Differential Induction of Complement Fragment C5a and Inflammatory Cytokines during Intramammary Infections with Escherichia coli and Staphylococcus aureus

The prompt recruitment of neutrophils to the site of infection is essential for the defense of the bovine mammary gland against invading pathogens and is determinant for the outcome of the infection. Escherichia coli is known to induce clinical mastitis, characterized by an intense neutrophil recruitment leading to the eradication of the bacteria, whereas Staphylococcus aureus induces subclinical mastitis accompanied by a moderate neutrophil recruitment and the establishment of chronic mastitis. To elicit the neutrophil recruitment into the udder, inflammatory mediators must be produced after recognition of the invading pathogen. To our knowledge, those mediators have never been studied during S. aureus mastitis, although understanding of the neutrophil recruitment mechanisms could allow a better understanding of the differences in the pathogeneses elicited by E. coli and S. aureus. Therefore, we studied, at several time points, the accumulation of neutrophils and the presence of the chemoattractant complement fragment C5a and of the cytokines interleukin-1β (IL-1β), tumor necrosis factor alpha, and IL-8 in milk after inoculation of E. coli or S. aureus in lactating bovine udders. The low levels of C5a and the absence of cytokines in milk from S. aureus-infected cows, compared to the high levels found in milk from E. coli-infected animals, mirror the differences in the severities of the two inflammatory reactions. The cytokine deficit in milk after S. aureus inoculation in the lactating bovine mammary gland could contribute to the establishment of chronic mastitis. This result could help in the design of preventive or curative strategies against chronic mastitis.     

Beta-hydroxybutyrate abrogates formation of bovine neutrophil extracellular traps and bactericidal activity against mammary pathogenic Escherichia coli

Escherichia coli is an important bacterial species isolated from bovine mastitis. The rate of neutrophil recruitment into the mammary gland and their bactericidal activity largely affect the severity and outcome of the disease. Ketosis is a common metabolic disease, and affected dairy cows are known to have increased risk for mastitis and other infectious conditions. The disease is associated with high blood and milk levels of beta-hydroxybutyrate (BHBA), previously shown to negatively affect neutrophil function by unknown mechanisms. We show here that the mammary pathogenic E. coli strain P4 activates normal bovine neutrophils to form neutrophil extracellular traps (NETs), which are highly bactericidal against this organism. Preincubation of these neutrophils with increasing concentrations (0.1 to 8 mmol/liter) of BHBA caused a fivefold decrease of E. coli P4 phagocytosis, though intracellular killing was unaffected. Furthermore, BHBA caused a 10-fold decrease in the NETs formed by E. coli P4-activated neutrophils and a similar decrease in NET bactericidal activity against this organism. These negative effects of BHBA on bovine neutrophils might explain the increased susceptibility of ketotic cows to mastitis and other infectious conditions.     

Complement fragment C5a and inflammatory cytokines in neutrophil recruitment during intramammary infection with Escherichia coli.

Generation of inflammatory mediators and leukocyte recruitment to infection at an epithelial surface were studied during Escherichia coli-induced mastitis. One uninfected gland of each of eight midlactation cows was challenged with only 30 CFU of E. coli McDonald strain 487, a serum-resistant isolate from a cow with mastitis. Bacteria grew logarithmically during the first 10 to 12 h after challenge, reaching concentrations of more than 10(5) CFU/ml with no detectable host response during this time. An intense inflammatory reaction began approximately 12 h after the challenge and was characterized by a breakdown in the blood-milk permeability barrier followed by pyrexia and a pronounced leukocytic influx. Coincident with the onset of mammary inflammation was the appearance of neutrophil chemotactic activity in the milk from infected glands. Factors able to upregulate CD18 expression on peripheral blood neutrophils also appeared in milk at this time. The lack of appearance of chemotactic and CD18-upregulating activities until 12 h after challenge indicated that delays in neutrophil recruitment resulted from an initial lack of bacterial recognition and inflammatory mediator production. Production of complement fragment C5a, tumor necrosis factor, and interleukin-1 (IL-1) occurred earlier than production of IL-6 or IL-8. The early and intense production of C5a indicates that this chemoattractant may be more important than IL-8 during the initial recruitment and activation of neutrophils to a developing E. coli infection.     

Chemotactic Activities in Nonmastitic and Mastitic Mammary Secretions: Presence of Interleukin-8 in Mastitic but Not Nonmastitic Secretions

Due to its association with low-quality milk and a decrease in milk production in bovines, mastitis is a major cause of economic loss. Additionally, mastitis can be harmful to suckling newborns and can cause damage to the mammary gland. In mastitic mammary secretions there is a substantial increase in somatic cells, specifically neutrophils. In this study we examined the ability of mastitic and nonmastitic mammary secretions to cause in vitro neutrophil chemotaxis using a microchemotaxis assay. Also, the role of the inflammatory chemokine interleukin-8 (IL-8) in neutrophil recruitment during mastitis was addressed in these in vitro experiments. We found that both nonmastitic and mastitic mammary secretions were chemotactic, not chemokinetic, for neutrophils. The neutrophil chemotactic activity in mastitic, but not nonmastitic, mammary secretions was blocked by anti-IL-8 antibodies. Molecular mass separation of the active components showed that the chemotactic activity of the mastitic secretions was present in the 10-kDa-or-less fraction and was blocked by anti-IL-8 antibodies. These results indicate that IL-8 plays a major role in neutrophil recruitment during mastitis. An understanding of its role will be of help in designing strategies for immunomodulatory therapies for mastitis.     

Kinetics of cells and cytokines during immune-mediated inflammation in the mammary gland of cows systemically immunized with Staphylococcus aureus α-toxin

OBJECTIVE: To examine changes in inflammatory mediators, lymphocyte subpopulations and neutrophil activation that occur during an immune-mediated recruitment of neutrophils in the mammary gland. SUBJECTS: 11 clinically healthy cows. TREATMENT: 5 cows received 2 subcutaneous injections of 30 microg of alpha-toxin of Staphylococcus aureus, two months apart. Three months after the last immunization, 5 microg of alpha-toxin were injected, via the teat end, in one randomly selected quarter of the 5 immunized cows and of the 6 unimmunized cows (control group). METHODS: Blood and milk samples were collected at several times during 4 days post-challenge. Blood and milk cells were purified to be stained with specific mAbs and analysed by flow cytometry, or to be used for cytokine RT-PCR. Bovine serum albumin, haptoglobin, cytokines and C5a were also analysed in milk or plasma samples using radial immunodiffusion assay or ELISA. RESULTS: Large amounts of cells (> 1 million/ml of milk) were recruited in the quarters of the immunized cows, whereas no recruitment occurred in the control group. In blood of immunized animals, haptoglobin was present and expression of surface adhesion molecules on neutrophils was modified whereas no change was observed concerning the lymphocyte subpopulations. On milk-derived neutrophils, the expression of CD11b and CD18 was upregulated compared to blood, in contrast to CD62L that was downregulated. The CD8+ cells were recruited as soon as 12 h post-challenge, in contrast to the CD4+ cells, 96 h post-challenge. No IL-1beta, TNF-alpha, IL-8 and C5a were detected using ELISA. mRNA of IL-1alpha, IL-1beta, IL-6, TNF-alpha, IL-8 and IL-12 were found in almost all the samples. CONCLUSIONS: The immunization triggered an early and massive neutrophil recruitment from the blood into the milk compartment as well as the recruitment of a cytotoxic/suppressor lyphocyte population during the early acute phase response. These results could help to devise new vaccinal strategies to fight against staphylococcal mastitis.     

Escherichia coli and Staphylococcus aureus elicit differential innate immune responses following intramammary infection

Staphylococcus aureus and Escherichia coli are among the most prevalent species of gram-positive and gram-negative bacteria, respectively, that induce clinical mastitis. The innate immune system comprises the immediate host defense mechanisms to protect against infection and contributes to the initial detection of and proinflammatory response to infectious pathogens. The objective of the present study was to characterize the different innate immune responses to experimental intramammary infection with E. coli and S. aureus during clinical mastitis. The cytokine response and changes in the levels of soluble CD14 (sCD14) and lipopolysaccharide-binding protein (LBP), two proteins that contribute to host recognition of bacterial cell wall products, were studied. Intramammary infection with either E. coli or S. aureus elicited systemic changes, including decreased milk output, a febrile response, and induction of the acute-phase synthesis of LBP. Infection with either bacterium resulted in increased levels of interleukin 1beta (IL-1beta), gamma interferon, IL-12, sCD14, and LBP in milk. High levels of the complement cleavage product C5a and the anti-inflammatory cytokine IL-10 were detected at several time points following E. coli infection, whereas S. aureus infection elicited a slight but detectable increase in these mediators at a single time point. Increases in IL-8 and tumor necrosis factor alpha were observed only in quarters infected with E. coli. Together, these data demonstrate the variability of the host innate immune response to E. coli and S. aureus and suggest that the limited cytokine response to S. aureus may contribute to the well-known ability of the bacterium to establish chronic intramammary infection.     

Effect of interleukin-8 and granulocyte colony-stimulating factor on priming and activation of bovine neutrophils

Neutrophils are important effector cells in innate and acquired immunity, but the magnitude and character of their phagocytic and bactericidal responses depend on cues derived from mediators in the local microenvironment. This study investigated the effect of bovine interleukin-8 (IL-8) and granulocyte colony-stimulating factor (G-CSF) on priming and activation of bovine neutrophils in vitro and in vivo. Neutrophils were isolated from blood and cultured for up to 18 h, with or without cytokines, and then Mannheimia haemolytica-induced oxidative burst and phagocytosis of Staphylococcus aureus were measured by flow cytometry. Neither IL-8 nor G-CSF directly triggered an oxidative burst, but incubation with these cytokines for 18 h primed neutrophils for a greater oxidative burst triggered by M. haemolytica and for enhanced uptake of S. aureus. The maximal response was observed when neutrophils were incubated with both cytokines together, at concentrations of 200 ng/ml for G-CSF and 400 ng/ml for IL-8. The IL-8-induced priming effect was reduced by treatment with a neutralizing antibody to IL-8, and was not attributed to endotoxin contamination. Instillation of IL-8 into the lung using a bronchoscope induced neutrophil recruitment within 18 h. Neutrophils from IL-8-treated lung showed dose-dependent enhancement of the oxidative burst triggered by M. haemolytica. Histologically, neutrophils filled alveoli and bronchioles, and scattered macrophages contained neutrophils with morphological features of apoptosis. Thus, prolonged in vitro or in vivo exposure to IL-8 and/or G-CSF enhances the subsequent oxidative burst and phagocytic responses of bovine neutrophils.     

Milk complement and the opsonophagocytosis and killing of Staphylococcus aureus mastitis isolates by bovine neutrophils

Phagocytosis of bacteria by bovine polymorphonuclear neutrophils (PMN) has long been regarded as essential for host defense against mastitis infection. Complement-mediated opsonisation by complement component 3 (C3) binding is an important component of the innate immune system. We investigated the role of milk complement as an opsonin and its involvement in the phagocytosis and killing of Staphylococcus aureus isolates from cases of bovine mastitis by bovine blood PMN. We show that deposition of milk C3 component occurred on six different isolates of S. aureus and that the alternative pathway was the sole complement pathway operating in milk of uninflamed mammary gland. This deposition was shown to occur at the same location as the capsule, but not on capsular antigen. Milk complement enhanced the chemiluminescence response of PMN induced by S. aureus. Nevertheless, the association of S. aureus to cells and the overall killing of bacteria by bovine PMN were not affected by the presence of milk complement. Therefore, as all milk samples contained antibodies to capsular polysaccharide type 5 and to other surface antigens, it is likely that milk antibodies were responsible for these two phagocytic events. Results of this study suggest that the deposition of milk complement components on the surface of S. aureus does not contribute to the defence of the mammary gland against S. aureus.     

Identification and characterization of a new interleukin-8 receptor in bovine species

Mastitis is an inflammation of the mammary gland, most of the time caused by invading pathogens. Phagocytosis by neutrophils is a crucial defense of the mammary gland and the prompt recruitment of these phagocytes from blood to milk compartments is essential for the outcome of the infection. ELR+ CXC chemokines, ligands of the two interleukin-8 receptors (IL-8R), CXCR1 and CXCR2, are likely to be involved in the initiation of the inflammatory response and also in the migration of neutrophils. Recently, the polymorphism of bovine CXCR2 has been associated with resistance to mastitis. However, as the bovine IL-8R are not functionally defined, their contribution to the recruitment of neutrophils remains undetermined. In this study, the RNA ligase-mediated (RLM)-RACE method was used to clone a novel bovine interleukin-8 receptor (nIL-8R) of the bovine species. We showed that both bovine IL-8R (nIL-8R and the published CXCR2) are functional since bovine IL-8 induced migration of HEK-293 cells expressing either IL-8R. In addition, comparisons of full-length sequences suggested that the published CXCR2 sequence was improperly annotated and that the sequences of the nIL-8R and the published CXCR2 are homologous to human CXCR2 and CXCR1, respectively. This was confirmed by binding assays with labeled IL-8 and GRO-beta and calcium (Ca) flux responses of transfected cells. Moreover, the C-terminal of both bovine IL-8R showed 100% identity, whereas they differ in most other species, suggesting that the two bovine IL-8R initiate similar signal transduction. These results constitute a basis to improve our understanding of the molecular mechanisms implicated in the recruitment of bovine neutrophils.     

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.     

Enzyme-linked immunosorbent assay for detection of leukocidin toxin from Staphylococcus aureus in bovine milk samples.

An enzyme-linked immunosorbent assay was developed for the detection of leukocidin toxin from Staphylococcus aureus. The minimum concentration of leukocidin detectable with the assay was 30 ng/ml. The enzyme-linked immunosorbent assay was found to be a more sensitive method, by a mean of 45-fold, for leukocidin detection than was observation of cytolytic effects of the toxin on bovine neutrophils. A mean toxin concentration of 974 ng/ml was required to produce observable cytolytic effects on neutrophils. Although the enzyme-linked immunosorbent assay was able to detect leukocidin in milk samples from toxin-infused mammary glands, the toxin was detectable in only 2 of 27 S. aureus-infected milk samples (7%) from cows with chronic staphylococcal mastitis. To determine whether leukocidin antibodies in the mastitic milk samples were preventing toxin detection, leukocidin was mixed with milk with a high antileukocidin antibody titer (from a vaccinated cow) and evaluated with the immunoassay. Leukocidin was readily detected in this sample, indicating that milk antileukocidin antibodies were not sufficient to prevent detection of any leukocidin present in the mastitic milk samples. Failure to detect leukocidin in most mastitic milk samples with this assay indicated that, if leukocidin is produced in the bovine mammary gland during chronic staphylococcal mastitis, the concentration of the toxin may be too low to produce cytolytic effects on neutrophils.     

Purified Staphylococcus aureus leukotoxin LukM/F' does not trigger inflammation in the bovine mammary gland

An early recruitment of neutrophils in mammary tissue and milk is considered an important component of the defense of the mammary gland against Staphylococcus aureus. We investigated whether the leukotoxin LukM/F′, which is produced by a proportion of mastitis-causing strains of S. aureus, would be able to trigger inflammation in the udder. Infusion of purified LukM/F′ toxin in lactating mammary glands did not cause neutrophil influx in milk, showing that the toxin was not able to cause mastitis on its own. Purified LukM/F′ did not kill or stimulate mammary epithelial cells in culture. As expected, LukM bound to mammary macrophages and the complete LukM/F′ toxin killed these cells, but subcytotoxic LukM/F′ concentrations did not induce secretion of IL-8, TNF-α, IL-1β or IL-6 by macrophages. On the contrary, the production of these pro-inflammatory mediators by adhesion-stimulated macrophages was reduced. Overall, these results indicate that purified leukotoxin LukM/F′ is not likely to contribute to the initiation of the inflammatory response and could even play an anti-inflammatory role in the mammary gland by inactivating macrophages.     

Bovine TLR2 and TLR4 properly transduce signals from Staphylococcus aureus and E. coli, but S. aureus fails to both activate NF-kappaB in mammary epithelial cells and to quickly induce TNFalpha and interleukin-8 (CXCL8) expression in the udder

Staphylococcus aureus, but not E. coli pathogens frequently cause subclinical, chronic infections of the mammary gland. We examined here, if inadequate activation of the bovine TLR2 and TLR4 pathogen receptors by ligands derived from S. aureus pathogens might contribute to molecular mechanisms underpinning the escape strategies from mammary immune defence of this pathogen. We show that infections with live E. coli, but not S. aureus pathogens induce strongly IL-8 and TNFalpha gene expression in the udders. Yet, preparations of heat-killed bacteria from both pathogens activate equally well bovine TLR2 and TLR4 receptors to induce NF-kappaB activation, as shown in the HEK293 reconstitution system of TLR-signal transduction. LTA prepared from the S. aureus strain used to infect the cows activates the bovine TLR2 as strongly as the entire, heat-killed pathogen. Both pathogens induce in primary bovine mammary epithelial cells (pbMEC) IL-8 and TNFalpha gene expression, but S. aureus to less than 5% of the degree caused by E. coli. This impaired proinflammatory activation is paralleled by a complete lack of NF-kappaB activation in pbMEC by S. aureus or LTA. In contrast, E. coli and LPS activate strongly NF-kappaB in these cells. A large proportion of this activation is attributable to TLR-mediated signalling, since a dual transdominant negative DN-MyD88-DN-TRIF factor blocks >80% of the pathogen-related NF-kappaB activation in pbMEC. Our results prove that impaired binding of TLR-ligands from the pathogenic S. aureus strain are not the cause for the inadequate mammary immune response elicited by this pathogen. Rather, the pathogen causing subclinical mastitis impairs NF-kappaB activation in MEC thereby severely weakening the immune response in the udder.     

Recombinant soluble CD14 reduces severity of intramammary infection by Escherichia coli

The interaction among gram-negative bacteria, the innate immune system, and soluble CD14 (sCD14) has not been well documented. The effect of recombinant bovine sCD14 (rbosCD14) on milk somatic cell count (SCC), bacterial clearance, and cytokine production was investigated by using a bovine intramammary Escherichia coli infection model. We first determined whether rbosCD14 would increase the SCC during a lipopolysaccharide (LPS) challenge. Three quarters of each of six healthy lactating cows were injected with either 0.3 microg of LPS, 0.3 microg of LPS plus 100 micro g of rbosCD14, or saline. In comparison with quarters injected with LPS alone, the SCC was twofold higher (P < 0.05) in quarters injected with LPS plus rbosCD14 after the challenge. We therefore hypothesized that when E. coli bacteria invade the mammary gland, sCD14 in milk would interact with LPS and rapidly recruit neutrophils from the blood to eliminate the bacteria before establishment of infection. To test this hypothesis, two quarters of each of nine healthy cows were challenged with either 50 CFU of E. coli plus saline or 50 CFU of E. coli plus 100 microg of rbosCD14. Quarters challenged with E. coli plus rbosCD14 had a more rapid recruitment of neutrophils, which was accompanied by a faster clearance of bacteria, lower concentrations of tumor necrosis factor alpha and interleukin-8 in milk, and milder clinical symptoms, than challenged quarters injected with saline. Results indicate that increasing the concentration of sCD14 in milk may be a potential strategy with which to prevent or reduce the severity of infection by coliform bacteria.     

Experimental staphylococcal mastitis in the mouse: the induction of chronic mastitis and its response to antibiotic therapy

Two strains of Staphylococcus aureus derived from chronic bovine mastitis caused an acute reaction when inoculated into the mammary glands of mice. The response in mice was converted to a chronic mastitis if a neutrophil population was elicited in the alveolar lumen by intramammary inoculation of endotoxin 6 h before staphylococcal challenge. Chronic mastitis was established in this way with each of the strains of S. aureus and in each case the infection failed to respond to intramammary therapy with sodium cloxacillin. The results are discussed in relation to bovine mastitis.     

Roles of alpha-toxin and beta-toxin in virulence of Staphylococcus aureus for the mouse mammary gland.

Mutants of Staphylococcus aureus which fail to express alpha-toxin (Hly), beta-toxin (Hlb), or both have been constructed by site-specific mutagenesis. The virulence of the mutants was compared with that of wild-type toxigenic strains by intramammary inoculation of lactating mice. A bovine strain, M60, and a laboratory strain, 8325-4, caused acute mastitis and death within 48 h for 60% of the mice inoculated. Animals inoculated with Hly mutants also developed acute mastitis, but no deaths occurred. Comparisons of Hly- or Hlb-positive strains with the double mutation Hly Hlb showed that both toxins led to a significantly higher recovery of S. aureus from the gland 48 h postinfection. Histopathological examination of mammary glands showed that phagocytosis of bacteria occurred irrespective of toxigenicity, but toxigenic strains, particularly those which were Hly+, continued to multiply, invaded the interalveolar tissues, and produced severe lesions. Stimulation of an inflammatory response by inoculation of the mammary gland with endotoxin prior to challenge with S. aureus reduced recovery of the bacteria 10- to 100-fold and, under these conditions, neither alpha-toxin nor beta-toxin contributed significantly to growth and survival.     

The effect of inoculation of Pasteurella haemolytica into the lactating mammary gland of mice, rats, rabbits, sows and cows.

An isolate of Pasteurella haemolytica (A9), which consistently produced severe mastitis in ewes, was inoculated into the lactating mammary glands of a variety of species. Mastitis did not develop after the inoculation of log-phase bacteria into the mammary gland of lactating mice, rats, rabbits or sows but did so in the mammary gland of two cows. Another A9 isolate from a ewe with mastitis and an A1 isolate from a bovine pneumonic lung also induced mastitis in cows. Thus, in this study, P. haemolytica produced mastitis only in ruminant animals.     

Interferon-gamma (IFN-gamma) and macrophage inflammatory proteins (MIP)-1 and -2 are involved in the regulation of the T cell-dependent chronic peritoneal neutrophilia of mice infected with mycobacteria.

In mycobacterial infections of mice there is a chronic, immune-mediated mobilization of neutrophils to the infectious site. In this study we evaluated the role played by cytokines in the chronic peritoneal neutrophilia which occurs in mice intraperitoneally infected with Mycobacterium bovis BCG or M. avium. Antibodies to IFN-gamma and to MIP-1 and -2 were effective in reducing peritoneal neutrophilia when given during the infection. Whereas the former antibody was only effective when given early, the latter two were effective when administered late in infection, suggesting the MIPs were direct mediators of neutrophil recruitment. Recombinant IFN-gamma given intraperitoneally induced the accumulation of neutrophils and primed the peritoneal cells for an enhanced recruitment of neutrophils. Our data show that chronic neutrophilia during mycobacterial infection is regulated by different cytokines acting at different stages and levels of neutrophil recruitment.     

Inducible and constitutive in vitro neutrophil chemokine expression by mammary epithelial and myoepithelial cells.

Previously, our laboratory showed that bovine and caprine mammary secretions are chemotactic and that chemoattractants found in these secretions are qualitatively different according to infection status and/or lactation stage. However, the cellular source of the chemoattractants has not been defined. In this study we used a modified Boyden chamber assay to examine the ability of previously established caprine mammary epithelial cell (CMEC) and myoepithelial cell (CMMyoEC) lines to produce chemoattractants for neutrophils. We found that CMEC culture supernatants, but not those of CMMyoEC cultures, induced in vitro neutrophil chemotaxis. Further characterization showed that chemotactic activity was produced when the cells underwent contact-induced differentiation. Neutrophil migration was chemotactic, not chemokinetic, and was augmented when the epithelial and myoepithelial cells were cocultured. Additionally, chemotactic activity was inducible by Staphylococcus aureus plus alpha-toxin, Escherichia coli, and interleukin-1beta (IL-1beta) in CMEC cultures. However, CMMyoEC cultures could not be induced to produce chemotactic activity. Anti-IL-8 antibody was able to block some constitutively produced chemotactic activity and chemotactic activity induced by IL-1beta and S. aureus plus alpha-toxin. These results indicate that epithelial cells may play a major role in producing chemoattractants, specifically IL-8, in the mammary gland.