Bovine Mononuclear Phagocytes Research Articles

Review Paper: Modulation of Mononuclear Phagocyte Function by Mycobacterium avium subsp. paratuberculosis

Pathogenic mycobacteria are highly adapted for survival within host mononuclear phagocytes. This is largely due to the organism's capacity to prevent macrophage activation, block phagosome acidification and maturation, and attenuate presentation of antigens to the immune system. Mycobacterium avium subsp. paratuberculosis (MAP) is one such organism that modulates the ruminant innate immune response. It is the causative agent in paratuberculosis, a chronic progressive granulomatous enteritis in ruminants. MAP initially interacts with cell membrane receptors on bovine mononuclear phagocytes and initiates cell signaling responses and phagocytosis. Mannosylated liparabinomannan (Man-LAM) is a major component of the MAP cell wall that interacts with the cell membrane of mononuclear phagocytes and may be a major virulence factor. Toll-like receptor 2 (TLR2) has been incriminated as major signaling receptor that binds to MAP and initiates signaling though the mitogen-activated protein kinase (MAPK)-p38 pathway. This pathway induces transcription of interleukin (IL)-10. Early production of IL-10 suppresses proinflammatory cytokines, chemokines, IL-12, and major histocompatability factor class-II expression. Both IL-10 dependent and IL-10 independent mechanisms appear to be involved in attenuation of phagosome acidification and phagolysosome fusion. Many of the suppressive effects of MAP on bovine mononuclear phagocytes can be reproduced by exposure of bovine monocytes to Man-LAM. Therefore, MAP Man-LAM-induced TLR2-MAPK-p38 signaling with resultant excessive IL-10 expression has emerged as one of the mechanisms by which MAP organisms suppress inflammatory, immune, and antimicrobial responses and promote their survival within host mononuclear phagocytes.

Bovine monocyte TLR2 receptors differentially regulate the intracellular fate ofMycobacterium aviumsubsp. paratuberculosis andMycobacterium aviumsubsp.avium

Pathogenic mycobacterial organisms have the capacity to inhibit macrophage activation and phagosome maturation. Although the mechanism is complex, several studies have incriminated signaling through TLR2 receptors with subsequent activation of the MAPK pathway p38 (MAPKp38) and overproduction of IL-10 in the survival of pathogenic mycobacterial organisms. In the present study, we compared the response of bovine monocytes with infection by Mycobacterium avium subspecies paratuberculosis (MAP), the cause of paratuberculosis in ruminants, with the closely related organism M. avium subspecies avium (Maa), which usually does not cause disease in ruminants. Both MAP and Maa induced phosphorylation of MAPKp38 by bovine monocytes; however, addition of a blocking anti-TLR2 antibody partially prevented MAPKp38 phosphorylation of MAP-infected monocytes but not Maa-infected monocytes. Addition of anti-TLR2 antibody enhanced phagosome acidification and phagosome-lysosome fusion in MAP-containing phagosomes and enabled monocytes to kill MAP organisms. These changes were not observed in Maa-infected monocytes. The effect on phagosome maturation appears to occur independently from the previously described inhibitory effects of IL-10 on phagosome acidification and organism killing, as IL-10 production was not affected by addition of anti-TLR2 antibody to monocyte cultures. Therefore, signaling through the TLR2 receptor appears to play a role in phagosome trafficking and antimicrobial responses in MAP-infected bovine mononuclear phagocytes.

Role of the mitogen-activated protein kinase pathway in the differential response of bovine monocytes to Mycobacterium avium subsp. paratuberculosis and Mycobacterium avium subsp. avium

We compared the kinetics of activation and antimicrobial activities of MAPK-p38 and MAPK-ERK in bovine monocytes infected with Mycobacterium avium subsp. paratuberculosis ( MAP) and Mycobacterium avium subsp. avium ( Maa). Monocytes were incubated with MAP or Maa organisms with or without a specific inhibitor of the MAPK-p38 pathway (SB203580), and MAPK phosphorylation and antimicrobial functions of monocytes were evaluated. At early time points MAPK-p38 phosphorylation was greater in MAP-infected bovine monocytes than in Maa-infected monocytes. At later time points MAPK-p38 phosphorylation by both organisms was similar. MAPKp38 phosphorylation in MAP-infected monocytes was similar to negative control cells, whereas in Maa-infected this activation remained greater than negative control cells. Increase phosphorylation MAPK-ERK was similar at all time points for both organisms. Bovine monocytes had minimal capacity to kill MAP organisms, to acidify MAP-containing phagosomes, or to form phagolysosome. Alternatively, bovine monocytes were able to kill Maa organisms. Addition of SB203580 to monocyte cultures increased phagosome acidification, phagolysosome formation, and killing of MAP and Maa organisms. Taken together these data indicate that early transient activation of MAPK-p38 in bovine mononuclear phagocytes by MAP organisms may be a key mechanism involved in the capacity of MAP to survive in bovine monocytes.

Cell membrane receptors on bovine mononuclear phagocytes involved in phagocytosis of Mycobacterium avium subsp paratuberculosis

To determine cell membrane receptors involved in phagocytosis of Mycobacterium avium subsp paratuberculosis (MAP) organisms. Monocytes were obtained from healthy adult Holstein dairy cows that were test negative for MAP infection on the basis of bacteriologic culture of feces and serologic test results. Monocytes or bovine macrophage cell line (BoMac) cells were incubated with MAP organisms for 30, 60, or 120 minutes with or without inhibitors of integrins, CD14, or mannose receptors. Phagocytosis was evaluated by light microscopy or by flow cytometry. CD11a/CD18, CD11b, and CD14 expression on monocytes and BoMac cells was evaluated by use of flow cytometry. Monocytes and BoMac cells rapidly phagocytized MAP organisms. However, compared with BoMac cells, monocytes had a greater total capacity to phagocytize MAP organisms. Addition of neutralizing anti-integrin antibodies (anti-CD11a/CD18 and anti-CD11b) substantially inhibited phagocytosis by monocytes during the first 60 minutes of incubation with MAP organisms, but were less effective at 120 minutes of incubation. Anti-CD11a/CD18 and anti-CD11b antibodies were less effective in inhibiting phagocytosis by BoMac cells. Addition of inhibitors of CD14 or mannose receptors also inhibited phagocytosis of MAP by monocytes. Addition of a combination of integrin and mannose inhibitors had an additive effect in reducing phagocytosis, but addition of integrin and CD14 inhibitors did not have an additive effect. Multiple receptors are involved in phagocytosis of MAP organisms. Although CD11/CD18 receptors appear to be the major receptors used by MAP at early time points, mannose receptors and CD14 also contribute substantially to phagocytosis.

Extracellular ATP Is Cytotoxic to Mononuclear Phagocytes but Does Not Induce Killing of IntracellularMycobacterium aviumsubsp.paratuberculosis

Mycobacterium avium subsp. paratuberculosis is the etiologic agent of Johne's disease, a chronic granulomatous enteritis in ruminants. ATP has been reported to induce cell death of macrophages and killing of Mycobacterium species in human and murine macrophages. In this study we investigated the short-term effect of ATP on the viability of M. avium subsp. paratuberculosis-infected bovine mononuclear phagocytes and the bacilli within them. Addition of 5 mM ATP to M. avium subsp. paratuberculosis-infected bovine monocytes resulted in 50% cytotoxicity of bovine monocytes at 24 h. Addition of 2'(3')-O-(4-benzoylbenzoyl) ATP triethylammonium salt (Bz-ATP), which is a longer-lived ATP homologue and purinergic receptor agonist, significantly increased the uptake of YO-PRO, which is a marker for membrane pore activation by P2X receptors. Addition of Bz-ATP also stimulated lactate dehydrogenase release and caspase-3 activity in infected bovine monocytes. Neither ATP nor Bz-ATP reduced the survival of M. avium subsp. paratuberculosis in bovine mononuclear phagocytes. Likewise, addition of ATP or Bz-ATP was cytotoxic to murine macrophage cell lines (RAW 264.7 and J774A.1 cells) but did not affect the intracellular survival of M. avium subsp. paratuberculosis, nor were the numbers of viable Mycobacterium avium subsp. avium or Mycobacterium bovis BCG cells altered in bovine mononuclear phagocytes or J774A.1 cells following ATP or Bz-ATP treatment. These data suggest that extracellular ATP does not induce the killing of intracellular M. avium subsp. paratuberculosis in bovine mononuclear phagocytes.

IL-4 and IL-10 inhibition of IFN-γ- and TNF-α-dependent nitric oxide production from bovine mononuclear phagocytes exposed to Babesia bovis merozoites

The requirement for IFN-γ and/or TNF-α as co-stimulants with Babesia bovis merozoites for nitric oxide (NO) production was examined, as well as the regulatory role of IL-4 and IL-10. Purified B. bovis merozoites did not induce the production of NO in undifferentiated monocytes without addition of exogenous IFN-γ and TNF-α unless the monocytes taken ex vivo were producing TNF-α endogenously. Under the latter condition, the NO production resulting from merozoite stimulation remained IFN-γ-dependent. There was no evidence for endogenous synthesis of TNF-α in monocyte-derived macrophages (MDM), and merozoites alone were incapable of inducing TNF-α mRNA in MDM. However, while merozoites plus IFN-γ induced TNF-α mRNA expression in MDM, NO was not produced. Both IL-4 and IL-10 inhibited expression of iNOS and production of NO in merozoite-stimulated monocytes.

Intracellular survival ofHaemophilus somnusin bovine blood monocytes and alveolar macrophages

The mechanisms used byHaemophilus somnusto survive and multiply within bovine mononuclear phagocytes are not fully understood. In order to study the interaction between bovine mononuclear phagocytes andH. somnus, a colorimetric assay using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was developed to assess the survival ofH. somnuswithin cultured bovine blood monocytes (BBM). Using this system, it was found thatH. somnuswas able to survive within BMMin vitro, and the kinetics of its survival were similar to that seen in BBM isolated from experimentally infected cattle. Using ultrastructural studies, it was possible to demonstrate the survival ofH. somnusin freshly isolated bovine mononuclear phagocytes in membrane-bound vacuoles. To determine if activation of macrophage function would result in elimination of intracellularH. somnus, BBM were treated withE. colilipopolysaccharide (LPS) or recombinant bovine (rBo) cytokines, interferon-γ(IFN-γ), granulocyte macrophage colony stimulating factor (GM-CSF), tumour necrosis factor-α (TNF-α) or interleukin-1β (IL-1β). Treatment of BBM with rBoIFN-γ, rBoGM-CSF orE. coliLPS resulted in decreased intracellular survival ofH. somnusat 18 and 48 h, whereas BBM treated with rBoTNF-α or rBoIL-1β had reduced intracellular survival ofH. somnusonly at 18 h. However, none of these treatments resulted in complete elimination of the intracellular bacteria. The ability ofH. somnusto survive and multiply in both freshly isolated and cytokine-treated cultured BBM demonstrated the capability ofH. somnusto escape from macrophage killing mechanisms. This capability may play a role in the dissemination ofH. somnusinfection in the body.

The role of IL-10 in iNOS and cytokine mRNA expression during in vitro differentiation of bovine mononuclear phagocytes.

In the study reported here, we used RT-PCR with primers specific for interleukin-1 (IL-1), IL-6, IL-10, tumor necrosis factor-alpha (TNF-alpha), and nitric oxide synthase (iNOS) to assess the cytokine mRNA expression associated with bovine blood monocytes during their differentiation to macrophages cultured on plastic (1 week). In addition, we used RT-PCR to assess the contribution of gammadelta T cells as a source of interferon-gamma (IFN-gamma), the induction signal for iNOS. Further, we evaluated cytocentrifuge preparations from the cultures for the production of IL-10 using specific antibody. We previously demonstrated that iNOS can be induced in cultured bovine monocytes in response to IFN-gamma and TNF-alpha but lose this capability in a short period of time. However, we demonstrate here that iNOS induction from monocytes cultured with IFN-gamma secreting gammadelta T cells is prolonged, suggesting that this source of IFN-gamma primes the monocytes before exogenous stimulation. Based on mRNA expression, placement of monocytes in culture resulted in activation, followed by quiescence. By 6 days in culture, the iNOS message was reduced below the basal level. In addition, the TNF-alpha message was substantially reduced, and IL-1 and IL-6 messages were reduced below detectable levels. This correlated with an increase in IL-10 message. Downregulation of these same cytokine messages as well as IFN-gamma message occurred within a 20-h period when IL-10 was added exogenously to cultures of total leukocytes. At the same time, there was an increase in the number of IL-10-positive cells and an increase in the intensity of anti-IL-10 staining within adherent cells. These results provide evidence for IL-10 regulation of some bovine mononuclear phagocyte effector functions.

Effect ofHaemophilus somnuson nitric oxide production and chemiluminescence response of bovine blood monocytes and alveolar macrophages

Haemophilus somnusis able to survive and multiply in bovine blood monocytes (BBM) and alveolar macrophages (BAM), but the mechanisms used byH. somnusto evade killing mechanisms of bovine mononuclear phagocytes are not completely understood. To study the bactericidal ability of bovine mononuclear phagocytes following interaction withH. somnus,in vitroassay systems were developed to detect the luminol-dependent chemiluminescence response (LDCL) and nitric oxide (NO) production of BBM and BAM. Live logarithmically growing or stationary phaseH. somnusinhibited the LDCL of BBM and BAM costimulated with opsonizedStaphylococcus aureus. Inhibition of the LDCL response of BBM and BAM was not mediated by liveH. somnusopsonized with hyperimmune serum, or by killed bacteria.H. somnusstimulated both BBM and BAM to produce NO at levels comparable withEscherichia colilipopolysaccharide. While NO was being produced, viableH. somnuscould still be isolated from the cell cultures. The ability ofH. somnusto inhibit LDCL of both BBM and BAM, and resistance to NO killing may be an important mechanism that contributes to survival of the organism following ingestion by bovine mononuclear phagocytes.

Serum factors, cell membrane CD14, and beta2 integrins are not required for activation of bovine macrophages by lipopolysaccharide.

The role of serum factors such as lipopolysaccharide (LPS)-binding protein (LBP) and of macrophage-expressed CD14 and beta2 integrins in the activation of bovine macrophages by LPS was investigated. Macrophage activation was determined by measuring tumor necrosis factor production, NO generation, and upregulation of procoagulant activity by LPS (Escherichia coli O55:B5) at concentrations of 100 pg/ml to 100 ng/ml. The 50% effective dose for LPS was 1 order of magnitude higher than that for activating human macrophages. Macrophages were activated by LPS in the presence of serum or in the presence of albumin demonstrated to be free of LBP. The capacity to react to LPS in the absence of LBP was not due to the acquisition of LBP during a previous culture in serum. It was then established which CD14-specific antibodies block LPS binding to monocytes. Among the CD14-specific antibodies recognizing bovine mononuclear phagocytes (60bca, 3C10, My4, CAM36, VPM65, CMRF31, and TUK4), the first four blocked the binding of LPS-fluorescein isothiocyanate to bovine monocytes at low concentrations. Anti-CD14 antibodies did not block LPS-mediated activation of bovine bone marrow-derived macrophages, monocyte-derived macrophages, and alveolar macrophages. This was observed in experiments in which anti-CD14 concentrations exceeded the 50% inhibitory dose by >30-fold (3C10 and My4) or >300-fold (60bca), as defined in the binding assay described above. Monocyte-derived macrophages from an animal deficient in beta2 integrins and control macrophages were activated by similar concentrations of LPS, suggesting that beta2 integrins are not important bovine LPS receptors. Thus, in bovine macrophages, LPS recognition pathways which are independent of exogenous LBP, of membrane-expressed CD14, and of beta2 integrins may exist.

Modulation of phagocytic function of bovine mononuclear phagocytes by Haemophilus somnus

The interactions between bovine mononuclear cells and Haemophilus somnusare known to be complex. To study this interaction, a flow cytometric assay was developed to assess the effect of H. somnuson phagocytosis of killed opsonized Staphylococcus aureusby bovine alveolar macrophages and blood monocytes. Using this in vitrosystem, it was found that log phase H. somnussignificantly inhibited the phagocytosis of killed opsonized S. aureusby bovine alveolar macrophages obtained both from healthy calves and from cattle experimentally infected with H. somnus. However, killed log-phase H. somnus, in vitropassaged and stationary phase H. somnushad no effect on the phagocytic activity of these cells. In contrast to bovine alveolar macrophages, blood monocytes showed a significant increase in their phagocytic activity following in vitroexposure to either log or stationary phase H. somnus. Using a lypophilic, non-toxic fluorophore PKH2 to label live H. somnus, it was possible to simultaneously measure the uptake of both S. aureusand H. somnus. Stationary and log phase H. somnuswere taken up by macrophages equally well, even though phagocytosis of S. aureuswas inhibited by only log phase H. somnus. These results demonstrate the ability of H. somnusto modulate bovine mononuclear phagocytic function which might contribute towards the pathogenesis of bovine hemophilosis.

Assessment of bovine mononuclear phagocytes and neutrophils for induced L-arginine-dependent nitric oxide production

Microbicidal activity of reactive oxygen intermediates and reactive nitrogen intermediates has been described from both murine and human cytokine activated macrophages. An L-arginine-dependent pathway of nitric oxide generation has recently been described from bovine bone marrow-derived and monocyte-derived macrophages in response to a phagocytic stimulus. We have investigated the induction and release of both reactive oxygen intermediates and reactive nitrogen intermediates from bovine neutrophils, and blood and spleen mononuclear phagocytes in response to either a phagocytic or cytokine stimulus. Mononuclear phagocytes were poor producers of hydrogen peroxide (a measure of reactive oxygen intermediate production) under conditions that readily caused release by neutrophils. In contrast, nitrite, as a measure of nitric oxide production, could not be induced from neutrophils under any stimulation conditions, while mononuclear phagocytes responded to both a phagocytic stimulus and cytokines with the induction of nitric oxide synthase message and production of nitric oxide. There appeared to be two populations of monocytes that differed both in their adherent characteristics and their level of cytokine-induced nitric oxide production. Both populations stained with a single monoclonal antibody. However, the population that had not adhered to plastic within 3 h responded to cytokine stimulation, producing up to 3 times more nitric oxide on a per cell basis than the readily adherent population. Cytokine induction required the presence of interferon-gamma and either tumor necrosis factor-alpha or lipopolysaccharide. L-arginine dependence was demonstrated by inhibition with an L-arginine analog and restoration with addition of excess L-arginine.

Induction of Nitric Oxide Synthase in Bovine Mononuclear Phagocytes is Differentiation Stage-Dependent

Bovine monocytes and monocyte-derived macrophages (MDM) activated by various means were assessed for induction of inducible nitric oxide synthase (iNOS), using the Griess assay, Northern blotting and reverse transcription/polymerase chain reaction (RT-PCR). Interferon-γ (IFN-γ) induced little, if any, iNOS expression and NO production in MDM, although these cells responded to IFN-γ in other regards. In contrast, MDM produced copious amounts of NO when stimulated with LPS or Salmonella dublin, and this was paralleled by high steady state levels of iNOS mRNA. Heat-killed Listeria monocytogenes induced more iNOS mRNA and nitrite than IFN-γ, but much less than L. monocytogenes and IFN-γ combined. Monocytes differed from MФ with respect to iNOS induction and nitrite production in several regards: (1) LPS and S. dublin induced only low levels of iNOS mRNA and nitrite in monocytes, although cells responded to these stimuli in various other ways: (ii) IFN-γ alone induced in monocytes 1NOS mRNA generation and NO formation, although to a low and variable degree; (iii) upon maximal stimulation (e.g. by L. monocytogenes and IFN-γ combined), monocytes produced much less nitrite than MDM, and mRNA levels were lower. Regulation of macrophage iNOS varies considerably between species. We provide the first evidence in any species that the steady state levels of iNOS mRNA and NO generation in monocytes and macrophages activated by various means depend on the stage of mononuclear phagocyte differentiation.

Functional characteristics of enhanced Fc receptor expression of beta 2 integrin-deficient bovine mononuclear phagocytes.

Fc receptor expression, cytoplasmic Ca2+ signaling, chemiluminescent (CL) response, and electron spin resonance (ESR) combined with spin trapping of blood mononuclear phagocytes from control heifers and a heifer with leukocyte adhesion deficiency (LAD) were evaluated to elucidate the relationships between complement receptor type 3 (CR3) and Fc receptor expression and their functional responses. The mean fluorescence intensity of fluorescein isothiocyanate (FITC)-conjugated anti-bovine IgG bound to mononuclear phagocytes from the heifer with LAD was 1.8-fold higher than that of control heifers. The mean increments of cytoplasmic Ca2+ concentrations of mononuclear phagocytes from the heifer with LAD stimulated with OPZ, Agg-IgG, and PMA were 39.4 (P < 0.05), 118, and 71.6% compared with those of control heifers. A 1.27-fold increase in the CL response relative to control heifers was detected when mononuclear phagocytes from the heifer with LAD were stimulated with Agg-IgG. The OPZ-induced CL response of mononuclear phagocytes from the heifer with LAD was significantly (P < 0.05) decreased, whereas the PMA-induced CL response was similar to that of control heifers. The ESR spectrum of mononuclear phagocytes from the heifer with LAD was increased when stimulated with Agg-IgG, and was impaired when stimulated by OPZ compared with that of control heifers. The ESR spectrum of mononuclear phagocytes stimulated with PMA was similar in control heifers and the heifer with LAD. Fc receptors on mononuclear phagocytes from the heifer with LAD were enhanced, and their cytoplasmic Ca2+ signaling, CL response, and ESR-spin trapping when stimulated with Agg-IgG and OPZ appeared to be associated with enhanced Fc receptors.

The long-term culture of bovine monocyte-derived macrophages and their use in the study of intracellular proliferation of Brucella abortus

Although the immune response to Brucella abortus is multifaceted, the key event in contending with this pathogen appears to be the interaction of the organism with cells of the mononuclear phagocyte system. A cell culture system was developed which allowed the long-term maintenance of blood monocyte-derived macrophages in Teflon culture vessels in a relatively unstimulated state. The assay system was optimized for timing of bacteria-macrophage interaction and numbers of bacteria and macrophages used in each assay. Interaction of B. abortus strain 2308 with bovine mononuclear phagocytes from animals phenotypically resistant and susceptible to infection with B. abortus was investigated. This cell culture and assay system should provide a useful model for the investigation of intracellular parasitism in cattle.

Effect of aflatoxin B 1 on in vitro production of interleukin-1 by bovine mononuclear phagocytes

In this study we examined the effects of preincubation with aflatoxin B 1 (AFB 1) on the ability of bovine monocytes to produce the immunological mediator interleukin-1 (IL-1). Monocytes preincubated for 6–24 h with 10 μg ml −1 AFB 1 demonstrated a diminished capacity to release IL-1 activity in response to bacterial lipopolysaccharide (LPS). Preincubation for less time, or with lower concentrations of AFB 1, did not affect IL-1 release. Pretreatment of monocytes with AFB 1 also resulted in diminished release of IL-1 activity in response to in vitro infection with Listeria monocytogenes. Incubation with AFB 1 reduced the amount of IL-1 β mRNA in LPS-stimulated bovine monocytes; however, this was observed only at high concentrations of AFB 1 that non-specifically reduced steady-state transcription of actin mRNA. We therefore concluded that AFB 1 does not specifically suppress monocyte release of IL-1, other than through its general inhibition of mRNA transcription.

Differentiation antigens on bovine mononuclear phagocytes identified by monoclonal antibodies

Five monoclonal antibodies (MAb) produced against cell surface antigens on bovine mononuclear phagocytes (MPh) were characterized. None of the MAb recognized erythrocytes, thrombocytes, B lymphocytes or resting or activated T lymphocytes. Two MAb (IL-A22 and IL-A24) reacted with the majority of monocytes and granulocytes in peripheral blood, with 20–40% bone marrow cells comprising myelo-monocytic cells, and with a proportion of mature macrophages. Reactivity of the remaining three MAb was restriced to MPh: one of these (IL-A25) was apparently specific for pulmonary macrophages, whereas the molecules recognized by the other two (IL-A23 and CH16A) were expressed on subpopulations of blood monocytes and tissue macrophages. None of the MAb inhibited adherence of MPh to plasma-coated gelating surfaces or Fc-mediated rosette formation. One of the MAb, IL-A24, which reacts with MPh and granulocytes, inhibited antigen-specific proliferative response or peripheral blood mononuclear leukocytes (PBM) to the soluble antigen, keyhole limpet hemocyanin (KLH) but did not inhibit responses to concanavalin A or allogeneic leukocytes. This MAb was shown to react with two polypeptides of approximately 75 kD and 110 kD on the surface of peripheral blood monocytes.

"Haemophilus somnus," a facultative intracellular pathogen of bovine mononuclear phagocytes.

We have reported previously that bovine neutrophils are unable to kill the bovine respiratory pathogen "Haemophilus somnus." In the present study we expanded our efforts and examined the interaction of bovine mononuclear phagocytes with this important veterinary pathogen. Bovine alveolar macrophages and blood monocytes ingested but did not kill opsonized "Haemophilus somnus" in vitro, whereas these same cells ingested and killed opsonized Escherichia coli. Because this suggested that "H. somnus" was a facultative intracellular pathogen, we developed an assay to monitor the intracellular fate of ingested "H. somnus" within bovine monocytes. Our results indicated that ingested "H. somnus" multiplied within bovine monocytes (1- to 2-log10 increase in 4 h); equivalent intracellular growth was noted for both a laboratory strain and a recent field isolate of "H. somnus." Bovine monocytes killed ingested E. coli (1- to 2-log10 decrease in 4 h) under the same assay conditions that were used to follow intracellular growth of "H. somnus," thus indicating that the assay conditions did not induce a generalized defect in monocyte antibacterial activity. Light and electron microscopic examination of "H. somnus"-infected monocytes confirmed that intracellular growth had occurred. We did not observe an obvious correlation between the release of superoxide anion from bovine mononuclear phagocytes that had ingested opsonized "H. somnus" and E. coli and the subsequent intracellular survival of the bacteria. The results of this study suggest that infected mononuclear phagocytes sustain "H. somnus" infections in cattle and thus contribute to the subacute to chronic clinical course that has been reported.

Growth inhibition of Babesia bovis in culture by secretions from bovine mononuclear phagocytes.

Bovine blood mononuclear phagocytes from babesia-free cattle were cultured in vitro. Cell monolayers were treated with culture-derived soluble Babesia bovis exoantigens, immune complexes, and bovine anti-B. bovis immune serum. Subsequently, the monolayers were washed free of the reagents and allowed to develop further in the presence of standard culture medium. Transfer of supernatant media from these cultures to those of B. bovis revealed the presence of growth-inhibiting factors. These factors were thermostable, nondialyzable, and were degraded by freeze-thawing, and their action was concentration dependent. Supernatants from antigen- and immune complex-treated monolayers demonstrated greater inhibitory effects than did supernatants from antibody-treated or untreated monolayers. Erythrocytes incubated with supernatant medium from antigen-treated monolayers did not support growth of B. bovis as well as did erythrocytes incubated with supernatants from untreated monocyte monolayers. This result suggests that the mechanism of action of soluble factors could be through some modification of the erythrocyte such as the blockage of active transport of essential nutrients.

Evaluation of flow cytometry and fluorescence microscopy for the estimation of bovine mononuclear phagocytes

Bovine blood mononuclear cells were isolated by density gradient centrifugation on Ficoll-Paque. Phagocytic mononuclear cells were characterized functionally by ingestion of fluorescent latex beads. After incubation with beads the cells were treated with Triton X-100 and propidium iodide (PI) to stain DNA. Cells were analyzed with a FACS-III instrument connected to a Nuclear Data-6660 multiparameter computer system. The computer was used to evaluate the 2 parameter histograms in order to enumerate the percentage of cells with different numbers of associated beads. With this system we also obtained information about cell concentration and number of beads per cell. Results from flow cytometry and manual counting by fluorescence microscopy were compared and good correlation ( r = 0.91) was obtained. During the first hours of incubation latex beads adhered to cell surfaces as demonstrated by FCM histograms and fluorescence microscopy. Blood mononuclear phagocytes have to be incubated for several hours before significant phagocytotic activity can be detected.