemokines (CCl2, CCL3 and CCL5) or CXCL chemokines (CXCL1, CXCL12, and CXCL16) at 100ng/ml. Results: Significant cytotoxicity was notably observed when MDA-MB-231 cells were co-cultured with neutrophils purified from patients with invasive tumors. Pre-operative breast cancer patients (n=21) had a cell kill range of 0–30% (mean = 12.1%), whereas healthy subjects (n=9) had a cell kill range of 0.2-8% (mean = 2.6%), p&amp;lt;0.004. DCIS patients (N=3) had a cell kill range of 3–4% (mean = 2.7). The addition of select chemokines to neutrophils from a DCIS patient with low cytotoxicity (3.2%) resulted in significant increases in cytotoxicity. Table 1 indicates the relative cytotoxicity percentages from the addition of each chemokine. Conclusions: To date, this preliminary work is the first to demonstrate the cytotoxic role of select neutrophils in the peripheral blood of breast cancer patients as contrasted with those from women without breast cancer. We further demonstrate the novel induction of neutrophil cytotoxicity by select chemokines. Further studies are needed to evaluate the prognostic and therapeutic role of cytotoxic neutrophils as well as the role of chemokines in neutrophil cytotoxicity. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-01-03.

The novel cytotoxicity of neutrophils in the peripheral blood of breast cancer patients versus women without breast cancer.

44 Background: Using murine mammary tumor models, recent research conducted by our laboratory at the Sloan-Kettering Institute indicates that select neutrophils are mobilized and entrained by a primary breast tumor and uniquely have the capacity to inhibit metastatic seeding in the lung (Granot Z et al. unpublished). We sought to determine whether entrainment of cytotoxic neutrophils also occurs in blood samples from women with newly diagnosed breast cancer as contrasted to those garnered from healthy women. Methods: Subjects were 21 newly diagnosed pre-operative breast cancer patients without evidence of metastatic disease, 9 healthy female volunteers with no history of any cancer, and 3 patients with newly diagnosed DCIS. Neutrophils were purified from the blood samples. Cytotoxicity was evaluated by incubating isolated neutrophils with luciferase labeled MDA-MB-231 cells. Luciferase activity, as a reflection of % cell kill, was measured using a Bio-Tek microplate luminescence reader. Results: Significant cytotoxicity was notably observed when MDA-MB-231 cells were co-cultured with neutrophils purified from patients with invasive tumors. Pre-operative breast cancer patients (n=21) had a cell kill range of 0-30% (mean = 12.1%), whereas healthy subjects (n=9) had a cell kill range of 0.2-8% (mean = 2.6%), p&lt;0.004. DCIS patients (N=3) had a cell kill range of 3-4% (mean = 2.7). Conclusions: To date, this preliminary work is the first to demonstrate the cytotoxic role of select neutrophils in the peripheral blood of breast cancer patients as contrasted with those from women without breast cancer. Further studies are needed to evaluate the prognostic and therapeutic role of cytotoxic neutrophils.

The novel cytotoxicity of neutrophils in the peripheral blood of patients with breast cancer versus healthy women.

e21055 Background: Using murine mammary tumor models, recent research conducted by our laboratory at the Sloan-Kettering Institute indicates that select neutrophils are mobilized and entrained by a primary breast tumor and uniquely have the capacity to inhibit metastatic seeding in the lung (Granot Z et al. unpublished). We sought to determine whether entrainment of cytotoxic neutrophils also occurs in blood samples from women with newly diagnosed breast cancer as contrasted to those garnered from healthy women. Methods: Subjects were 14 newly diagnosed pre-operative breast cancer patients without evidence of metastatic disease, 9 healthy female volunteers with no history of any cancer and 2 patients with newly diagnosed DCIS. Neutrophils were purified from the blood samples. Cytotoxicity was evaluated by incubating isolated neutrophils with luciferase labeled MDA-MB-231 cells. Luciferase activity, as a reflection of % cell kill, was measured using a Bio-Tek microplate luminescence reader. Results: Significant cytotoxicity was notably observed when MDA-MB-231 cells were co-cultured with neutrophils purified from patients with invasive tumors. Pre-operative breast cancer patients (n=14) had a cell kill range of 0-30% (mean = 13.9%), whereas healthy subjects (n=9) had a cell kill range of 0.2-8% (mean = 2.6%), p<0.001. DCIS patients (N=2) had a cell kill range of 3-4% (mean = 3.65). Conclusions: To date, this preliminary work is the first to demonstrate the cytotoxic role of select neutrophils in the peripheral blood of breast cancer patients as contrasted with those from women without breast cancer. Further studies are needed to evaluate the prognostic and therapeutic role of cytotoxic neutrophils.

α-Enolase From Injured Patients and Stored Packed Red Blood Cells Activates Pulmonary Endothelium and Serves as the First Event In a Two-Event Model of Neutrophil Cytotoxicity

AbstractAbstract 3355A significant number of injured patients with intermediate injury severity scores (15<ISS<30) develop multiple organ failure (MOF), which clinically begins with acute lung injury (ALI). Transfusion of >6 units of stored PRBCs (≥28 days) is associated with the development of ALI/MOF on day 3 post injury (Am J Surg 178:502-4, 1999). The pro-inflammatory mediators, e.g. cytokines, responsible for MOF/ALI in the injured have remained elusive; therefore, we hypothesize that “moonlighting” proteins, which have defined intracellular function when released in the circulation, activate innate immunity and are etiologic in the development of ALI/MOF post-injury. Methods:Proteomics on the field blood (plasma) of 3 patients with blunt trauma who later developed ALI/MOF and the plasma from 3 units of packed red blood cells (PRBCs) on day 1 and day 42 were completed using 2-dimensional gel electrophoresis/mass spectroscopy (MALDI/TOF) with computer analyses of the resultant peptides. The proteins from whole cell lysates from Human pulmonary microvascular endothelial cells (HMVECs) were separted by SDS-PAGE, transferred to nitrocellulse and immunoblotted with antibodies to protease activated receptor-1 (PAR-1) and PAR-2. HMVECs were also incubated for 6 hours and 1) ICAM-1 was measured by flow cytometry, 2) isolated neutrophils (PMNs) were added allowed to settle and in selected wells PMN adherence to these activated HMVECs was measured by myeloperoxidase content in the lysate, or 3) after the PMNs settled, lysophosphatidylcholines (lyso-PCs) [4.5μM], lipids from stored platelets implicated in TRALI, were added and the number of viable HMVECs/mm2 were counted by microscopy. Results:HMVECs display immunoreactivity for both PAR-1 and PAR-2. Of the 243 proteins identified in the injured patients and the stored vs. fresh PRBCs, α-enolase increased by 10.8-fold and 4.4-fold respectively (p<.05 & p<.005). Both thrombin and α-enolase induced ICAM-1 expression in HMVECs (Table 1) which was inhibited (60±8%) by pre-treatment with the anti-protease leupeptin. α-Enolase also induced significant PMN adhesion vs. media control: Media: 3.1±1.5; α-enolase (50 μg/ml): 14.4±4.7; LPS: 35.5±0.7*. The α-enolase-activated HMVECs vs. buffer-treated lyso-PCs induced significant PMN-mediated cytotoxicity (Table 2). We conclude that α-enolase from the injured and stored but not fresh PRBCs causes pro-inflammatory activation of HMVECs resulting in PMN adherence and PMN cytotoxicity in a two-event in vitro model through activation of PARs receptors. Moonlighting proteins like the glycolytic lyase α-enolase may have unexpected pro-inflammatory activity, which predispose the injured patient to increased morbidity and mortality. Disclosures:No relevant conflicts of interest to declare.

Epidermal expression of oncogenic ras subverts cytotoxic inflammation by skewing infiltrating myeloid cells towards an immunosuppressive phenotype. (100.16)

Abstract Cancer development is frequently accompanied by inflammation and immune activation. The nature of this inflammatory response, however, is non-uniform and can consist of cells with opposing functions. To characterize the innate immune response to neoplastic epithelial tissue, we used an epidermally-restricted transgenic mouse model of oncogenic H-Ras expression in which epidermal hyperplasia and premalignant progression induces aberrant myelopoesis and cutaneous hyperinflammation associated with the formation of myeloid resident epithelial microabscesses. Antibody mediated depletion of neutrophils concurrent with transgene activation ablated neutrophil infiltration and microabscesses resulting in increased epidermal thickening relative to isotype controls. Neutrophils isolated from mice expressing ras for 1 week killed keratinocytes in vitro, suggesting that the in vivo myeloid response to oncogenic ras expression is initially a cytotoxic one. Simultaneously, there is cutaneous upregulation of genes implicated in the activation, function and recruitment of myeloid derived suppressor cells (MDSC) such as GMCSF, GCSF, MMP9, VEGF, S100A8/9, CCL2, and IL1β. This coincides with upregulation of IL4Rα on myelocytes, increased percentage of Tregs, and decreased effector T cell responses from 1 to 2 weeks post H-Ras expression. These data suggest that the acute cytotoxic neutrophil response is replaced by a suppressor myeloid phenotype that could enhance neoplastic progression.

Evaluation of the anti-inflammatory and cytotoxic effects of anthraquinones and anthracenes derivatives in human leucocytes.

A variety of anthracene- and anthraquinone-related derivatives, modified from three types of lead structures, including 9-acyloxy 1,5-dichloroanthracene (type I), 1,5-bisacyloxy-anthraquinones with O-linked substituents (type II) and 1,5-bisacyloxy-anthraquinones with S-linked substituents (type III), were synthesized and evaluated by an in-vitro bioassay for their anti-inflammatory and cytotoxic effects in human leucocytes. Among these derivatives, type I compounds displayed potent anti-inflammatory activity against phorbol-12-myristate-13-acetate (PMA)-induced superoxide anion production, a bio-marker of inflammatory mediator production by neutrophils, with 50% inhibition (IC50) concentrations (microM) for compounds 1f, 1g, 1h and 1m being 13.8 +/- 3.0, 6.3 +/- 4.1, 33.2 +/- 1.3 and 33.9 +/- 5.7, respectively. Type II and type III derivatives (i. e., 1,5-bisacyloxy anthraquinone-related compounds) and the reference compound, emodin, exhibited relatively minor (20-40%) inhibitory effect against superoxide production by neutrophils. Furthermore, none of these compounds showed a significant cytotoxic effect in human neutrophils. In conclusion, these results suggest that compounds modified from 9-acyloxy 1,5-dichloroanthracence (type I) are more powerful than the other two types as anti-inflammatory drugs. This is the first demonstration that derivatives modified from anthracenes or anthraquinones possess anti-inflammatory activity with no significant cytotoxicity in human neutrophils.

Dual mechanism in induction of human neutrophil cytotoxicity: activation of protein kinase C and elevation in intracellular calcium

Human neutrophils can damage the non-immunized K562 cell line when stimulated by phorbol myristate acetate (PMA). The combination of the activation of protein kinase C by phorbol and the increase of free intracellular calcium by ionophore potentiates the lytic reaction. The OKT9-immunized target cells are not able to induce by themselves the lytic response in neutrophils, but by combining the two signals, the antigenic stimulus and PMA, a high level of cytolytic response is attained. The addition of EGTA does not affect neutrophil cytotoxicity against antibody-coated targets, while it markedly reduced the lytic reaction against non-immunized targets; in contrast, the addition of EGTA together with the ionophore ionomycin completely suppresses the lysis of immunized and non-immunized targets. The treatment of neutrophils with the protein kinase C inhibitor H-7 causes a dose-related inhibition of the lytic functions that is greater on unsensitized K562. Thus the interaction of Fc receptors with immunized targets is required for reaching the maximal cytolysis. The enhanced lytic activity that occurs in the presence of immunized targets is mediated by calcium flux, as detected by using the monoclonal antibody AB8.28 which binds to FcIII receptors (FcRIII), thus supporting that both signals are involved.

Requirement for Vav Proteins in Post-Recruitment Neutrophil Cytotoxicity in IgG but Not Complement C3-Dependent Injury

The signals linking neutrophil opsonic receptors, FcgammaRs and complement receptor 3 (Mac-1) to cellular cytotoxic responses are poorly understood. Furthermore, because a deficiency in activating FcgammaRs reduces both IgG-mediated neutrophil recruitment and tissue injury, the role of FcgammaRs specifically in mediating neutrophil cytotoxicity in vivo remains unclear. In this study, we demonstrate that neutrophil Vav 1 and 3, guanine exchange factors for Rac GTPases, are required for IgG/FcgammaR-mediated hemorrhage and edema in the reverse passive Arthus in the lung and skin. Rac GTPases are also required for development of the reverse passive Arthus reaction. A deficiency in Vav 1 and 3 does not affect neutrophil accumulation at the site of immune complex deposition, thus uncoupling neutrophil recruitment and tissue injury. Surprisingly, Vav and Rac proteins are dispensable for the development of the local Shwartzman reaction in vivo and phagocytosis of complement-opsonized RBC in vitro, processes strictly dependent on Mac-1 and complement C3. Thus, FcgammaR signaling through the Vav and Rac proteins in neutrophils is critical for stimulating immune complex disease while Vav- and Rac-independent pathways promote Mac-1/complement C3-dependent functions.

The anti-tumor agent, ingenol-3-angelate (PEP005), promotes the recruitment of cytotoxic neutrophils by activation of vascular endothelial cells in a PKC-delta dependent manner.

The modes of action of the novel anti-skin tumor agent ingenol-3-angelate (PEP005) are incompletely understood. Crucially, the cytotoxic functions of neutrophils recruited to the tumor in response to topical application of PEP005 are necessary for effective ablation of the treated lesion. Here, we investigated the hypothesis that the phorbol ester-like properties of PEP005 and its ability to activate PKC could directly activate endothelial cells (EC) so that they support the recruitment of neutrophils. Exposure of EC to PEP005 induced mRNA and/or protein for E-selectin, ICAM-1 and IL-8 in a dose dependent manner, while in a flow based adhesion assay, PEP005 treated EC supported the recruitment of neutrophils at levels comparable to EC stimulated with TNF-alpha. Neutrophil adhesion was inhibited by antibody against E-selectin but not P-selectin. Activation of EC was inhibited by the PKC inhibitor bisindolylmaleimide-1 and confocal immuno-fluorescent studies demonstrated translocation of PKC-delta from the cytosol to the peri-nuclear membrane in response to PEP005. Importantly, the knock down of PKC-delta using siRNA completely abolished neutrophil recruitment to EC subsequently treated with PEP005. Thus, we describe a novel route by which the anti-tumor agent PEP005 regulates the recruitment of cytotoxic leukocytes by directly activating EC in a PKC-delta dependent manner.

A PEGylated Fab′ Fragment against Tumor Necrosis Factor for the Treatment of Crohn Disease

Antibodies, having a high specificity for their particular target, are increasingly being used as therapeutic agents with functions including agonist, antagonist, and targeted drug delivery. The use of many biologic therapies, including antibody fragments, is generally limited by their rapid clearance from plasma. A commonly used approach to extend exposure to biologic therapies is the attachment of polyethylene glycol.Tumor necrosis factor (TNF)-alpha is a multifunctional cytokine involved in the regulation of immune responses. Elevated levels of TNFalpha are found in a wide range of diseases, including the chronic inflammatory conditions rheumatoid arthritis, psoriasis, and Crohn disease (CD). Anti-TNFalpha antibodies have proved highly efficacious in the treatment of these conditions. In addition, they have proved invaluable for investigating the role of TNFalpha in disease etiology. Based on evidence showing that neutralizing antibodies to TNFalpha were effective in animal models of CD, anti-TNFalpha antibody treatments were assessed in clinical trials. Interestingly, the anti-TNFalpha antibody etanercept proved ineffective at achieving remission in active CD despite potently neutralizing soluble TNFalpha. This indicated that an additional mode of action is also involved in the efficacy of the anti-TNFalpha agents adalimumab, certolizumab pegol, and infliximab in CD; one suggestion was apoptosis. However, etanercept, like adalimumab and infliximab, can induce apoptosis. Furthermore, certolizumab pegol (which has demonstrated efficacy in CD) does not cause complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity, apoptosis, or necrosis of neutrophils, all measured in vitro. These functional differences observed with certolizumab pegol stem from its unique structure that does not include the crystallizable fragment (Fc) portion present in the other anti-TNFalpha agents, and the way in which it signals through membrane TNF. It is well established that bacteria are a major part of the inflammatory process in CD. The property identified that reflected the efficacies of the anti-TNFalpha agents etanercept, adalimumab, certolizumab pegol, and infliximab in CD was the ability to inhibit the cytokine production by monocytes that is induced by bacterial lipopolysaccharide. It may therefore be the case that this mode of action is important for efficacy in CD.

In vivo Role of NAD(P)H:Quinone Oxidoreductase 1 in Metabolic Activation of Mitomycin C and Bone Marrow Cytotoxicity

NAD(P)H:quinone oxidoreductase 1(-/-) (NQO1(-/-)), NQO1(+/-) along with NRH:quinone oxidoreductase 2(-/-) (NQO2(-/-)), and wild-type (WT) mice were exposed to five once weekly doses of mitomycin C. The mice were euthanized 15 weeks after the first dose. Blood cell counts and histologic analyses were done. WT and NQO2(-/-) mice showed hypocellularity and a significant increase in adipocytes in bone marrow. They also showed anemia because of the loss of RBC and hemoglobin. The neutrophils and platelets were reduced, whereas other blood cell types and tissues were normal. Interestingly, NQO1(-/-) mice showed a complete resistance to mitomycin C-induced bone marrow cytotoxicity and reduction in RBC, hemoglobin, and neutrophils. NQO1(+/-) mice also showed limited resistance to mitomycin C-induced bone marrow cytotoxicity. These data show a major in vivo role of NQO1 in metabolic activation of mitomycin C with implications in mitomycin C chemotherapy.

Reduced inflammatory response and increased microcirculatory disturbances during hepatic ischemia-reperfusion injury in steatotic livers of ob/ob mice

Steatosis is a major risk factor for complications after liver surgery. Since neutrophil cytotoxicity is critical for ischemia-reperfusion injury in normal livers, the aim of the present study was to evaluate whether an exaggerated inflammatory response could cause the increased injury in steatotic livers. In C57Bl/6 mice, 60 min of warm hepatic ischemia triggered a gradual increase in hepatic neutrophil accumulation during reperfusion with peak levels of 100-fold over baseline at 12 h of reperfusion. Neutrophil extravasation and a specific neutrophil-induced oxidant stress (immunostaining for hypochlorous acid-modified epitopes) started at 6 h of reperfusion and peaked at 12-24 h. Ob/ob mice, which had a severe macrovesicular steatosis, suffered significantly higher injury (alanine transaminase activity: 18,000 +/- 2,100 U/l; 65% necrosis) compared with lean littermates (alanine transaminase activity: 4,900 +/- 720 U/l; 24% necrosis) at 6 h of reperfusion. However, 62% fewer neutrophils accumulated in steatotic livers. This correlated with an attenuated increase in mRNA levels of several proinflammatory genes in ob/ob mice during reperfusion. In contrast, sham-operated ob/ob mice had a 50% reduction in liver blood flow and 35% fewer functional sinusoids compared with lean littermates. These deficiencies in liver blood flow and the microcirculation were further aggravated only in ob/ob mice during reperfusion. The attenuated inflammatory response and reduced neutrophil-induced oxidant stress observed in steatotic livers during reperfusion cannot be responsible for the dramatically increased injury in ob/ob mice. In contrast, the aggravated injury appears to be mediated by ischemic necrosis due to massive impairment of blood and oxygen supply in the steatotic livers.

Granulocyte Macrophage Colony–Stimulating Factor Expression in Human Herpetic Stromal Keratitis: Implications for the Role of Neutrophils in HSK

Granulocyte macrophage colony-stimulating factor (GM-CSF) is thought to play a key role in chronic inflammatory diseases by governing the survival and function of infiltrating neutrophils. The objective of this study was to determine the putative role of GM-CSF in the pathogenesis of human herpetic stromal keratitis (HSK). Primary human corneal fibroblast (HCF) cultures and a telomerase-immortalized human corneal epithelial (HCE) cell line representative of native HCE were stimulated with the known HSK-inducing cytokines interferon (IFN)-gamma, interleukin (IL)-1beta, and tumor necrosis factor (TNF)-alpha. Alternatively, the T-cell cytokine IL-17 was added solely or simultaneously. Human neutrophils were incubated with conditioned medium (CM) of the HCF and HCE stimulated with the aforementioned cytokines, or recombinant GM-CSF, and their viability or activation status was determined by flow cytometry. GM-CSF and IL-8 secretion levels in the CM were determined by ELISA. The antibody-dependent cellular cytotoxicity (ADCC) of neutrophils toward herpes simplex virus (HSV)-infected HCFs was determined by flow cytometry. The expression of GM-CSF was determined in HSK and control corneal buttons by real-time RT-PCR and immunohistology. Compared with IFN-gamma, CM of either cell type stimulated with IL-1beta, or in the case of HCE cells, stimulated with TNF-alpha or IL-17, delayed neutrophil apoptosis significantly. Only in HCFs did IL-17 exhibit a synergistic effect with TNF-alpha. The antiapoptotic activity was attributable in part to the GM-CSF secreted by the activated HCFs and HCE cells. GM-CSF stimulation of neutrophils induced their activation and the secretion of IL-8. GM-CSF did not increase significantly the ADCC reaction of neutrophils toward HSV-infected HCFs. Finally, GM-CSF was expressed in corneas of the patients with HSK but not in control subjects. The data suggest that GM-CSF, expressed by cornea-resident cells such as HCFs and HCE cells, may play a role in the immunopathogenesis of HSK by prolonging the survival and modulating the effector function of corneal infiltrating neutrophils.

Effect of indole‐3‐acetic acid administration by gavage and by subcutaneous injection on rat leukocytes

This study was done to investigate the effect of indole-3-acetic acid (IAA) administered subcutaneously and by gavage on neutrophil function and cytotoxicity in neutrophils and lymphocytes. A gavage administration resulted in an increase in phagocytic capacity in neutrophils in a dose depended manner for 1 mg, 2 mg, 18 mg, and 40 mg of IAA per kg of body mass, respectively, compared with the control. Similarly, subcutaneous administration of IAA at 2, 18, and 40 mg per kg of body mass promoted a significant rise in phagocytosis by neutrophils. H2O2 production in neutrophils from treated rats by gavage was similar to those receiving subcutaneous IAA treatment, and did not show a significant difference between treatments and control. IAA treatment, whether by gavage or subcutaneous, did not produce an alteration in antioxidant enzyme activities or in glucose-6-phosphate dehydrogenase activity of either neutrophils or lymph nodes. Subcutaneous IAA administration did not alter the neutrophil and lymphocyte death as deduced by unaltered membrane integrity, DNA fragmentation and mitochondrial transmembrane potential, compared with controls. In conclusion, IAA administration either subcutaneously or by gavage could increase the phagocytic capacity by neutrophils and this acid administration did not have prooxidant effects or cytotoxic effects on neutrophils and lymphocytes.

The effect of organosulfur compounds on peroxynitrite‐induced cytotoxicity: Attenuation or Potentiation?

The present study was aimed to evaluate the effect of naturally occurring organosulfur compounds namely allyl isothiocyanate (AITC) and benzyl isothiocyanate (BITC) on peroxynitrite-induced cytotoxicity in milk-derived polymorphonuclear neutrophils (PMNs), utilizing the tetrazolium salt XTT assay to measure cell survival. 3-Morpholinsydnonimine (SIN-1), a peroxynitrite donor, induced the loss of PMNs viability in a concentration-dependent manner with estimated EC50 value of 375 microM. Pretreatment with AITC or BITC further enhanced cytotoxicity in SIN-1-treated cells; however, AITC or BITC alone at concentration ≤3 microM did not cause the cytotoxic effect as compared to untreated control cells. On the contrary, glutathione (GSH), melatonin and uric acid markedly and concentration dependently attenuated the cytotoxic effect of SIN-1. These data suggest that these organosulfur compounds could render cells more susceptible to injury and death mediated by oxidants. This work was supported by the Commission of Higher Education, Ministry of Education and the Thailand Research Fund.

Null mutation of myeloperoxidase in mice prevents mechanical activation of neutrophil lysis of muscle cell membranes in vitro and in vivo

Membrane lysis is a common and early defect in muscles experiencing acute injuries or inflammation. Although increased mechanical loading of muscles can induce inflammation and membrane lysis, whether mechanical loads applied to muscle can promote the activation and cytolytic capacity of inflammatory cells and thereby increase muscle damage is unknown. We tested whether mechanical loads applied to mouse muscle cells in vitro can increase membrane lysis, and whether neutrophil-mediated lysis of muscle cells is promoted by mechanical loads applied in vitro and in vivo. Cyclic loads applied to muscle cells for 24 h in vitro produced little muscle cell lysis. Similarly, the addition of neutrophils to muscle cell cultures in the presence of superoxide dismutase (SOD) produced little muscle cell lysis. However, when cyclic mechanical loads were applied to neutrophil-muscle co-cultures in the presence of SOD, there was a synergistic effect on muscle cell lysis, suggesting that mechanical loading activates neutrophil cytotoxicity. However, application of mechanical loads to co-cultures of muscle cells and neutrophils that are null mutants for myeloperoxidase (MPO) showed no mechanical activation of neutrophil cytotoxicity. This indicates that loading promotes neutrophil cytotoxicity via MPO. Activity assays confirmed that mechanical loading of neutrophil-muscle co-cultures significantly increased MPO activity. We further tested whether muscle membrane lysis in vivo was mediated by neutrophils when muscle was subjected to modified loading by using a mouse model of muscle reloading following a period of unloading. We observed that MPO-/-soleus muscles showed a significant 52% reduction in membrane lysis compared to wild-type mice, although the mutation did not decrease inflammatory cell extravasation. Together, these in vitro and in vivo findings show that mechanical loading activates neutrophil-mediated lysis of muscle cells through an MPO-dependent pathway.