Gland Macrophages Research Articles

Activated Nuclear Factor Kappa B and Airway Inflammation After Smoke Inhalation and Burn Injury in Sheep

In a recent study, we have shown a rapid inflammatory cell influx across the glandular epithelium and strong proinflammatory cytokine expression at 4 hours after inhalation injury. Studies have demonstrated a significant role of nuclear factor kappa B in proinflammatory cytokine gene transcription. This study examines the acute airway inflammatory response and immunohistochemical detection of p65, a marker of nuclear factor kappa B activation, in sheep after smoke inhalation and burn injury. Pulmonary tissue from uninjured sheep and sheep at 4, 8, 12, 24, and 48 hours after inhalation and burn injury was included in the study. Following immunostaining for p65 and myeloperoxidase, the cell types and the percentage of bronchial submucosal gland cells staining for p65 and the extent of myeloperoxidase stained neutrophils in the bronchial submucosa were determined. Results indicate absence of detection of P65 before 12 hours after injury. At 12 hours after injury, strong perinuclear staining for p65 was evident in bronchial gland epithelial cells, macrophages, and endothelial cells. Bronchial submucosal gland cells showed a significant increase in the percentage of cells stained for p65 compared with uninjured animals and earlier times after injury, P < .05. At 24 and 48 hours after injury, p65 expression was evident in the bronchiolar epithelium, Type II pneumocytes, macrophages, and endothelial cells. Quantitation of the neutrophil influx into the bronchial submucosa showed a significant increase compared with uninjured tissue at 24 and 48 hours after injury, P < .05. In conclusion, immunohistochemical detection of activated p65 preceded the overall inflammatory response measured in the lamina propria. However, detection of p65 did not correlate with a recent study showing rapid emigration of neutrophils at 4 hours postinjury. Together, these results suggest that p65 immunostaining may identify cells that are activated to produce proinflammatory cytokines after injury; however, the immunoexpression may not adequately reflect the temporal activation of gene transcription that may occur with proinflammatory cytokine production with inhalation injury.

Mammary Gland Macrophages: Pleiotropic Functions in Mammary Development

Mammary gland development is a complex process involving epithelial cells and supporting stromal cells. Macrophages (MØs) are an important component of the mammary gland stroma and are critical for normal mammary gland development; however, the mechanisms by which macrophages regulate these processes are not well understood. MØs are known to interact with numerous cell types, including epithelial cells, fibroblasts, adipocytes, and endothelial cells, all of which are significant components of mammary gland development. Therefore, the purpose of this review is to describe the interactions between MØs and these various cell types and use this knowledge to identify potential functions of MØs in the mammary gland.

Immune surveillance of mammary tissue by phagocytic cells.

The leukocytes in milk consist of lymphocytes, neutrophil polymorphonuclear leukocytes (PMN) and macrophages. Lymphocytes together with antigen-presenting cells function in the generation of an effective immune response. Lymphocytes can be divided into two distinct subsets, T- and B-lymphocytes, that differ in function and protein products. The professional phagocytic cells of the bovine mammary gland are PMN and macrophages. In the normal mammary gland macrophages are the predominate cells which act as sentinels to invading mastitis causing pathogens. Once the invaders are detected, macrophages release chemical messengers called chemoattractants that cause the directed migration of PMN into the infection. Migration of neutrophils into mammary tissue provides the first immunological line of defense against bacteria that penetrate the physical barrier of the teat canal. However, their presence is like a double-edged sword. While the PMN are phagocytosing and destroying the invading pathogens, they inadvertently release chemicals which induces swelling of secretory epithelium cytoplasm, sloughing of secretory cells, and decreased secretory activity. Permanent scarring will result in a loss of milk production. Resident and newly migrated macrophages help reduce the damage to the epithelium by phagocytosing PMN that undergo programmed cell death through a process called apoptosis. Specific ligands on the neutrophil surface are required for directed migration and phagocytosis. In response to infection, freshly migrated leukocytes express greater numbers of cell surface receptors for immunoglobulins and complement and are more phagocytic than their counterparts in blood. However, phagocytic activity rapidly decreases with continued exposure to inhibitory factors such as milk fat globules and casein in mammary secretions. Compensatory hypertrophy in non-mastitic quarters partially compensates for lost milk production in diseased quarters. Advances in molecular biology are making available the tools, techniques, and products to study and modulate host-parasite interactions. For example the cloning and expression of proteins that bind endotoxin may provide ways of reducing damaging effects of endotoxin during acute coliform mastitis. The successful formation of bifunctional monoclonal antibodies for the targeted lysis of mastitis causing bacteria represents a new line of therapeutics for the control of mastitis in dairy cows.

Expression of CD3 and CD11b antigens on blood and mammary gland leukocytes and bacterial survival in milk of cows with experimentally induced Staphylococcus aureus mastitis

To differentiate early (1 to 8 days) from late (9 to 14 days) inflammatory phases and assess relationships between leukocyte phenotype and bacterial recovery in cows with Staphylococcus aureus-induced mastitis. 10 first-lactation Holstein cows. Blood and milk samples were collected from 4 or 6 cows before and after intramammary infusion of sterile broth or S. aureus, respectively. Flow cytometric expression of CD3 and CD11b antigens on blood and milk leukocytes, leukocyte differential counts, bacterial counts in milk, and somatic cell counts were determined longitudinally. Density of CD3 molecules decreased on blood lymphocytes and increased on milk lymphocytes after infusion of bacteria. Density of CD11b molecules on lymphocytes and phagocytes and percentage of CD11b+ lymphocytes in milk increased significantly after infusion; maximum values were achieved during the early inflammatory phase. Density of CD3 and CD11b molecules on milk lymphocytes and macrophages, respectively, 1 day after inoculation were negatively correlated with bacterial recovery on day 1 and days 9 to 14, respectively. Density of CD11b molecules on milk macrophages and the ratios of phagocyte to lymphocyte percentages and polymorphonuclear cell to macrophage percentages in milk differentiated the early from the late inflammatory phase. Activation of bovine mammary gland macrophages and T cells in response to intramammary infusion of S. aureus was associated with an inability to culture this bacterium from milk. Identification of specific inflammatory phases of S. aureus-induced mastitis in cows may allow for the design of more efficacious treatment and control programs.

Johne's Disease and Milk: Do Consumers Need to Worry?

Mycobacterium paratuberculosis, an acid-fast bacillus that causes enteritis in ruminants, has been suggested as an etiological agent of Crohn's disease in humans. The mode of transmission is unclear; however, some evidence suggests that humans may become infected via contaminated milk. Currently, it is not known whether commercial pasteurization effectively kills M. paratuberculosis in contaminated raw milk. Using a laboratory-scale pasteurizer unit designed to simulate the high-temperature, short-time method (72°C, 15sec) currently used by commercial dairies, we previously demonstrated that treatment of raw milk inoculated with 104 to 106 cfu of M. paratuberculosis/ml reduced numbers to an undetectable level. However, M. paratuberculosis is an intracellular pathogen that resides within the macrophages of the host and evades destruction. We subsequently performed further experiments examining heat treatment of milk inoculated with mammary gland macrophages containing ingested M. paratuberculosis. Heat treatment of these samples under high-temperature, short-time conditions demonstrated that the macrophage does not protect the organism because we were unable to recover any viable M. paratuberculosis from the samples. Conversely, other researchers have demonstrated that a residual population of M. paratuberculosis may survive heat treatment of milk. In addition, a recent news report stated that viable M. paratuberculosis organisms have been cultured from retail-ready milk in Ireland. A summary of past and current studies concerning this issue along with a discussion of methodologies used to recover M. paratuberculosis from experimentally inoculated milk will be presented in this paper.

Differential distribution of Fc gamma RIIIa in normal human tissues and co-localization with DAF and fibrillin-1: implications for immunological microenvironments.

Fc gamma RIIIa is a cytokine-inducible IgG Fc receptor implicated in the activation of macrophages by immune complexes. Differential expression of Fc gamma RIIIa by macrophages in different tissues may therefore modulate local immune responsiveness. Fc gamma RIIIa expression in normal human tissues was assessed semiquantitatively using microdensitometry. Synovial intimal, serosal, alveolar, salivary gland and placental macrophages, Kupffer cells, and macrophages in mechanically stressed dermis expressed high levels of Fc gamma RIIIa. Less consistent expression was seen in skeletal muscle and lymphoid organs. No significant expression was observed in brain, thyroid, spine, intestine, myocardium, prostate, uterus, flexor forearm dermis, uterus, or kidney. Staining for Fc gamma RIII was also observed on extracellular matrix, and co-localized with both complement decay-accelerating factor and fibrillin-1. It is proposed that differential levels of both cellular and extracellular Fc gamma RIIIa, by modulating the response to immune complexes, may contribute to relative tissue susceptibility to infection and autoimmune disease.

Distinct populations of macrophages in the adult rat adrenal gland: a subpopulation with neurotrophin-4-like immunoreactivity

Macrophages are widely distributed in lymphohaemopoietic and many other mammalian tissues, where they are mainly involved in host defence mechanisms, phagocytosis, wound repair, and secretion of growth factors. Increasing evidence suggests that secretory products of macrophages can influence adrenal gland functions. In the present study, we have used specific antibodies to ED1 (cytoplasmic antigen), ED2 (membrane antigen), ED8 (membrane antigen), and OX-6 (MHC class II/membrane antigen) as markers for macrophages to examine their distribution within the adult rat adrenal gland. ED2 and OX-6 recognize distinct subpopulations of adrenal gland macrophages, whereas macrophages immunoreactive (-ir) for ED1 and ED8 could not be detected. OX-6-ir macrophages were most numerous in the cortical reticularis and glomerulosa zones, while only few cells were found in the zona fasciculata and in the adrenal medulla. Macrophages immunoreactive for ED2 were restricted to the adrenal medulla. The majority of these macrophages were associated with vascular sinuses or chromaffin cells. By double-immunolabelling we found that most of ED2-ir medullary macrophages contain neurotrophin-4 (NT-4)-like ir. Attempts to clarify whether macrophages take up NT-4 from NT-4-ir chromaffin cells indicated that medullary macrophages are immunonegative for chromogranin A and neuropeptide Y, two major secretory products of chromaffin cells. In situ hybridizations and immunofluorescence showed expression of the neurotrophin receptor TrkA, but not TrkB in the adrenal medulla. In vitro studies indicated that NT-4, similar to nerve growth factor, can induce c-fos-ir in chromaffin cells. We conclude that chromaffin cells are putative targets for adrenal medullary NT-4, whose functions remain to be clarified.

Glucocorticoids modulate the cellular disposition of lipocortin 1 in the rat brain in vivo and in vitro.

The role of glucocorticoids in the regulation of lipocortin 1 (LC1) mRNA/protein expression in the brain is uncertain. This study has examined the influence of dexamethasone on the disposition of LC1 protein in various central and peripheral tissues in the rat. LC1 was readily detectable in all tissues studied by Western blot analysis. Exposure to dexamethasone in vitro (0.1 microM, 3 h) or in vivo (20 micrograms/100 g i.p., 24 h before autopsy) had no discernible effects on intracellular LC1 levels but increased the amount of LC1 attached to the outer surface of cells (pericellular LC1) in cortex, hippocampus, hypothalamus, pituitary gland and peritoneal macrophages (in vitro only). The results suggest that in central tissues, as in the periphery, glucocorticoids promote the translocation of LC1 from intracellular to pericellular sites.

Phagocytosis of Streptococcus uberis by bovine mammary gland macrophages

The ability of macrophages isolated from the bovine mammary gland to phagocytose and kill Streptococcus uberis was investigated. The strains of S uberis used were selected on the basis of their known resistance (C197C) or sensitivity (EF20) to phagocytosis by bovine peripheral blood neutrophils in the same assay system. Macrophages isolated from mammary secretions collected during the mid-dry period were capable of phagocytosing both strains of S uberis in the presence of serum and skimmed milk. The removal of complement from serum by heat-inactivation did not influence the opsonization of S uberis for phagocytosis by mammary macrophages and both IgGI and IgG2 isotypes were found to opsonise both strains. The uptake of S uberis into the cells was confirmed by electron microscopy. Potential mechanisms by which S uberis could resist phagocytosis by neutrophils but not by macrophages are discussed.

Influence of Streptococcus dysgalactiae surface hydrophobicity on adherence to mammary epithelial cells and phagocytosis by mammary macrophages.

Bacterial surface hydrophobicity (SH) plays a role in adhesion of bacteria to host surfaces and ingestion by phagocytic cells. Streptococcus dysgalactiae (n = 60) isolated from bovine intramammary infections were examined for expression of SH after growth in Todd-Hewitt broth (THB) and THB supplemented with skim milk, whey, lactose, and casein. Strains were significantly more hydrophobic after growth in THB and THB plus whey and more hydrophilic after growth in THB plus skim milk. Both trypsin and proteinase K abolished SH in three strains tested. Mild pepsin treatment had little effect on SH, while heat treatment at 70 degrees or 80 degrees C abolished SH in two strains tested. A hydrophilic strain of S. dysgalactiae did not adhere as well to bovine mammary epithelial cells as a hydrophobic strain. Trypsin treatment significantly reduced adherence of a hydrophobic strain of S. dysgalactiae to epithelial cells while adherence of a hydrophilic strain remained unaltered. A hydrophilic strain of S. dysgalactiae was significantly more resistant to phagocytosis by bovine mammary gland macrophages than a hydrophobic strain. Differences in expression of SH may play an important role in determining the ability of S. dysgalactiae to establish successfully within the mammary gland.

Effects of in vitro supplementation of bovine mammary gland macrophages and peripheral blood lymphocytes with α-tocopherol and sodium selenite: implications for udder defences

Vitamin E and selenium have been observed to enhance the functions of bovine mammary gland macrophages and peripheral blood lymphocytes. In vitro supplementation with these compounds enhanced the production of neutrophil chemotaxins by macrophages stimulated with opsonised Staphylococcus aureus. Supplementation also enhanced the proliferation of peripheral blood lymphocytes in response to stimulation with concanavalin A but not to phytohaemagglutinin or pokeweed mitogen. There was no evidence of additive or synergistic effects of vitamin E and selenium. Results suggest that supplementation of cattle may optimise resistance to mastitis by enhancing the functions of resident macrophage and lymphocyte populations.

Effect of Tumor Necrosis Factor-α, Interleukin-1β, and Antibiotics on the Killing of Intracellular Staphylococcus aureus

A bovine blood polymorphonuclear neutrophil system was used to determine the ability of cytokines, alone or in combination with various antibiotics, to enhance the intracellular killing of Staphylococcus aureus sequestered within these phagocytes. Pretreatment of blood polymorphonuclear neutrophils with human recombinant tumor necrosis factor-α, bovine macrophage supernatant tumor necrosis factor-α, or human recombinant interleukin-1β enhanced the killing of S. aureus by the phagocytes compared with that of untreated polymorphonuclear neutrophils. This enhancement was dose-dependent and required 30min of contact between the cytokine and the blood polymorphonuclear neutrophils. However, mammary gland polymorphonuclear neutrophils, macrophages, and blood monocytes showed no enhanced killing activity following pretreatment with tumor necrosis factor. Pretreatment of polymorphonuclear neutrophils with tumor necrosis factor and subsequent exposure to various antibiotics after infection with S. aureus enhanced intracellular bacterial killing by certain antibiotics, including paldimycin, rifampin, and ciprofloxacin (antibiotics that normally kill intracellular S. aureus). However, no enhancement occurred with antibiotics that do not normally kill intracellular S. aureus. Cytokine therapy may be a useful adjunct to antibiotic therapy in the treatment of bovine staphylococcal infection and mastitis.

Regulation of mammary gland macrophage tumour necrosis factor-α production with interferon-γ

Recombinant bovine interferon (rBOIFN)-γ was found to modify the production of tumour necrosis factor ( TNF-α) production by lipopolysaccharide (LPS) stimulated mammary gland macrophages. Alveolar macrophages isolated from bovine mammary glands were incubated with 0, 1, 10, 100 or 1000 U of rBOIFN-γ either three hours before or three hours following stimulation with LPs. The level of TNF-α production was not affected when macrophages were treated with rBoiFN before LPs stimulation. In contrast, macrophages treated with 10,100 or 1000 U of rBOIFN after stimulation with LPs produced significantly lower concentrations Of TNF-α when compared with non-treated cultures and those treated with only 1 U of interferon (IFN)-γ. These results indicate that IFN-γ is capable of down-regulating the production of TNF-α by a local macrophage population. The ability of IFN-γ to modify the production of TNF-α is dependent on the timing of administration relative to LPs exposure. The significance of these findings is that under some circumstances, low dose administration of IFN-γ may be directly responsible for down-regulation of acute phase inflammatory responses which may prevent pathological alterations to host tissues during endotoxaemia.

Infection of bovine monocyte/macrophage populations with Theileria annulata and Theileria parva

Infection and transformation of cells of the bovine immune system by Theileria annulata and T. parva were compared. Preliminary experiments with mammary gland macrophages indicated that they were permissive to infection by T. annulata but only to a limited extent by T. parva. Further experiments involved several purified subpopulations of bovine cells including bovine monocytes, T cells and MHC class II positive and negative populations. These subpopulations were incubated with T. annulata or T. parva sporozoites in limiting dilution cultures. T. annulata preferentially infected macrophage type cells and also MHC class II positive cells, whereas the frequency of MHC class II negative cells infected by this parasite was negligible. T cells also showed a very low level of infection. In complete contrast, T. parva preferentially infected T cells and did not infect cells phenotypically defined as monocytes at all. These results suggested that class II expression was necessary for T. annulata infection and not necessary for, though not a barrier to T. parva infection. T. annulata infected cell lines all expressed class II molecules to varying degrees. Other available phenotypic markers were only expressed at very low levels or no longer expressed. The immunological significance of the different cell preferences and phenotypes of infected cell lines of T. annulata and T. parva is discussed.

Day-night variability in acid phosphatase activity of pineal gland macrophages

Acid phosphatase (ACP) activity was investigated in the photoreceptive pineal gland of the goldfish by both light and electron microscopy. Intraluminal macrophages exhibited the greatest ACP reactivity that was generally confined to lysosomal bodies containing membranous debris. Quantitative light microscopic evaluation revealed significantly higher ACP reactivity in animals collected at mid-light compared to those collected at mid-dark suggesting that functional activities of macrophages in this photoneuroendocrine organ are cyclical over the 24-h photoperiod. Day-night differences in macrophage ACP activity may reflect rhythmic renewal and shedding of photoreceptor outer segment membranes.

Evaluation of antibiotic effectiveness against Staphylococcus aureus surviving within the bovine mammary gland macrophage.

Bovine mastitis due to Staphylococcus aureus may become chronic and refractory to antibiotic therapy because of the organism's ability to survive within the mammary gland macrophages and/or polymorphonuclear neutrophils (PMNs). Therefore, phagocytosis and killing of S. aureus by bovine udder macrophages, udder and blood neutrophils and blood monocytes were studied. Gland and blood PMNs were about equally effective at phagocytosing (2.5 log reduction in supernatant) and killing the bacteria (92% reduction of viable bacteria by two hours). Gland macrophages phagocytosed at a lower rate (1.5 log reduction) and were less effective at killing the bacteria (73% reduction by two hours). Blood monocytes phagocytosed and killed S. aureus at the lowest rate. An udder macrophage monolayer system was developed and used to evaluate the ability of antibiotics to kill surviving intracellular S. aureus. This assay was similar to our previously described system with blood PMNs. Several classes of antibiotics were investigated. These included naphthalenic ansamycin, lincosaminide, tetracycline, coumarin, peptide, paulomycin, quinolone, macrolide, cephalosporin, and penicillin-class antibiotics. The activity of these compounds was compared to positive (rifampicin), negative (cloxacillin), and nonantibiotic treated controls. Only naphthalenic ansamycin class antibiotics, paulomycin, paldimycin and ciprofloxacin caused significant reduction in viable intracellular bacteria in the macrophage system. These results were similar to those obtained in the blood PMN monolayer system. Because a low intraphagolysosomal pH could affect an antibiotic's ability to kill intracellular bacteria by affecting the drug itself or inhibiting bacterial growth, the effect of low pH on the minimum inhibitory concentration and the minimum lethal concentration of clindamycin and rifampicin against three strains of S. aureus was also tested. While the activity of clindamycin at pH 5.0 compared to pH 7.0 was not affected greatly, the activity of rifampicin was greatly enhanced at acidic pH. These results suggest that at least some of the excellent activity of rifampicin for intracellular S. aureus is due to potentiation of its activity in the intracellular acidic compartment of the phagolysosome.

Fine Structure of Melanocytes and Macrophages in the Harderian Gland of the Mouse

The presence of dendritic cells containing melanin granules has been demonstrated employing silver impregnation and electron microscopy in the interstitial tissue of the Harderian gland of the mouse. Two types of melanocytes, either with or without the various developmental stages of melanin granules, were found in the gland. Cells with developing granules were more dendritic and contained a large number of cytoplasmic organelles. The other cells were ellipsoidal or slender in shape and contained few cytoplasmic organelles and a large number of fully melanized granules, but no developing granules. In general, the granules of the Harderian gland melanocytes resembled granules from other organs (particularly the skin of the eyelids). The general size range of the granules was 0.2-0.9 micron. Each granule was enclosed by a membrane. The Harderian gland macrophages contained fully pigmented melanin granules of various sizes. The granules were enclosed by a membrane either singly or in groups. Some of the melanin granules within the phagosomes showed signs of degradation, revealing the underlying matrix.

Chemotactic factors for bovine neutrophils in relation to mastitis

The chemotactic effect of a variety of agents for bovine blood polymorphonuclear neutrophils (PMN) was evaluated in vitro in assays of migration under agarose. Activated serum and leukotriene B4 showed significant chemotactic activity whereas various bacterial products, formyl peptides, casein and platelet activating factor failed to attract bovine PMN. In vitro cultures of bovine mammary gland macrophages released chemotactic activity for homologous PMN when stimulated by addition of opsonised, killed Staphylococcus aureus, Escherichia coli or Zymosan or sterile E. coli culture filtrates or endotoxin. No significant activity was produced by unstimulated macrophages. Pharmacological levels of various inhibitors or arachidonic acid oxygenation had no significant effect on the generation of PMN chemoattractants by mammary macrophages.

Recruitment of neutrophils by an encapsulated coagulase-negative strain of Staphylococcus simulans in the mammary gland of the mouse.

The encapsulated coagulase-negative strain of Staphylococcus simulans (strain 76) was inoculated into the mammary glands of lactating mice. In contrast to the coagulase-positive encapsulated Staphylococcus aureus (strain M), which elicited a neutrophil response within 18 hr, strain 76 organisms multiplied in the glands but failed to elicit a neutrophil response for 3 days; they were then eliminated from the gland by 8 days. When strain 76 was inoculated into mice whose offspring had been removed 3 days earlier, a neutrophil response was induced within 18 hr and, although phagocytic ingestion was resisted for at least 42 hr, the organisms were eliminated from most glands within 5 days. Inoculation of a naturally occurring double-stranded RNA (BRL 5907) into the mammary gland induced a macrophage infiltration of the alveolar lumen that was qualitatively similar to 3 days involution. Intramammary inoculation of strain 76 18 hr after BRL 5907 resulted in a neutrophil infiltration within 6 hr. In vitro, strain 76 stimulated the release of chemotactic factors for neutrophils from bovine mammary gland macrophages. The results suggest that the recruitment of neutrophils by strain 76 in the mammary gland of the mouse is mediated by macrophages.

Constant light exposure induces damage and squamous metaplasia in Harderian glands of albino mice

Harderian glands from control albino mice kept in a cyclical light/dark environment had tubulo-alveoli comprised of lipid-filled glandular epithelial cells. The porphyrin content of the gland measured 122 μg/100 mg gland. Constant light exposure for 24 hr caused exopthalmos grossly. Histologically most of the secretory cells were swollen and the lumens of many tubulo-alveoli were obliterated; a few areas of the gland showed damage. The porphyrin content had decreased to 116 μg/100 mg gland. After 3 days of constant light exposure the tubulo-alveoli were markedly altered. Lipid and cellular debris filled the lumens, and lining cells were highly irregular, ranging in shape from columnar to squamous. The porphyrin content had decreased to 72 μg/100 mg gland and leukocytes and macrophages were abundant. Despite this extensive damage a number of tubulo-alveolar epithelial cells were observed undergoing mitosis. After 7 days of constant exposure to light, some tubulo-alveolar epithelial cells had undergone squamous metaplasia, and the porphyrin content had dropped markedly to 50 μg/100 mg gland. These pronounced cellular changes are believed to result from a direct effect of light on the gland.