Lysozyme mRNA Levels Research Articles

Individual and combined effects of the dietary Spirulina platensis and Bacillus licheniformis supplementation on growth performance, antioxidant capacity, innate immunity, relative gene expression and resistance of goldfish, Carassius auratus to Aeromonas hydrophila

This study evaluated the individual and combined effects of the dietary Spirulina platensis (SP) and probiotic bacterium Bacillus licheniformis (BL) on the growth performance, immune responses, and disease resistance in goldfish (Carassius auratus). A total of 216 fish (3.39 ± 0.24 g) were randomly distributed in 12 tanks with 18 fish per tank (4 treatments with 3 replications) and fed with diets containing 0% S. platensis and B. licheniformis (T0), 108 CFU/g B. licheniformis (T1), 2.5% S. platensis (T2), and 108 CFU/g B. licheniformis + 2.5% S. platensis (T3(. There were no significant differences in growth parameters. The alternative complement pathway (ACH50) and lysozyme activity were significantly increased in T2 and T3 treatments. No marked differences were observed in total immunoglobulin and protease activity among treatments (P > 0.05). The relative expression of IGF-1 was not affected by experimental diets (P > 0.05). Ghrelin gene showed significantly higher mRNA levels in fish fed with SP and BL (P < 0.05). The relative expression of catalase (CAT), and glutathione reductase (GSR) significantly increased in fish fed with the SP and BL (P < 0.05). No marked difference in glutathione peroxidase (GPX) gene expression was seen between the treatments (P > 0.05). The mRNA levels of lysozyme, IL6, IL-1β, TGF, and TNF2 transcription were higher in fish fed with SP and BL (P < 0.05). No notable difference was observed in TNF1 and IL10 gene expression between treatments (P > 0.05). Moreover, the result of the challenge test with A. hydrophila showed that goldfish fed with SP and BL had a lower mortality rate than the control. In conclusion, the supplementation of SP and BL can be used as feed additives to enhance disease resistance against A. hydrophila infection by stimulating the immune system in goldfish.

Lactobacillus johnsonii Attenuates Citrobacter rodentium–Induced Colitis by Regulating Inflammatory Responses and Endoplasmic Reticulum Stress in Mice

BackgroundProbiotics are beneficial in intestinal disorders. However, the benefits of Lactobacillus johnsonii in experimental colitis remain unknown. ObjectivesThis study aimed to investigate the benefits of L. johnsonii against Citrobacter rodentium–induced colitis. MethodsThirty-six 5-wk-old female C57BL/6J mice were randomly assigned to 3 groups (n = 12): control (Ctrl) group, Citrobacter rodentium treatment (CR) group (2 × 109 CFU C. rodentium), and Lactobacillus johnsonii and Citrobacter rodentium cotreatment (LJ + CR) group (109 CFU L. johnsonii with C. rodentium). Colon length, mucosal thickness, proinflammatory cytokine genes, and endoplasmic reticulum stress were tested. ResultsThe CR group had greater spleen weight, mucosal thickness, and Ki67+ cells (0.4–4.7 times), and a 23.8% shorter colon length than the Ctrl group, which in the LJ + CR group were 22.4%–77.6% lower and 30% greater than in the CR group, respectively. Relative to the Ctrl group, serum proinflammatory cytokines and immune cell infiltration were greater by 0.3–1.6 times and 6.2–8.8 times in the CR group, respectively; relative to the CR group, these were 19.9%–61.9% and 69.5%–84.2% lower in the LJ + CR group, respectively. The mRNA levels of lysozyme (Lyz) and regenerating islet-derived protein III were 22.7%–36.5% lower and 1.5–2.7 times greater in the CR group than in the Ctrl group, respectively, whereas they were 22.2%–25.7% greater and 57.2%–76.9% lower in the LJ + CR group than in the CR group, respectively. Cell apoptosis was 11.9 times greater in the CR group than in the Ctrl group, and 87.4% lower in the LJ + CR group than in the CR group. Consistently, the protein abundances of C/EBP homologous protein (CHOP), cleaved caspase 1 and 3, activating transcription factor 6α (ATF6A), and phospho-inositol-requiring enzyme 1α (P-IRE1A) were 0.3–2.1 times greater in the CR group and 31.1%–60.4% lower in the LJ + CR group. All these indexes did not differ between the Ctrl and LJ + CR groups, except for CD8+ T lymphocytes and CD11b+ and F4/80+ macrophages (1–1.5 times greater in LJ + CR) and mRNA concentration of Lyz2 (20.1% lower in LJ + CR). ConclusionsL. johnsonii supplementation is a promising nutritional strategy for preventing C. rodentium–induced colitis in mice.

Dietary Soy Saponin Improves Antioxidant and Immune Function of Layer Hens.

The objective of this study was to determine the effects of dietary soy saponin (SS) on the antioxidant and immune functions of laying hens. Two hundred seventy 22-week-old Hy-line gray layers were randomly allocated into three treatment groups: a control group (Control) fed a basal diet with low soybean meal and groups supplemented with 50 and 500 mg/kg SS (50 SS and 500 SS). After ten weeks, eight chickens from each treatment group were anesthetized and sacrificed to collect tissue samples. In the 50 and 500 SS groups, results showed that the levels of superoxide dismutase (SOD) in serum and spleen were elevated, and the content of malondialdehyde (MDA) in serum decreased. The mRNA levels of genes such as NF-E2-related factor 2 (Nrf-2) in the ileum and Nrf-2 and SOD in the spleen were also upregulated. In addition, the skin irritation index of phytohemagglutinin (PHA), the number of serum white blood cells, and lymphocytes were elevated in the two groups. At the same time, the number of monocytes in the blood increased in the 50 SS group, and it was significantly higher in the 500 SS group. In addition, the mRNA levels of lysozyme (LYZ) and IFN-γ in the spleen were upregulated, similar to the mRNA levels of zinc finger protein A20 (A20) in the ileum. Furthermore, the mRNA levels of NF-κB and IL-6 in the ileum were downregulated. In conclusion, with supplementation of 50 and 500 mg/kg SS in low soybean meal diets, the antioxidant, and immune functions of laying hens were improved. More importantly, the target for SS to exert biological effects on laying hens may be in the intestine and spleen tissues.

Identification and characterization of an antimicrobial peptide, lysozyme, from Suncus murinus.

Lysozyme is one of the most prominent antimicrobial peptides and has been identified from many mammalian species. However, this enzyme has not been studied in the order Insectivora, which includes the most primitive placental mammals. Here, we done the lysozyme cDNA from Suncus murinus (referred to as suncus, its laboratory name) and compare the predicted amino acid sequence to those from other mammalian species. Quantitative PCR analysis revealed a relatively higher expression of this gene in the spleen and gastrointestinal tract of suncus. The lysozyme-immunopositive (ip) cells were found mainly in the red pulp of the spleen and in the mucosa of the whole small intestine, including the follicle-associated epithelium and subepithelial dome of Peyer's patches. The lysozyme-ip cells in the small intestine were mostly distributed in the intestinal crypt, although lysozyme-expressing cells were found not only in the crypt but also in the villi. On the other hand, only a few lysozyme-ip cells were found in the villi and some granules showing intense fluorescence were located toward the lumen. As reported for other mammals, Ki67-ip cells were localized in the crypt and did not co-localize with the lysozyme-ip cells. Moreover, fasting induced a decrease in the mRNA levels of lysozyme in the intestine of suncus. In conclusion, we firstly identified the lysozyme mRNA sequence, clarified expression profile of lysozyme transcripts in suncus and found a unique distribution of lysozyme-producing cells in the suncus intestine.

Effect of dietary Bacillus coagulans supplementation on growth performance and immune responses of broiler chickens challenged by Salmonella enteritidis.

This study was conducted to evaluate the protective efficacy of dietary Bacillus coagulans (B. coagulans) supplementation in birds receiving Salmonella enteritidis (SE). Two hundred and forty 1-day-old Cobb broilers were randomly assigned to 2 × 2 factorial arrangements of treatments with 2 levels of dietary B. coagulans (0 or 400mg/kg) and 2 levels of SE challenge (0 or 1 × 109 SE between d 9 to 11). Results showed that SE infection did not affect growth performance, but caused intestinal inflammation and barrier function impairment by reducing intestinal goblet cells and beneficial bacteria numbers, increasing cecal Salmonella colonization and liver Salmonella invasion, downregulating jejunal mucin-2 (at 7 and 17 d post-infection, DPI), TLR2 (at 7 and 17 DPI), TLR4 (at 17 DPI), TNFSF15 (at 7 and 17 DPI) gene mRNA levels, and upregulating jejunal IFN-γ mRNA levels (at 17 DPI) compared to uninfected birds. Moreover, SE infection also elevated the concentration of jejunal anti-Salmonella IgA and sera anti-Salmonella IgG compared to uninfected birds. However, chickens received B. coagulans diets showed significant increase in body weight gain and weight gain to feed intake ratio from d 15 to 21, alkaline phosphatase activity (at 7 DPI), cecal Lactobacilli and Bifidobacterium numbers (at 7 DPI; at 17 DPI), villous height: crypt ratio (at 17 DPI), and goblet cell numbers (at 7 and 17 DPI), whereas exhibiting reduced jejunal crypt depth (at 17 DPI), cecal Escherichia coli (at 7, 17, and 31 DPI), and Salmonella (at 7 and 17 DPI) levels compared with the non-supplemented birds, regardless of SE infection. In addition, B. coagulans supplement upregulated lysozyme mRNA levels (at 17 DPI), downregulated IFN-γ mRNA levels (at 7 and 17 DPI), showed an increased trend in Fowlicidin-2 mRNA levels (at 7 DPI) and a reduced trend in liver Salmonella load compared to the non-supplemented control. These data indicated that B. coagulans has a protective effect in SE infected broilers.

Molecular cloning, expression and antibacterial activity of goose-type lysozyme gene in Microptenus salmoides

It is well known that lysozymes are key proteins to teleosts in the innate immune system and possess high bactericidal properties. In the present study, a g-type lysozyme gene was cloned from Microptenus salmoides. The g-type sequence consisted of 582 bp, which translated into a 193 amino acid (AA) protein (GenBank accession no: MH087462). The predicted molecular weight and theoretical isoelectric point were 21.36 kDa and 6.91 respectively and no signal peptide was observed. The qRT-PCR analysis showed that the g-type lysozyme gene was differentially expressed in various tissues under normal conditions and the highest g-type lysozyme level was observed in liver, gill and spleen while there seemed to be low expression in the muscle, heart and head-kidney. The expression of g-type lysozyme was differentially upregulated in the spleen, gill and intestine after stimulation with heat stress and Aeromonas hydrophila (A. hydrophila). Under heat stress and A. hydrophila injection, the g-type lysozyme mRNA levels all in spleens, gill and intestine tissues increased significantly (P < 0.05), with the maximum levels attained at 12 h, 24 h (or 12 h) and 24 h. Thereafter, they all decreased significantly (P < 0.01) and the expression in gill returned to nearly the basal value within 72 h. Those results suggested that g-type lysozyme was involved in the immune response to heat stress and bacterial challenge. The cloning and expression analysis of the g-type lysozyme provide theoretical basis to further study the mechanism of anti-adverseness in Microptenus salmoides. The g-type lysozyme gene perhaps also played an important role in the immune responses against bacterial invasion.

Efficacy of PLGA microparticle-encapsulated formalin-killed Aeromonas hydrophila cells as a single-shot vaccine against A. hydrophila infection

Control and prevention of disease is a high priority in aquaculture, and vaccination is important to prevent outbreaks. Here, poly(d,l-lactide-co-glycolic acid) (PLGA) microparticles (MPs) approximately 36μm in diameter were used to encapsulate and deliver Aeromonas hydrophila formalin-killed cells (FKC) as an antigen, and the innate and adaptive immune responses of cyprinid loaches and common carp were assessed following vaccination. The antigen was confirmed to be well encapsulated by scanning electron microscopy analysis of PLGA MP sections. Blood and head kidney specimens were collected and analyzed for bacterial agglutination activity and relative mRNA expression of immune-related genes (IL-1β, IL-10, TNF-α, lysozyme C, TGF-β, and IgM) at 2, 4, 6, and 8weeks post vaccination (wpv). For both fish species, the curve of antibody titer over time was shallower in the PLGA group than the FKC group. These titers in loaches and carp were very similar in the two vaccination groups until 8 and 6 wpv, respectively, but differences were subsequently noted in both species until the end of experiment. Loaches and carp were then challenged with A. hydrophila at 12 and 20 wpv, and 10 and 14 wpv, respectively, and relative survival rates were calculated. For both species, the PLGA groups demonstrated higher survival rates at all time points. Relative expression of IL-1β and TNF-α mRNA was significantly upregulated in the PLGA group at 2 and 4 wpv. Moreover, PLGA-MP vaccination increased relative mRNA levels of lysozyme C and IgM, which were significantly higher than those observed with FKC treatment at 2 wpv and 4, 6, and 8 wpv, respectively. In conclusion, PLGA-MP vaccines have the potential to induce longer and more potent immune responses than FKCs alone, and protect both cyprinid loaches and common carp with greater efficiency.

Differential proteome profile of skin mucus of gilthead seabream (Sparus aurata) after probiotic intake and/or overcrowding stress

Gilthead seabream (Sparus aurata L.) is the major cultured fish species in the Mediterranean area. High density stocking causes stress and increases the impact of diseases leading to economic losses. Probiotics could represent a solution to prevent diseases through several mechanisms such as improving the immune status and/or mucosal microbiota or competing with pathogens. The probiotic Shewanella putrefaciens, also known as Pdp11, was firstly isolated from the skin of healthy gilthead seabream. Our study focuses on the skin mucus proteome after dietary probiotic Pdp11 intake in fish maintained under normal or overcrowding conditions. 2-DE of skin mucus followed by LC–MS/MS analysis was done for each experimental group and differentially expressed proteins were identified. The results showed differentially expressed proteins especially involved in immune processes, such as lysozyme, complement C3, natural killer cell enhancing factor and nonspecific cytotoxic cell receptor protein 1, whose transcript profiles were studied by qPCR. A consistency between lysozyme protein levels in the mucus and lysozyme mRNA levels in skin was found. Further research is necessary to unravel the implications of skin mucosal immunity on fish welfare and disease. Biological significanceThe present work reveals the proteomic changes, which are taking place in the skin mucus of stressed and non-stressed gilthead seabream after Pdp11 probiotic intake. The study contributes to improving the knowledge on skin mucosal immunology of this relevant farmed fish species. Furthermore, the paper shows for the first time how a suitable proteomic methodology, in this case 2-DE followed by LC–MS/MS is useful to perform a comparative study with a non-invasive technique of skin mucus of gilthead seabream.

Partial Enteral Nutrition Preserves Elements of Gut Barrier Function, Including Innate Immunity, Intestinal Alkaline Phosphatase (IAP) Level, and Intestinal Microbiota in Mice

Lack of enteral nutrition (EN) during parenteral nutrition (PN) leads to higher incidence of infection because of gut barrier dysfunction. However, the effects of partial EN on intestina linnate immunity, intestinal alkaline phosphatase (IAP) and microbiota remain unclear. The mice were randomized into six groups to receive either standard chow or isocaloric and isonitrogenous nutritional support with variable partial EN to PN ratios. Five days later, the mice were sacrificed and tissue samples were collected. Bacterial translocation, the levels of lysozyme, mucin 2 (MUC2), and IAP were analyzed. The composition of intestinal microbiota was analyzed by 16S rRNA pyrosequencing. Compared with chow, total parenteral nutrition (TPN) resulted in a dysfunctional mucosal barrier, as evidenced by increased bacterial translocation (p < 0.05), loss of lysozyme, MUC2, and IAP, and changes in the gut microbiota (p < 0.001). Administration of 20% EN supplemented with PN significantly increased the concentrations of lysozyme, MUC2, IAP, and the mRNA levels of lysozyme and MUC2 (p < 0.001). The percentages of Bacteroidetes and Tenericutes were significantly lower in the 20% EN group than in the TPN group (p < 0.001). These changes were accompanied by maintained barrier function in bacterial culture (p < 0.05). Supplementation of PN with 20% EN preserves gut barrier function, by way of maintaining innate immunity, IAP and intestinal microbiota.

Differential Expression of Immune-Related Genes in Larval and Adult Apis mellifera Honeybees Challenged with Korean Sacbrood Virus

Recently, the Korean sacbrood virus (KSBV) caused great loss of honeybee colonies in Korean Apis mellifera. Three antimicrobial peptide genes (AMPs) (abaecin (ABA), defensin (DEF) and hymenoptaecin (HYM)) and two immune-related enzyme genes (phenol-oxidase (PO) and lysozyme (LYS)) were investigated using qRT-PCR as a measure of the defense reactions of honeybee larvae and adults challenged with KSBV in vitro. Transcript levels of three AMPs were upregulated in adults exposed to KSBV, compared to control adults. ABA and DEF transcripts were 22.7 and 21.9 fold higher (P<0.05), respectively, after 24h exposure while HYM transcript did not change significantly. At this time point, PO and LYS mRNA levels were 18.08 and 16.03 fold higher, respectively, in KSBVinfected adult bees compared to control adults (P<0.05). By contrast, the larvae infected with KSBV had downregulated expression of AMPs and slight up-regulation of PO and LYS compared to the controls. These results suggest that the differential expression of the genes studied could indicate their involvement in defense processes against KSBV in adult and larva bees.

Differential expression of vitellogenin in honey bees (Apis mellifera) with different degrees of Nosema ceranae infection

SummaryNosema apis and Nosema ceranae are causative agents of Nosemosis in the honey bee Apis mellifera, although N. ceranae may cause a more virulent disease. Selection of colonies with high tolerance to N. ceranae could be important for reducing problems caused by this pathogen. The aim of the present work was to evaluate the existence of honey bee colonies with different degrees of N. ceranae infection and test if this difference could be related to the immune response or vitellogenin expression. Healthy honey bee colonies were relocated to a plantation of Eucalyptusgrandisto favour natural infection of N. ceranae. Fifteen and thirty days after relocation, the proportion of infected bees and the number of N. ceranae spores per field were quantified. The colonies with higher and lower levels of infection (HL and LL, respectively) were selected. Newly emerged bees from both colonies were artificially infected with N. ceranaeand seven days after infection the expression of immune related genes and vitellogenin was evaluated by real time PCR. No significant differences were observed in expression of abaecin, hymenoptaecin, defensin, glucose dehydrogenase or lysozyme mRNA levels between infected bees from HL and LL colonies or between control bees from both colonies. Vitellogenin expression was higher in bees from the LL colony than in bees from the HL colony, when infected or control bees were compared between them. This protein possesses pleiotropic effects and is a central element in the life-history of honey bees. For that reason, its differential expression could be associated with resistance to N. ceranae.

AML1/ETO-Mediated Lysozyme Repression Is Independently Relieved by Inhibitors of DNA Methylation and Histone Deacetylation.

The human lysozyme (LZM) gene, a marker gene for myeloid-specific development, is highly methylated in immature myeloid and in non-myeloid cells (all LZM-negative), and unmethylated in LZM-expressing mature phagocyte cells. Thus this gene provides an excellent model for investigating differentation-associated DNA methylation changes during myelopoiesis. There is now increasing evidence that LZM (containing five perfect consensus binding sites for AML1/RUNX1 in its 5′ region) is repressed by the AML1/ETO chimeric transcription factor (Fliegauf et al, Oncogene 23:9070–81, 2004), and this repression can be relieved by siRNA-mediated AML1/ETO depletion in AML1/ETO-positive Kasumi-1 cells (Dunne et al., Oncogene, 2006). Recently, AML1/ETO has also been implicated in gene-specific epigenetic repression of interleukin-3 (Liu et al, Cancer Res 65, 1277–84, 2005).By extensive methylation analyses of the LZM gene including bisulfite sequencing, we now demonstrate marked demethylation in both the CpG-poor 5′ region and the exonic CpG island after treatment of Kasumi-1 cells with non-cytotoxic concentrations of the DNA methyltransferase (DNMT) inhibitor 5-aza-2′-deoxycytidine (5-azaCdR), which was not associated with cellular differentiation. By Northern blot analysis, LZM mRNA levels in Kasumi-1 cells but not in AML1/ETO-negative HL-60 and U-937 cell lines were specifically and independently upregulated upon treatment with 5-azaCdR and, to a lesser extent, with the histone deacetylase (HDAC) inhibitor Trichostatin A (TSA). Combined treatment with subliminal concentrations of 5-azaCdR and TSA applied in different schedules did not reveal synergistic effects on LZM transcription. Relative chromatin accessibility of the LZM 5′ region, as detected by “MspI protection” assay, and associated with partial demethylation in several myeloid cell lines, was increased in Kasumi-1 with 5-azaCdR-induced further DNA demethylation, but not by TSA. As shown by chromatin immunoprecitation, TSA increased the acetylation of histones H3 and H4 both in the 5′ flanking region and exonic CpG island. In a U-937 inducible model, antagonization of AML1/ETO-mediated repression of LZM was achieved by TSA, implying that the histone deacetylation in this region of the human LZM gene is mediated by AML1/ETO protein.In conclusion, we demonstrate functional interactions between DNA methylation and histone modifications in mediating LZM gene repression which implicate AML1/ETO as one component involved in local chromatin remodelling. Interestingly, inhibitors of DNA methylation and histone deacetylation independently relieve repression of this CpG-poor gene in AML1/ETO-positive cells.

Molecular cloning, expression and evolution of the Japanese flounder goose-type lysozyme gene, and the lytic activity of its recombinant protein

In this study, we cloned the goose-type (g-type) lysozyme gene from the Japanese flounder genomic DNA library, the first such data in fish and only the second after the chicken g-type lysozyme gene. The Japanese flounder g-type lysozyme gene was 1252 bp in length from the transcription site to the polyadenylation site, coded for 758 bp of mRNA and 195 deduced amino acids, which contain five exons and four introns. A phylogenetic analysis based on amino acid sequences showed that the flounder gene was closer to g-type lysozyme, followed by phage-type lysozyme and then chicken-type (c-type) lysozyme. Although exon 1 of the flounder gene differs from exons 1 and 2 of the chicken g-type lysozyme gene, three catalytic residues, as well as their neighboring amino acids were conserved between the Japanese flounder and the four avian g-type lysozymes. In a Southern blot analysis using the genomic DNA of homo-cloned Japanese flounder, the flounder g-type lysozyme gene showed a simple pattern, suggesting that it is encoded by a single copy gene. A Northern blot analysis showed that this gene was expressed in all tissues of Japanese flounder that we examined in this study and showed major differences from those expressed tissues of the chicken g-type gene. Japanese flounder g-type lysozyme mRNA levels in the intestine, heart and whole blood increased after injecting the fish with Edwardsiella tarda. Recombinant flounder g-type lysozyme, which has an optimal pH and temperature of pH 6.0 and 25°C, possessed lytic activity against Micrococcus lysodeikticus and several fish pathogenic bacteria. This is the first report of a g-type lysozyme gene other than for reported avian species.

Monocyte chemoattractant protein 1 is responsible for macrophage recruitment following injury to sciatic nerve.

Following injury to the peripheral nervous system, circulating monocytes/macrophages are recruited to the damaged tissue, where they play vital roles during both nerve degeneration and subsequent regeneration. Monocyte chemoattractant protein-1 (MCP-1), a member of the C-C or beta-chemokine family, is a powerful leukocyte recruitment/activation factor that is relatively specific for monocytes/macrophages. Because these are the predominant leukocyte type recruited by injured nerve, we hypothesized that upregulation of MCP-1 expression is involved in recruitment of these cells. Indeed, assay of steady-state levels of MCP-1 mRNA in rat sciatic nerve during tellurium-induced primary demyelination indicated up-regulation of this chemokine with a peak after 3 days of tellurium exposure, preceding the peak of accumulation of phagocytic macrophages (assayed as lysozyme mRNA levels) by 6 days. Increasing levels of MCP-1 mRNA expression, induced by increasing levels of tellurium exposure, resulted in corresponding increases in subsequent recruitment of macrophages. In situ hybridization suggested that MCP-1 mRNA was localized in Schwann cells. No expression of MIP-2, which is a C-X-C or alpha-chemokine that is specific for recruitment of neutrophils, was detected, consistent with the lack of recruitment of significant numbers of these cells. In addition, we also investigated the response seen following nerve transection (axonal degeneration and secondary demyelination with no subsequent regeneration) and nerve crush (degeneration followed by regeneration). In these latter two nerve injury models, there was also a marked, early up-regulation of MCP-1 mRNA, with a time course that is compatible with a role for this chemokine in macrophage recruitment. We conclude that MCP-1 is involved in recruiting monocytes/macrophages to injured peripheral nerve and that the specificity of leukocyte types recruited results from specificity of chemokine production.

Regulation of the secretory phenotype of human airway epithelium by retinoic acid, triiodothyronine, and extracellular matrix.

The purpose of our studies was to identify factors which regulate the composition of airway secretions produced by normal human tracheobronchial epithelial (NHTBE) cells. Individual factors were removed from the culture media of NHTBE cells grown in air-liquid interface (ALI) cultures (which support mucociliary differentiation) and the effects on mucin, lysozyme (LZ), and secretory leukocyte protease inhibitor (SLPI) secretion and gene expression were examined. Deletion of hydrocortisone, epinephrine, transferrin, or gentamycin-amphotericin from the media had no reproducible effects; deletion of insulin was incompatible with culture growth. We identified 3 factors, namely retinoic acid (RA), triiodothyronine (T3) and collagen gel substratum, which had a major impact on the profile of NHTBE secretions. Removal of RA from the media caused a drastic decrease in mucin secretion and a decrease in expression of the mucin genes MUC2 and MUC5AC.LZ and SLPI secretions were increased in these cultures. Paradoxically LZ mRNA was decreased, while SLPI mRNA levels were increased. Removal of T3 selectively increased mucin secretion, MUC2 gene expression was not affected, but MUC5AC mRNA levels reproducibly increased, suggesting that the expression of these two mucin genes is differentially regulated. LZ and SLPI secretion levels were not significantly affected by deletion of T3 from the culture media; however, LZ mRNA levels were increased in the absence of T3 while SLPI transcript levels were not affected. Omission of the attachment substratum, type I collagen gel, resulted in significant increases in all 3 secretory products. MUC2 and MUC5AC steady state mRNA levels were not consistently affected. In contrast LZ and SLPI gene expression were reproducibly increased. Our studies show that individual factors in the epithelial environment can regulate expression of specific secretory cell gene products in a highly selective manner.

Lysozyme is an inducible marker of macrophage activation in murine tissues as demonstrated by in situ hybridization.

This study demonstrates the induction of lysozyme mRNA expression in situ in tissue macrophages (M phi) of mice following in vivo stimulation. The resting resident tissue M phi of most tissues do not contain enough lysozyme mRNA to be detected by in situ hybridization using 35S-labeled RNA probes. Following Bacille Calmette Guerin or Plasmodium yoelli infection, however, M phi recruited to liver and spleen hybridize strongly to the lysozyme probe. Within 24 h of infection, cells found in the marginal zone of the spleen begin to produce lysozyme mRNA. This response is also evoked by a noninfectious agent (intravenously injected sheep erythrocytes), and is possibly the result of an early phagocytic interaction. Later in the infection, other cells in the red and white pulp of the spleen, and cells in granulomas in the liver, become lysozyme-positive. Kupffer cells are rarely lysozyme-positive. Lysozyme mRNA levels in liver granulomas remain relatively constant during the infection, and lysozyme is produced by most granuloma cells. This contrasts with tumor necrosis factor alpha (TNF alpha) mRNA, which is produced by fewer cells in the granuloma, and which can be massively induced by lipopolysaccharide administration. The production of lysozyme, previously considered a constitutive function of M phi, is therefore an indicator of M phi activation in vivo, where immunologically specific and nonspecific stimuli both stimulate lysozyme production at high levels in subpopulations of cells occupying discrete anatomical locations.