Inflammatory effect of cleaved bovine lactoferrin by elastase on staphylococcal mastitis.

Elastase activity and concanavalin A (Con A) low affinity bovine lactoferrin (bLf) molecule were detected in mammary gland secretions (MGSs) from mammary glands (MGs) with clinical staphylococcal mastitis. Changes in clinical symptoms correlated with increases in both elastase activity and the concentration of Con A low-affinity Lf in MGSs from mastitic MGs. Bovine Lf treated with elastase (elastase-Lf) showed various small bLf molecules and the same image on Con A two-dimensional immunoelectrophoresis as low Con A affinity bLf in MGSs. We confirmed the presence of four common bLf peptides for the elastase-bLf and low Con A affinity bLf molecules in mastitic MGSs, and synthesized four peptides. Strong mRNA expression of interleukin-6 (IL-6), tumor necrosis factor alpha (TNFalpha), interleukin-8 (IL-8), and monocyte chemotactic protein-1 (MCP-1) was induced in bovine mammary epithelial cells on stimulation with low Con A affinity bLf, elastase-bLf, and GQRDLLFKDSAL, a synthesis bLf peptide based on nuclear factor kappa B (NFkappaB) activation. These results suggest that bLf was cleaved by elastase, and that this cleavage changed the physical function of Lf. Our results indicate that elastase induced production of low Con A affinity bLf, including the bLf peptide GQRDLLFKDSAL, and had an inflammatory effect on staphylococcal mastitis.

Effect of combination therapy with lactoferrin and antibiotics against staphylococcal mastitis on drying cows.

We examined combination therapy with both lactoferrin (Lf) and antibiotics on clinical mastitis due to Staphylococcus aureus (S.aureus) on drying cows. The clinical symptoms of mastitic quarters were cured 81% of combination therapeutic quarters at 7 days post injection (dpi). Moreover, most of mammary gland secretions (MGSs) in combination therapeutic quarters were normal at 7 days after parturition. In the quarters with combination therapy, S.aureus counts, Lf concentrations and content rate of concanavalin A (Con A) low-affinity Lf decreased and were lower than in the quarters treated with Lf or antibiotics alone. The mRNA expression of tumor necrosis factor alpha (TNFalpha) of the quarters with combination therapy also decreased and was lower than that of the Lf or antibiotics treated. The mRNA expression of pro-inflammatory cytokines and chemokines in bovine mammary gland epithelial lined cells (BMEC) stimulated with Lf were lower than those of Con A low-affinity Lf stimulated BMEC. Moreover, Lf showed an inhibitory effect to the induction of pro-inflammatory cytokine mRNA expression when co-stimulated with Lf and Con A low-affinity Lf. Nuclear factor kappa B (NFkappaB) activation was also induced with Con A low-affinity Lf, and the inhibitory effects of Lf were also confirmed on BMEC co-stimulated with Lf and Con A low-affinity Lf. These results indicated that the efficacy of combination therapy with antibiotics and Lf caused antibacterial effect of antibiotics and inhibition of pro-inflammatory cytokine and chemokine production with Lf via the inhibition of NFkappaB activation.

A new diagnostic indicator using concanavalin a low-affinity lactoferrin levels in mammary gland secretion in mastitic drying cows.

We examined the effective diagnostic indicator using the concanavalin A (Con A) low-affinity lactoferrin (Lf) to mastitic drying cows. The concentrations of both Lf and Con A low-affinity Lf in mammary gland secretions (MGSs) were lower than normal MGSs at the early and middle dry periods and colostrums. On the other hand, the levels of Con A low-affinity Lf in MGSs increased following the appearance of mastitis symptoms, and decreased when the mastitic symptoms were cured. Moreover, IgG1 concentrations of colostrums decrease on the quarters where a high level of Con A low-affinity Lf was determined after the onset of dry period. These results suggest that this method could be used as a useful indicator to mastitic drying cows.

Small molecule lactoferrin with an inflammatory effect but no apparent antibacterial activity in mastitic mammary gland secretion.

We have identified various lactoferrin (Lf) molecules in mastitic mammary gland secretions (MGSs), and these Lf molecules were examined for their physiological function in MG. These Lf molecules were isolated by Con A affinity chromatography, and then analyzed by various electrophoresis methods and N-terminal amino acid sequencing. The low Con A affinity Lf was found to have low molecular peptides as compared with the 86 kDa of the high Con A affinity Lf, which is usually detected in healthy MGSs. The N-terminal amino acid sequence of each of the small molecular Lfs were confirmed as fragments of 86 kDa Lf. This low Con A affinity Lf stimulated spleen adherent cells to produce more O(2)(-) than 86 kDa Lf. Furthermore, the low Con A affinity Lf showed low antibacterial activity against E. coli and S. aureus, and had decreased iron-binding capacity in comparison with 86 kDa Lf. Moreover, the 86 kDa Lf could stimulate bovine T cells or macrophages to produce IFN-gamma, IL-4, IL-6, and IL-1alpha. However low Con A affinity Lf induced the production of TNFalpha, but not physiological T cell or macrophage cytokines. It was also found that when the healthy MGs of dry cows were injected with the low Con A affinity Lf, there was an increase in polymorphonuclear cells together with TNFalpha, MCP-1, and IL-8 production. These results suggested that low Con A affinity Lf in mastitic MGSs differed from 86 kDa Lf in physiological characteristics, and, that it induced an inflammatory reaction in MGs.

Critical roles of platelets in lipopolysaccharide-induced lethality: effects of glycyrrhizin and possible strategy for acute respiratory distress syndrome.

Within a few minutes of an intravenous injection of a lipopolysaccharide (LPS) into mice, platelets accumulate, largely in the lung. At higher doses, LPS induces rapid shock (within 10 min), leading to death within 1 h. This type of shock differs from so-called endotoxin shock, in which shock signs and death occur several hours or more later. Here, we found that platelet depletion (by a monoclonal anti-platelet antibody) prevented LPS-induced rapid shock, but increased delayed lethality. In Japan, glycyrrhizin (GL), a compound isolated from licorice, is daily and slowly infused intravenously into chronic hepatitis C patients. A single bolus intravenous injection into mice of GL (200 mg/kg or less) shortly before (or simultaneously with) LPS injection reduced the pulmonary platelet accumulation and the severity of the rapid shock, and prevented death in both the early and later periods. GL itself, at 400 mg/kg, produced no detectable abnormalities in the appearance or activity of mice. Intraperitoneal injection of aspirin or dexamethasone had only marginal effects on LPS-induced platelet responses and lethality. These results suggest that platelets play important roles in the development of both the rapid and delayed types of shock induced by LPS. Although the mechanism by which GL suppresses platelet responses and delayed lethality remains to be clarified, GL might provide a strategy for alleviating the acute respiratory distress syndrome seen in sepsis. Our results may also support the proposal by Cinatl et al. [Cinatl J, Morgenstern B, Bauer G, Chandra P, Ravenau H, Doerr HW. Glycyrrhizin, an active component of liquorice roots, and replication of SARS-associated coronavirus. Lancet 2003; 361: 2045-6.] that GL may be an effective drug against severe acute respiratory syndrome.

Induction of nitric oxide production mediated by tumor necrosis factor alpha on staphylococcal enterotoxin C-stimulated bovine mammary gland cells.

Mammary gland (MG) secretions (MGS) derived from secretory cows infected with coagulase-negative staphylococci (CoNS) showed somatic cell counts and lactoferrin similar to levels found in the MGS of secretory cows infected with Staphylococcus aureus. However, nitrite and nitrate (NOx) and staphylococcal enterotoxin C (SEC) were found in MGS infected with S. aureus at much higher levels than in cows infected with CoNS. These results suggested that NOx could be intimately correlated with the production of SEC in secretory cows infected with S. aureus. Therefore, we examined the production of NOx and the expression of proinflammatory cytokines and microsomal cytochrome P450 (CYP450) after injection of SEC into the MGS of secretory cows. We were able to detect NOx and the proinflammatory cytokine tumor necrosis factor alpha (TNF-alpha) on MG cells of SEC-injected MGS. It was also found that CYP450 in the MG cells from SEC-injected MGS was down-regulated by approximately one-third in comparison with the cells from phosphate-buffered saline-injected MGS. This in vitro system also showed that NOx could be induced in the culture of bovine macrophage-lined cells (FBM-17) with the supernatants of SEC-stimulated bovine peripheral blood lymphocytes (BoPBLs) but not in the culture of peripheral mononuclear cells with SEC-stimulated BoPBLs. The expression of the mRNA for both inducible nitric oxide synthase and TNF-alpha in FBM-17 was enhanced by culturing with the supernatant of SEC-stimulated BoPBLs, although CYP450 was down-regulated. These results indicate that the down-regulation of CYP450 was caused by the production of TNF-alpha in SEC-stimulating MG cells containing macrophages and via NOx production. Therefore, we suggest that NOx released from activated MG cells via the superantigenic activity of SEC caused oxidative damage to the MG in S. aureus-induced mastitis.

Anti-inflammatory effects of intramammary infusions of glycyrrhizin in lactating cows with mastitis caused by coagulase-negative staphylococci.

OBJECTIVE: To determine the anti-inflammatory effects of glycyrrhizin (GL) in lactating cows with mastitis attributable to naturally occurring infection with coagulase-negative staphylococci (CNS). ANIMALS: 12 lactating Holstein cows with mastitis attributable to infection with CNS and 2 healthy cows without mastitis. PROCEDURE: Clinical signs, number of bacteria in milk, somatic cell count (SCC) in milk, concentrations of alpha-lactalbumin and lactoferrin in milk, and concentration of histamine in milk were investigated before and after intramammary infusion of GL (6 cows) or antimicrobials (6 cows). Glands of 2 healthy cows were infused with staphylococcal enterotoxin; milk leukocytes were then harvested and incubated with various doses of GL. RESULTS: In cows infected with CNS that had a low bacterial concentration in milk, infusion of GL alone resulted in significant improvements in swelling, firmness of glands, and number of clots in milk, and it decreased the SCC, but not significantly. Percentage of neutrophils decreased significantly (to < 30%) by 2 days after infusion. Use of lactoferrin as a marker of inflammation in mammary glands revealed a decrease in concentrations, whereas use of alpha-lactalbumin as a marker of recovery for mammary glands revealed significant increases in concentrations in the GL-infused group. Accompanying these anti-inflammatory effects, a decrease in the concentration of histamine in milk was observed in the GL-infused group. Glycyrrhizin decreased histamine production by milk leukocytes in a concentration-dependent manner. CONCLUSIONS AND CLINICAL RELEVANCE: Infusion of GL may regulate intramammary inflammation through modulation of inflammatory mediators such as histamine.

Inflammatory responses of bovine polymorphonuclear neutrophils induced by staphylococcal enterotoxin C via stimulation of mononuclear cells.

To elucidate the pathological roles of staphylococcal enterotoxin C (SEC) in bovine staphylococcal mastitis, a histopathological analysis of SEC-inoculated mammary glands was performed. SEC-inoculated mammary glands exhibited interstitial inflammation, and the leukocytes that migrated into the gland were predominantly polymorphonuclear neutrophils (PMN). In the gland cistern tissues dissected from SEC-inoculated mammary glands, epithelial cellular degeneration was observed. We also investigated the physiological effects of SEC on PMN in vitro. PMN migration was induced by culture supernatant of SEC-stimulated peripheral blood mononuclear cells (S-PBMC sup) but not by that of nonstimulated PBMC (N-PBMC sup). The concentration of interleukin-8 was significantly (P < 0.05) higher in S-PBMC sup than N-PBMC sup, and a significantly (P < 0.05) higher mRNA expression of growth-regulated oncogenes was detected in SEC-stimulated PBMC than in nonstimulated PBMC. Milk PMN collected from SEC-inoculated mammary glands produced more than 2 times the amount of superoxide at 1 day postinoculation (dpi) than at 0 dpi in the presence of phorbol 12-myristate 13-acetate (PMA). PMN cultured with S-PBMC sup for 24 h also produced significantly (P < 0.05) larger amounts of superoxide than those cultured with N-PBMC sup in the presence of PMA. Moreover, S-PBMC sup induced the long-time survival of PMN. These results indicate that SEC induces the activation of PMN via the stimulation of mononuclear cells.

Concentrations and specific antibodies to staphylococcal enterotoxin-C and toxic shock syndrome toxin-1 in bovine mammary gland secretions, and inflammatory response to the intramammary inoculation of these toxins.

To investigate the pathological role of staphylococcal enterotoxins (SEs) and toxic shock syndrome toxin-1 (TSST-1) in bovine mastitis, the production of SEs and TSST-1 was investigated in staphylococci isolated from 120 mammary gland secretions (MGS, 51 from no clinical sign-mammary glands and 69 from staphylococcal mastitic-mammary glands) collected from dairy farms where staphylococcal mastitis frequently occurred in Miyagi and Yamagata prefectures from 1997 to 1998. Concentrations of these toxins and specific antibody titers in each MGS were also measured. Furthermore, SEC and TSST-1 were inoculated into lactating mammary glands and inflammatory responses were analyzed. A high percentage of staphylococci including Staphylococcus aureus and coagulase-negative staphylococci isolated from both no clinical sign- and mastitic-MGS produced both SEC and/or TSST-1. The concentration of SEC increased with the severity of the mastitis, and was significantly higher (P<0.05) in acute mastitic-than in no clinical signs-MGS. Titers of specific antibodies to TSST-1 in MGS were significantly higher (P<0.05) than those to SEC, regardless of whether or not the cows were lactating or mastitic. Specific antibodies purified from MGS neutralized each toxin in vitro. A significant increase (P < 0.05) in somatic cell counts was induced by the intramammary inoculation of SEC but not TSST-1. These findings indicated that SEC rather than TSST-1 plays an important role in the pathology of staphylococcal bovine mastitis. The inflammatory activity of TSST-1 was probably neutralized by specific antibodies in MGS.

Elevation of histidine decarboxylase activity in the stomach of mice by ulcerogenic drugs.

Histamine is involved in the development of gastric lesions. To examine the contribution of the histamine-forming enzyme, histidine decarboxylase, to drug-induced gastric lesions, we compared the effects of aspirin, indomethacin and dexamethasone on histidine decarboxylase activity in mice. Administration of these drugs, orally or intraperitoneally, elevated histidine decarboxylase activity in the stomach but not in the liver, lung or spleen, dexamethasone being the most potent. In contrast, acetaminophen (a non-ulcerogenic drug) was inactive. These results and our previously reported findings (elevation of histidine decarboxylase activity by lipopolysaccharide, interleukin-1 and tumour necrosis factor, and by different types of stress) suggest that an elevation of histidine decarboxylase activity in the stomach may be a common feature of the responses to ulcerogenic stimuli. The possible participation of histidine decarboxylase in gastric lesions is discussed on the basis of the known actions of histamine, our findings and the effect of histamine H(2) receptor antagonists on histidine decarboxylase activity.

Differential gene expression of cytokine and cell surface molecules in T cell subpopulation derived from mammary gland secretion of cows.

PROBLEM: As T cell subpopulations in the mammary gland secretion (MGS) of cows dynamically vary through the lactation cycle, their functional analysis is important to understand the mammary immune responses. METHOD OF STUDY: T cell subpopulations were positively selected from MGS during lactation period and non-lactation period (dry period) by a magnetic cell sorter. The messenger RNA (mRNA) expression of cytokine and cell surface molecules in the subpopulations stimulated with anti-CD3 was investigated using reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: CD4+ T cells from MGS significantly expressed mRNA of interferon (IFN)-gamma, interleukin (IL)-2, granulocyte-macrophage colony-stimulating factor (GM-CSF), transforming growth factor (TGF)-beta, tumor necrosis factor (TNF)-alpha, IL-4, CD40 ligand (CD40L), Fas ligand (FasL) and IL-2 receptor (IL-2R) during dry period, and mRNA of IFN-gamma, IL-2 and TGF-beta during lactation period. Their expression during lactation period was always less than that during dry period. CD8+ T cells from MGS substantially expressed mRNA of IFN-gamma, IL-2, GM-CSF, TGF-beta, TNF-alpha, FasL and IL-2R during dry period and mRNA of IFN-gamma, GM-CSF, TGF-beta, TNF-alpha and c-kit during lactation period. The TGF-beta, TNF-alpha, c-kit and IL-2R mRNA expression of T cells in MGS during lactation period mostly depended on gammadelta T cells. Interestingly, c-kit mRNA was exclusively expressed in gammadelta T cells. CONCLUSIONS: The cytokine expression of T cells in MGS of cows depended on the T cell subpopulations. The present findings suggested that the activation of gammadelta T cells via c-kit receptor participated in the suppressed expression of cytokine mRNA in T cells during lactation period.

Effects of bovine lactoferrin by the intramammary infusion in cows with staphylococcal mastitis during the early non-lactating period.

To evaluate the clinical effects of bovine lactoferrin on staphylococcal mastitis in Holstein cows during the early non-lactating period, 41 mammary quarters were selected randomly from 36 cows on 3 dairy farms. Twelve quarters were infused intramammarily with bovine lactoferrin. Twenty-nine quarters were infused with antibiotic as a control. In the bovine lactoferrin-infused group, 91.7% of mastitic quarters were cured at 7 days after calving, compared with 48.3% in the control group. Furthermore, the changes in mammary secretion induced by the infusion of bovine lactoferrin were investigated. Mean numbers of staphylococci in mammary gland secretions were significantly decreased in both 5 bovine lactoferrin-infused quarters and 5 antibiotic-infused control quarters (p<0.05). Unlike in the control quarters, the mean total cell concentration in the mammary gland secretions increased in bovine lactoferrin-infused quarters. Similar results were obtained in 6 healthy quarters which were infused with bovine lactoferrin. In these quarters, the cell population contained mainly phagocytes such as polymorphonuclear leukocytes and cells positive for CD11b which is known as a complement receptor. The mean concentration of C3 in mammary gland secretions was significantly increased in 5 mastitic quarters infused with bovine lactoferrin (p<0.05), but showed no significant change in 5 mastitic control quarters. These results suggested that bovine lactoferrin treatment for staphylococcal mastitis in the early non-lactating period might increase the rate of cure through the induction of innate immunity in the host.

Decreased IL-10 production by psoriatic peripheral blood mononuclear cells stimulated with streptococcal superantigen.

The strong association of acute guttate psoriasis and streptococcal throat infection has suggested a role for streptococcal antigens in the pathogenesis of psoriasis. We have reported that psoriatic peripheral blood mononuclear cells (PBMCs) showed significantly lower responses to cytoplasmic membrane-associated protein (CAP) isolated from group A beta-hemolytic streptococci, a kind of streptococcal superantigen. The objectives were to evaluate the abnormal cytokine production by psoriatic PBMCs to streptococcal superantigen, CAP. We compared the production of four different cytokines, i.e. IL-4, IL-5, IL-10, and IFN-gamma, by PBMCs between psoriatic patients and healthy controls after stimulation with CAP or two different staphylococcal superantigens, staphylococcal enterotoxin A (SEA) or E (SEE). When PBMCs were stimulated with CAP, the production of IL-10 was significantly lower by psoriatic PBMCs than by those from healthy controls, whereas those of IL-4, IL-5, or IFN-gamma were not different between the two groups. Such a significant decrease in IL-10 production by psoriatic PBMCs was not observed when they were stimulated with staphylococcal superantigens. Flow cytometric analysis of intracytoplasmic IL-10 demonstrated defective IL-10 production by psoriatic PBMCs in both CD3+ T cells and CD14+ monocytes. There was a significant positive correlation between IFN-gamma production by PBMCs and the proliferation of Vbeta8+ T cells preferentially stimulated by CAP. These data demonstrating the defective IL-10 production by psoriatic PBMCs stimulated with streptococcal superantigen seem to explain why only psoriatic patients evolve sustained and Th-1 deviated skin lesions after streptococcal upper respiratory infection.

Lactoferrin stimulates A Staphylococcus aureus killing activity of bovine phagocytes in the mammary gland.

Lactoferrin (Lf) may play a key role in the clearance of microorganisms from a host. To study in vitro the bactericidal mechanisms of Lf during nonlactating periods, we investigated whether the effects of Lf were influenced by bovine mammary gland secretory cells (MGSC) and fresh normal bovine serum (NBS) as a source of complement. Phagocytic killing tests demonstrated that a phagocytic mixture of unopsonized Staphylococcus aureus (S. aureus) and MGSC in the presence of Lf reduced bacterial growth, compared with that of unopsonized S. aureus and MGSC without Lf. The opsonization with Lf and fresh NBS together resulted in more than a 95% reduction in CFU. The activation of complement induced by Lf also resulted in increased deposition of C3 on S. aureus, and the phagocytic activity of MGSC was augmented by opsonization with Lf and fresh NBS. Inhibition of C3 deposition by Lf was not induced in the presence of Mg-EGTA, but was induced by the addition of bovine Lf antiserum. These results strongly suggest that Lf induces the activation of complement in fresh NBS mainly through an alternative pathway. The results demonstrated a Lf-dependent, antibody-independent and complement-mediated phagocytic killing of S. aureus, and implied that Lf was synergistically capable of activating both the alternative pathway of the bovine complement cascade and phagocytosis by phagocytes.