Social enrichment alters the response of brain leukocytes to chemotherapy and tumor development in aged mice.

Aging is a risk factor for the development of breast cancer. Foundational science studies have supported associations among neuroinflammation, breast cancer, and chemotherapy, but to date, these associations are based on studies using young adult rodents. The current study examined the neuroinflammatory effects of chemotherapy in aged, tumor-naïve and tumor-bearing mice with or without social enrichment. Mice received two intravenous injections of doxorubicin (A) and cyclophosphamide (C) at a two-week interval. Brain immune cells were enriched/assessed via flow cytometry, seven days following the second chemotherapy injection. Social enrichment enhanced peripheral immune cell trafficking in aged tumor-naive mice treated with AC. Group housed aged tumor bearing mice receiving AC had reduced percentage of IL-6+ monocytes and granulocytes relative to their singly housed counterparts. Notably, group housing aged experimental mice with young cage partners significantly reduced TNF + monocytes, tumor volume, and tumor mass. These data illustrate the importance of social enrichment in attenuating neuroinflammation and are the first to demonstrate that social support with young housing partners reduces tumor growth in aged mice.

Cell Sorter Cleaning Practices and Their Impact on Instrument Sterility.

Cells isolated using electrostatic cell sorters are subsequently evaluated in a variety of in vitro and in vivo applications. Thus, manipulations to the cells during the pre- and post-sort processing as well as when the cells are being analyzed by and passing through the sorter fluidics has the potential to affect the experimental results. There are many variables to consider when seeking to preserve cellular integrity and function during the cell-sorting process. A previous study by the Association of Biomolecular Resource Facilities Flow Cytometry Research Group (FCRG) investigated downstream effects on different cell types as a function of sorting variables such as pressure, nozzle size, and temperature. This multisite study revealed site-to-site variability based on differential gene expression when the same cell type and sort conditions were used. These results indicated the possibility that environmental factors such as the presence of contaminants in the sorter fluidics could exhibit effects on downstream molecular assays (ie, endotoxins or RNases). In the study described here, the FCRG sought to better understand how sorters are maintained and evaluated for contaminants such as bacteria, endotoxin, and RNases. In addition, the efficacy of an endotoxin decontamination method was evaluated. The results demonstrated that the majority of sorters in shared resource laboratories are free of RNase activity and bacteria; however, many are contaminated with endotoxin. The efficacy of a hydrogen peroxide cleaning procedure was tested and found to exhibit only a short-term effectiveness in eliminating endotoxin contamination.

Myeloid-Derived Suppressor Cells Gain Suppressive Function during Neonatal Bacterial Sepsis.

Neonates are at an increased risk of an infectious disease. This is consistent with an increased abundance of myeloid-derived suppressor cells (MDSCs) compared with older children and adults. Using a murine model of neonatal bacterial sepsis, we demonstrate that MDSCs modulate their activity during an infection to enhance immune suppressive functions. A gene expression analysis shows that MDSCs increased NOS2, Arg-1 and IL-27p28 expression in vitro and in vivo in response to Escherichia coli O1:K1:H7 and this is regulated at the level of the gene expression. Changes in the effector gene expression are consistent with increased enzymatic activity and cytokine secretion. The neonatal MDSCs express toll-like receptor (TLR) 2, 4 and 5 capable of recognizing pathogen-associated molecular patterns (PAMPS) on E. coli. However, a variable level of effector expression was achieved in response to LPS, peptidoglycan or flagellin. Individual bacterial PAMPs did not stimulate the expression of Arg-l and IL-27p28 equivalently to E. coli. However, the upregulation of NOS2 was achieved in response to LPS, peptidoglycan and flagella. The increased immune suppressive profile translated to an enhanced suppression of CD4+ T cell proliferation. Collectively, these findings increase our understanding of the dynamic nature of MDSC activity and suggest that these cells abundant in early life can acquire activity during an infection that suppresses protective immunity.

Increased circulating microparticles in streptozotocin-induced diabetes propagate inflammation contributing to microvascular dysfunction.

KEY POINTS: Circulating microparticles (MPs) are elevated in many cardiovascular diseases and have been considered as biomarkers of disease prognosis; however, current knowledge of MP functions has been mainly derived from in vitro studies and their precise impact on vascular inflammation and disease progression remains obscure. Using a diabetic rat model, we identified a >130-fold increase in MPs in plasma of diabetic rats compared to normal rats, the majority of which circulated as aggregates, expressing multiple cell markers and largely externalized phosphatidylserine; vascular images illustrate MP biogenesis and their manifestations in microvessels of diabetic rats. Using combined single microvessel perfusion and systemic cross-transfusion approaches, we delineated how diabetic MPs propagate inflammation in the vasculature and transform normal microvessels into an inflammatory phenotype observed in the microvessels of diabetic rats. Our observations derived from animal studies resembling conditions in diabetic patients, providing a mechanistic insight into MP-mediated pathogenesis of diabetes-associated multi-organ microvascular dysfunction. ABSTRACT: In various cardiovascular diseases, microparticles (MPs), the membrane-derived vesicles released during cell activation, are markedly increased in the circulation. These MPs have been recognized to play diverse roles in the regulation of cellular functions. However, current knowledge of MP function has been largely derived from in vitro studies. The precise impact of disease-induced MPs on vascular inflammation and disease progression remains obscure. In this study we investigated the biogenesis, profile and functional roles of circulating MPs using a streptozotocin-induced diabetic rat model with well-characterized microvascular functions. Our study revealed a >130-fold increase in MPs in the plasma of diabetic rats compared to normal rats. The majority of these MPs originate from platelets, leukocytes and endothelial cells (ECs), and circulate as aggregates. Diabetic MPs show greater externalized phosphatidylserine (PS) than normal MPs. When diabetic plasma or isolated diabetic MPs were perfused into normal microvessels or systemically transfused into normal rats, MPs immediately adhered to endothelium and subsequently mediated leukocyte adhesion. These microvessels then exhibited augmented permeability responses to inflammatory mediators, replicating the microvascular manifestations observed in diabetic rats. These effects were abrogated when MPs were removed from diabetic plasma or when diabetic MPs were pre-coated with a lipid-binding protein, annexin V, suggesting externalized PS to be key in mediating MP interactions with endothelium and leukocytes. Our study demonstrated that the elevated MPs in diabetic plasma are actively involved in the propagation of vascular inflammation through their adhesive surfaces, providing mechanistic insight into the pathogenesis of multi-organ vascular dysfunction that commonly occurs in diabetic patients.

SOX9 inhibits ß-TrCP-mediated protein degradation to promote nuclear GLI1 expression and cancer stem cell properties.

The high mobility group box protein SOX9 and the GLI1 transcription factor play protumorigenic roles in pancreatic ductal adenocarcinoma (PDA). In Kras transgenic mice, each of these factors are crucial for the development of PDA precursor lesions. SOX9 transcription is directly regulated by GLI1, but how SOX9 functions downstream of GLI1 is unclear. We observed positive feedback, such that SOX9-deficient PDA cells have severely repressed levels of endogenous GLI1, attributed to loss of GLI1 protein stability. SOX9 associated with the F-box domain of the SKP1/CUL1/F-box (SCF) E3 ubiquitin ligase component, ß-TrCP (also known as F-box/WD repeat-containing protein 1A), and suppressed its association with SKP1 and GLI1, a substrate of SCF-ß-TrCP. SOX9 also tethered ß-TrCP within the nucleus and promoted its degradation. SOX9 bound to ß-TrCP through the SOX9 C-terminal PQA/S domain that mediates transcriptional activation. Suppression of ß-TrCP in SOX9-deficient PDA cells restored GLI1 levels and promoted SOX9-dependent cancer stem cell properties. These studies identify SOX9-GLI1 positive feedback as a major determinant of GLI1 protein stability and implicate ß-TrCP as a latent SOX9-bound tumor suppressor with the potential to degrade oncogenic proteins in tumor cells.

MicroRNAs 206 and 21 cooperate to promote RAS-extracellular signal-regulated kinase signaling by suppressing the translation of RASA1 and SPRED1.

Despite the low prevalence of activating point mutation of RAS or RAF genes, the RAS-extracellular signal-regulated kinase (ERK) pathway is implicated in breast cancer pathogenesis. Indeed, in triple-negative breast cancer (TNBC), there is recurrent genetic alteration of pathway components. Using short hairpin RNA (shRNA) methods, we observed that the zinc finger transcription factor Krüppel-like factor 4 (KLF4) can promote RAS-ERK signaling in TNBC cells. Endogenous KLF4 bound to the promoter regions and promoted the expression of two microRNAs (miRs), miR-206 and miR-21 (i.e., miR-206/21). Antisense-mediated knockdown (anti-miR) revealed that miR-206/21 coordinately promote RAS-ERK signaling and the corresponding cell phenotypes by inhibiting translation of the pathway suppressors RASA1 and SPRED1. In TNBC cells, including cells with mutation of RAS, the suppression of either RASA1 or SPRED1 increased the levels of GTP-bound, wild-type RAS and activated ERK 1/2. Unlike the control cells, treatment of RASA1- or SPRED1-suppressed cells with anti-miR-206/21 had little or no impact on the level of activated ERK 1/2 or on cell proliferation and failed to suppress tumor initiation. These results identify RASA1 and SPRED1 mRNAs as latent RAS-ERK pathway suppressors that can be upregulated in tumor cells by anti-miR treatment. Consequently, KLF4-regulated miRs are important for the maintenance of RAS-ERK pathway activity in TNBC cells.

Sibling donor and recipient immune modulation with atorvastatin for the prophylaxis of acute graft-versus-host disease.

PURPOSE: Graft-versus-host disease (GVHD) is major cause of morbidity and mortality after allogeneic hematopoietic cell transplantation (HCT). Atorvastatin is a potent immunomodulatory agent that holds promise as a novel and safe agent for acute GVHD prophylaxis. PATIENTS AND METHODS: We conducted a phase II trial to evaluate the safety and efficacy of atorvastatin administration for GVHD prophylaxis in both adult donors and recipients of matched sibling allogeneic HCT. Atorvastatin (40 mg per day orally) was administered to sibling donors, starting 14 to 28 days before the anticipated first day of stem-cell collection. In HCT recipients (n = 30), GVHD prophylaxis consisted of tacrolimus, short-course methotrexate, and atorvastatin (40 mg per day orally). RESULTS: Atorvastatin administration in healthy donors and recipients was not associated with any grade 3 to 4 adverse events. Cumulative incidence rates of grade 2 to 4 acute GVHD at days +100 and +180 were 3.3% (95% CI, 0.2% to 14.8%) and 11.1% (95% CI, 2.7% to 26.4%), respectively. One-year cumulative incidence of chronic GVHD was 52.3% (95% CI, 27.6% to 72.1%). Viral and fungal infections were infrequent. One-year cumulative incidences of nonrelapse mortality and relapse were 9.8% (95% CI, 1.4% to 28%) and 25.4% (95% CI, 10.9% to 42.9%), respectively. One-year overall survival and progression-free survival were 74% (95% CI, 58% to 96%) and 65% (95% CI, 48% to 87%), respectively. Compared with baseline, atorvastatin administration in sibling donors was associated with a trend toward increased mean plasma interleukin-10 concentrations (5.6 v 7.1 pg/mL; P = .06). CONCLUSION: A novel two-pronged strategy of atorvastatin administration in both donors and recipients of matched sibling allogeneic HCT seems to be a feasible, safe, and potentially effective strategy to prevent acute GVHD.

Prenatal cadmium exposure alters postnatal immune cell development and function.

Cadmium (Cd) is generally found in low concentrations in the environment due to its widespread and continual use, however, its concentration in some foods and cigarette smoke is high. Although evidence demonstrates that adult exposure to Cd causes changes in the immune system, there are limited reports of immunomodulatory effects of prenatal exposure to Cd. This study was designed to investigate the effects of prenatal exposure to Cd on the immune system of the offspring. Pregnant C57Bl/6 mice were exposed to an environmentally relevant dose of CdCl(2) (10ppm) and the effects on the immune system of the offspring were assessed at two time points following birth (2 and 7weeks of age). Thymocyte and splenocyte phenotypes were analyzed by flow cytometry. Prenatal Cd exposure did not affect thymocyte populations at 2 and 7weeks of age. In the spleen, the only significant effect on phenotype was a decrease in the number of macrophages in male offspring at both time points. Analysis of cytokine production by stimulated splenocytes demonstrated that prenatal Cd exposure decreased IL-2 and IL-4 production by cells from female offspring at 2weeks of age. At 7weeks of age, splenocyte IL-2 production was decreased in Cd-exposed males while IFN-γ production was decreased from both male and female Cd-exposed offspring. The ability of the Cd-exposed offspring to respond to immunization with a S. pneumoniae vaccine expressing T-dependent and T-independent streptococcal antigens showed marked increases in the levels of both T-dependent and T-independent serum antibody levels compared to control animals. CD4(+)FoxP3(+)CD25(+) (nTreg) cell percentages were increased in the spleen and thymus in all Cd-exposed offspring except in the female spleen where a decrease was seen. CD8(+)CD223(+) T cells were markedly decreased in the spleens in all offspring at 7weeks of age. These findings suggest that even very low levels of Cd exposure during gestation can result in long term detrimental effects on the immune system of the offspring and these effects are to some extent sex-specific.

Early Postnatal Ozone Exposure Alters Rat Nodose and Jugular Sensory Neuron Development.

Sensory neurons originating in nodose and jugular ganglia that innervate airway epithelium (airway neurons) play a role in inflammation observed following exposure to inhaled environmental irritants such as ozone (O(3)). Airway neurons can mediate airway inflammation through release of the neuropeptide substance P (SP). While susceptibility to airway irritants is increased in early life, the developmental dynamics of afferent airway neurons are not well characterized. The hypothesis of this study was that airway neuron number might increase with increasing age, and that an acute, early postnatal O(3) exposure might increase both the number of sensory airway neurons as well as the number SP-containing airway neurons. Studies using Fischer 344 rat pups were conducted to determine if age or acute O(3) exposure might alter airway neuron number. Airway neurons in nodose and jugular ganglia were retrogradely labeled, removed, dissociated, and counted by means of a novel technique employing flow cytometry. In Study 1, neuron counts were conducted on postnatal days (PD) 6, 10, 15, 21, and 28. Numbers of total and airway neurons increased significantly between PD6 and PD10, then generally stabilized. In Study 2, animals were exposed to O(3) (2 ppm) or filtered air (FA) on PD5 and neurons were counted on PD10, 15, 21, and 28. O(3) exposed animals displayed significantly less total neurons on PD21 than FA controls. This study shows that age-related changes in neuron number occur, and that an acute, early postnatal O(3) exposure significantly alters sensory neuron development.

Evaluating macrophages in immunotoxicity testing.

Macrophages are the heterogeneous grouping of cells that are derived from monocytes. They have a multitude of functions depending on their final differentiated state. These functions range from phagocytosis to antigen presentation to bone destruction, to name a few. Their importance in both the innate and acquired immune functions is undeniable. Xenobiotics that degrade their functional status can have grave consequences. In this chapter, we provide an overview of the types of macrophages, their hematopoietic origin and a general discussion of the many different assays that are used to assess their functional status.

Prenatal cadmium exposure dysregulates sonic hedgehog and Wnt/beta-catenin signaling in the thymus resulting in altered thymocyte development.

Cadmium (Cd) is both an environmental pollutant and a component of cigarette smoke. Although evidence demonstrates that adult exposure to Cd causes changes in the immune system, there are limited reports in the literature of immunomodulatory effects of prenatal exposure to Cd. The sonic hedgehog (Shh) and Wnt/beta-catenin pathways are required for thymocyte maturation. Several studies have demonstrated that Cd exposure affects these pathways in different organ systems. This study was designed to investigate the effect of prenatal Cd exposure on thymocyte development, and to determine if these effects were linked to dysregulation of Shh and Wnt/beta-catenin pathways. Pregnant C57Bl/6 mice were exposed to an environmentally relevant dose (10 ppm) of Cd throughout pregnancy and effects on the thymus were assessed on the day of birth. Thymocyte phenotype was determined by flow cytometry. A Gli:luciferase reporter cell line was used to measure Shh signaling. Transcription of target genes and translation of key components of both signaling pathways were assessed using real-time RT-PCR and western blot, respectively. Prenatal Cd exposure increased the number of CD4(+) cells and a subpopulation of double-negative cells (DN; CD4(-)CD8(-)), DN4 (CD44(-)CD25(-)). Shh and Wnt/beta-catenin signaling were both decreased in the thymus. Target genes of Shh (Patched1 and Gli1) and Wnt/beta-catenin (c-fos, and c-myc) were affected differentially among thymocyte subpopulations. These findings suggest that prenatal exposure to Cd dysregulates two signaling pathways in the thymus, resulting in altered thymocyte development.

Scalable analysis of flow cytometry data using R/Bioconductor.

Flow cytometry is one of the fundamental research tools available to the life scientist. The ability to observe multidimensional changes in protein expression and activity at single-cell resolution for a large number of cells provides a unique perspective on the behavior of cell populations. However, the analysis of complex multidimensional data is one of the obstacles for wider use of polychromatic flow cytometry. Recent enhancements to an open-source platform-R/Bioconductor-enable the graphical and data analysis of flow cytometry data. Prior examples have focused on high-throughput applications. To facilitate wider use of this platform for flow cytometry, the analysis of a dataset, obtained following isolation of CD4(+)CD62L(+) T cells from Balb/c splenocytes using magnetic microbeads, is presented as a form of tutorial. A common workflow for analyzing flow cytometry data was presented using R/Bioconductor. In addition, density function estimation and principal component analysis are provided as examples of more complex analyses. The compendium presented here is intended to help illuminate a path for inquisitive readers to explore their own data using R/Bioconductor (available as Supporting Information).

A review of the immunotoxicity of the pesticide 3,4-dichloropropionanalide.

The pesticide 3,4-dichloropropionanilide (propanil or, alternatively, DCPA) is a member of the acetanilide chemical family and is predominantly used for the control of weeds on commercial rice crops worldwide. This article was written to provide a brief review of the general toxicity of propanil followed by a detailed summary of the immunotoxicity studies that were performed to date in mammalian in vivo and in vitro models. Propanil affects the immune system at organ, cellular, and molecular levels. Studies demonstrated that it produces thymic atrophy and splenomegaly and decreases developing T- and B-cell populations in the thymus and bone marrow. Natural killer (NK) cells and macrophages are critical components of the innate immune system. NK cell cytotoxicity and the ability of macrophages to phagocytose, kill pathogenic bacteria, and produce inflammatory cytokines are suppressed by propanil. Propanil also affects the respiratory burst of macrophages, inhibiting reactive oxygen and nitrogen species production. Molecular mechanisms responsible for propanil's effects have begun to be elucidated and include alterations in nuclear factor (NF)-kappaB transcription factor activity and intracellular Ca(2+) signaling. Propanil exposure alters a number of functions of mature T lymphocytes and B lymphocytes that impacts the adaptive immune response. T-cell cytotoxic activity and cytokine production are major T-cell functions inhibited by propanil. The humoral antibody response to model antigens and intact bacteria is differentially affected after propanil exposure. How these changes in innate and adaptive immune responses impact the host response to bacterial challenge or vaccination has begun to be examined.

3,4-Dichloropropionanilide (DCPA) inhibits T-cell activation by altering the intracellular calcium concentration following store depletion.

Stimulation of T cells through the T-cell receptor results in the activation of a series of signaling pathways that leads to the secretion of interleukin (IL)-2 and cell proliferation. Influx of calcium (Ca(2+)) from the extracellular environment, following internal Ca(2+) store depletion, provides the elevated and sustained intracellular calcium concentration ([Ca(2+)](i)) critical for optimal T-cell activation. Our laboratory has documented that exposure to the herbicide 3,4-dichloropropionanilide (DCPA) inhibits intracellular signaling events that have one or more Ca(2+) dependent steps. Herein we report that DCPA attenuates the normal elevated and sustained [Ca(2+)](i) that follows internal store depletion in the human leukemic Jurkat T cell line and primary mouse T cells. DCPA did not alter the depletion of internal Ca(2+) stores when stimulated by anti-CD3 or thapsigargin demonstrating that early inositol 1,4,5-triphosphate-mediated signaling and depletion of Ca(2+) stores were unaffected. 2-Aminoethyldiphenol borate (2-APB) is known to alter the store-operated Ca(2+) (SOC) influx that follows Ca(2+) store depletion. Exposure of Jurkat cells to either DCPA or 50 microM 2-APB attenuated the increase in [Ca(2+)](i) following thapsigargin or anti-CD3 induced store depletion in a similar manner. At low concentrations, 2-APB enhances SOC influx but this enhancement is abrogated in the presence of DCPA. This alteration in [Ca(2+)](i), when exposed to DCPA, significantly reduces nuclear levels of nuclear factor of activated T cells (NFAT) and IL-2 secretion. The plasma membrane polarization profile is not altered by DCPA exposure. Taken together, these data indicate that DCPA inhibits T-cell activation by altering Ca(2+) homeostasis following store depletion.

Modulating temporal control of NF-kappaB activation: implications for therapeutic and assay selection.

The activation of transcription factor NF-kappaB (nuclear factor-kappaB) plays a central role in the induction of many inflammatory response genes. This process is characterized by either oscillations or stable induction of NF-kappaB nuclear binding. Changes in dynamics of binding result in the expression of distinct subsets of genes leading to different physiological outcomes. We examined NF-kappaB DNA binding activity in lipopolysaccharide (LPS)-stimulated IC-21 cells by electromobility shift assay and nonradioactive transcription factor assay and interpreted the results using a kinetic model of NF-kappaB activation. Both assays detected damped oscillatory behavior of NF-kappaB with differences in sensitivity and reproducibility. 3,4-Dichloropropionaniline (DCPA) was used to modulate the oscillatory behavior of NF-kappaB after LPS stimulation. DCPA is known to inhibit the production of two NF-kappaB-inducible cytokines, IL-6 and tumor necrosis factor alpha, by reducing but not completely abrogating NF-kappaB-induced transcription. DCPA treatment resulted in a potentiation of early LPS-induced NF-kappaB activation. The nonradioactive transcription factor assay, which has a higher signal/noise ratio than the electromobility shift assay, combined with in silico modeling, produced results that revealed changes in NF-kappaB dynamics which, to the best of our knowledge, have never been previously reported. These results highlight the importance of cell type and stimulus specificity in transcription factor activity assessment. In addition, assay selection has important implications for network inference and drug discovery.

Developmental immunotoxicity of atrazine in rodents.

There is a substantial literature reporting that the developing immune system is more sensitive to toxic insult and that the measurable phenotype resulting from prenatal/neonatal exposure often differs from that seen in adult exposure models (reviewed in Holladay and Steven, and Smialowicz et al.). Atrazine is a common herbicidal contaminant of groundwater in agricultural areas in the USA. The potential immunotoxicity of atrazine has been extensively studied using adult-exposure models; however, few studies have explored its immunotoxicity in a prenatal and/or lactational exposure model. Prenatal/lactational atrazine exposure affects the function of young adult rodent immune systems in both sex- and age-dependant manners. In our studies, the humoural and cell-mediated immune responses of offspring from atrazine-exposed dams were assessed at two ages, 3 and 6 months of age to test the hypothesis that prenatal/lactational atrazine exposure would cause greater health complications as the mice aged. Male offspring showed a significant immunopotentiation at three moa that was not apparent at 6 months. Three-month-old female offspring showed no significant difference in immune response from controls. However, at 6 months, female litter mates showed a significant depression in their immune function. These results indicate a decreasing trend in immune capacity. Rooney et al. showed a significant depression of the immune function of young male rat exposure prenatally and lactationally to atrazine. These results demonstrate a sex- and age-dependant effect of prenatal exposure to atrazine on the immune system of the adult offspring using two rodent strains.

In vitro atrazine-exposure inhibits human natural killer cell lytic granule release.

The herbicide atrazine is a known immunotoxicant and an inhibitor of human natural killer (NK) cell lytic function. The precise changes in NK cell lytic function following atrazine exposure have not been fully elucidated. The current study identifies the point at which atrazine exerts its affect on the stepwise process of human NK cell-mediated lyses of the K562 target cell line. Using intracellular staining of human peripheral blood lymphocytes, it was determined that a 24-h in vitro exposure to atrazine did not decrease the level of NK cell lytic proteins granzyme A, granzyme B or perforin. Thus, it was hypothesized that atrazine exposure was inhibiting the ability of the NK cells to bind to the target cell and subsequently inhibit the release of lytic protein from the NK cell. To test this hypothesis, flow cytometry and fluorescent microscopy were employed to analyze NK cell-target cell co-cultures following atrazine exposure. These assays demonstrated no significant decrease in the level of target cell binding. However, the levels of NK intracellular lytic protein retained and the amount of lytic protein released were assessed following a 4-h incubation with K562 target cells. The relative level of intracellular lytic protein was 25-50% higher, and the amount of lytic protein released was 55-65% less in atrazine-treated cells than vehicle-treated cells following incubation with the target cells. These results indicate that ATR exposure inhibits the ability of NK cells to lyse target cells by blocking lytic granule release without affecting the ability of the NK cell to form stable conjugates with target cells.

3,4-dichloropropionaniline suppresses normal macrophage function.

Macrophages are a critical part of the innate immune response and natural surveillance mechanisms. As such, proper macrophage function is crucial for engulfing bacterial pathogens through phagocytosis and destroying them by generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS). The production of a number of cytokines by macrophages, such as tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1beta, and IL-6, plays an important role in the initiation of the acquired immune response creating an inflammatory environment favorable for fighting a bacterial infection. 3,4-Dichloropropionaniline (DCPA) suppresses several inflammatory parameters, including TNF-alpha production through a mechanism where nuclear factor-kappaB (NF-kappaB)-DNA binding is inhibited but not entirely abrogated. The goal of the present study was to evaluate the effects of DCPA on the inflammatory mediators of macrophages, including ROS and RNS in both murine peritoneal exudate cells and the human monocytic cell line, THP-1. The ability to perform phagocytosis and directly kill Listeria monocytogenes was also assessed. The results indicate that DCPA decreases the ability of both types of macrophages to phagocytize beads and generate both types of reactive species, which was correlated with a decrement in listericidal activity. These results demonstrate that DCPA has profound effects on macrophage function and provide insight into the potential mechanisms of immunosuppression by DCPA.

Propanil exposure induces delayed but sustained abrogation of cell-mediated immunity through direct interference with cytotoxic T-lymphocyte effectors.

The postemergent herbicide propanil (PRN ; also known as 3,4-dichloropropionanilide) is used on rice and wheat crops and has well-known immunotoxic effects on various compartments of the immune system, including T-helper lymphocytes, B lymphocytes, and macrophages. It is unclear, however, whether PRN also adversely affects cytotoxic T lymphocytes (CTLs) , the primary (1 degrees ) effectors of cell-mediated immunity. In this study we examined both the direct and indirect effects of PRN exposure on CTL activation and effector cell function to gauge its likely impact on cell-mediated immunity. Initial experiments addressed whether PRN alters the class I major histocompatibility complex (MHC) pathway for antigen processing and presentation by antigen-presenting cells (APCs) , thereby indirectly affecting effector function. These experiments demonstrated that PRN does not impair the activation of CTLs by PRN-treated APCs. Subsequent experiments addressed whether PRN treatment of CTLs directly inhibits their activation and revealed that 1 degrees alloreactive CTLs exposed to PRN are unimpaired in their proliferative response and only marginally inhibited in their lytic activity. Surprisingly, secondary stimulation of these alloreactive CTL effectors, however, even in the absence of further PRN exposure, resulted in complete abrogation of CTL lytic function and a delayed but significant long-term effect on CTL responsiveness. These findings may have important implications for the diagnosis and clinical management of anomalies of cell-mediated immunity resulting from environmental exposure to various herbicides and other pesticides.

Immunomodulatory effects of maternal atrazine exposure on male Balb/c mice.

Atrazine is a widely used herbicide applied to corn, sugar and other crops as a broad leaf weed inhibitor. Using the Balb/c mouse model, we have determined that prenatal/lactational exposure to atrazine alters adult immune function. Pregnant Balb/c dams were exposed subcutaneously for 21 days via time release pellets to 700 microg per day of atrazine beginning between days 10 and 12 of pregnancy. Prenatal/Lactational exposure caused no overt physical malformations in the offspring and had no effect on the number of litters carried to term or the litter size. Upon reaching early adulthood (approximately 3 months of age), the state of their immune system was evaluated. There were no changes in body weight or in the organ to body weight ratio of the spleen. Additionally, no changes were observed in the number of CD8+ T cell, CD4+ T cell, or B220+ B cell subpopulations in the spleen. T cell function was assessed by measuring proliferation and cytolytic activity after in vitro allogeneic stimulation. Male mice which had been prenatally/lactationally exposed to atrazine had an increase in both T cell proliferation and cytolytic activity. The humoral immune response was assessed after immunization with heat killed Streptococcus pneumoniae (HKSP). There was a significant increase in the number of HKSP-specific IgM secreting B cells in the spleen of prenatal/lactational exposed male mice. Inasmuch as atrazine is a widespread environmental contaminant, this immunopotentiation raises concerns that it may potentiate clinical diseases, such as autoimmune disease and hypersensitivity, and needs to be carefully monitored and studied.