Glucocorticoids modulate the development of dendritic cells from blood precursors.

Dendritic cells (DC) are professional antigen-presenting cells, capable of priming naive T cell responses. Glucocorticoids (GC) are frequently used in asthmatic patients. In this study we describe the effects of GC on the development and function of monocyte-derived DC (MoDC) in vitro and in vivo. Monocytes from healthy individuals were isolated and incubated with granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4 for 6 days, to induce maturation into MoDC. To study the role of GC on DC differentiation in vitro cells were incubated with dexamethasone at different stages of MoDC development. At day 6 cells were characterized phenotypically by flow cytometry and functionally in an allogeneic mixed leucocyte reaction. To study the effect of GC in vivo patients with mild/moderate atopic asthma were selected. In one group no GC were used, whereas the other group used inhalation GC. MoDC from these patients were generated as described above and tested functionally. Incubation of MoDC or its peripheral blood precursors with dexamethasone decreased the accessory potency dose-dependently. The functional differences could not be explained by the changes in the expression of MHC II and the costimulatory molecules CD40 and CD86. The relevance of this mechanism was confirmed for the in vivo situation as well. MoDC from patients using inhalation GC showed a decreased accessory potency. These data suggest a modulatory effect of GC therapy at the level of the peripheral blood monocyte. The results indicate that GC influence DC development and function in vitro as well as in vivo.

Effects of stress on alveolar macrophages: a role for the sympathetic nervous system.

Alveolar macrophages (AMs) play an important role in the regulation of the local immune reactivity in the lung. It was previously shown that exposure of rats to mild inescapable electrical footshock stress (20 min, 4 shocks/min, 5 s/shock, 0.8 mAmp) leads to apparent changes in the activity of AMs upon stimulation, reflected by an enhanced interleukin-1beta and tumor necrosis factor-alpha secretion and decreased nitric oxide secretion compared with the secretion by AMs isolated from nonstressed rats. Here we show that in vivo blockade of the autonomic nervous system by intraperitoneal injection of the nicotinic receptor antagonist chlorisondamine leads to complete abrogation of these stress-induced alterations in AM activity. This role for the autonomic nervous system could further be attributed to sympathetic stimulation of beta-adrenergic receptors as shown by blockade of beta-adrenoceptors. Blockade of either alpha-adrenoceptors or parasympathetic output did not result in abrogation of the stress-induced changes in AM activity. The beta-adrenergic modulation of AM activity most likely is not due to a direct effect of catecholamines on AMs because mimicking the in vivo stress effects by in vitro preincubation of AMs with various doses of catecholamines followed by lipopolysaccharide stimulation did not result in an altered cytokine secretion by AMs.

Functional immaturity of rat alveolar macrophages during postnatal development.

Alveolar macrophages (AM) are important in the regulation of immune responses in the lung, through their role as scavenger cells and through the production of many bioactive factors. Because in early infancy pulmonary infections are a recurrent problem, we studied the postnatal functional maturation of AM in a rat model. AM were isolated from rat lungs by bronchoalveolar lavage at several time intervals after birth and tested for their ability to ingest Escherichia coli in the presence of surfactant protein A (SP-A). Furthermore, their capacity to produce nitric oxide (NO) and interleukin-1 beta (IL-1 beta) after in vitro lipopolysaccharide (LPS) stimulation was analysed, as well as their capacity to downregulate proliferation of T cells from both mature and neonatal rats. SP-A-mediated phagocytosis of E. coli by AM was reduced in 14-day-old neonatal rats, as compared with mature rats (P < or = 0.05). Also the IL-1 beta production by rat AM after LPS stimulation was impaired at 14 days of age, as compared with IL-1 beta production by AM from mature rats (P < or = 0.05). In contrast, the LPS-induced NO production by rat AM as well as the capacity to inhibit T-cell proliferation were well developed at all ages tested. In conclusion, during postnatal development the rat AM is functionally immature, with respect to phagocytosis and secretion of inflammatory mediators. These differences may underly the enhanced susceptibility to pulmonary infections as found in human neonates.

Acute and long-term effects of stressors on pulmonary immune functions.

To study the effects of different types or intensities of stressors on immune reactivity in the lungs, we studied the ex vivo production of nitric oxide (NO) and IL-1beta by alveolar macrophages (AM) after short exposure of rats to restraint stress or inescapable electric footshocks. Exposure to electric footshocks of various intensities resulted in an intensity-dependent decrease in NO production whereas the IL-1beta production by AM had increased. The secretory activity was similarly affected by restraint stress. When the time course of electric footshocks on secretory functions of AM was studied, it was found that the effects on NO and IL-1beta production by AM were normalized 3 days after the stress induction, but reappeared when cells were isolated 1 to 2 wk after stress exposure. Analysis of the effects of electric footshocks of various intensities on antibody production 10 days after the stress session and subsequent lung immunization with trinitrophenyl conjugated keyhole limpet hemocyanin (TNP-KLH), showed a footshock intensity-dependent response. Although exposure to stress induced an increase in plasma levels of adrenocorticotropic hormone (ACTH) and corticosterone (CORT), hormone levels did not differ between the various stress-exposed groups. This suggests that the observed stress effects on pulmonary immune functions were not mediated by ACTH or CORT but point to a direct involvement of the autonomic nervous system.

Alveolar macrophages are required for protective pulmonary defenses in murine Klebsiella pneumonia: elimination of alveolar macrophages increases neutrophil recruitment but decreases bacterial clearance and survival.

To study the in vivo role of alveolar macrophages (AM) in gram-negative bacterial pneumonia in mice, AM were eliminated by the intratracheal (i.t.) administration of dichloromethylene diphosphonate encapsulated liposomes. Subsequently, the AM-depleted mice were infected i.t. with 100 CFU of Klebsiella pneumoniae, and the effects of AM depletion on survival, bacterial clearance, and neutrophil (polymorphonuclear leukocyte [PMN]) recruitment were assessed. It was shown that depletion of AM decreases survival dramatically, with 100% lethality at day 3 postinfection, versus 100% long-term survival in the control group. This increased mortality was accompanied by 20- to 27- and 3- to 10-fold increases in the number of K. pneumoniae CFU in lung and plasma, respectively, compared to those in nondepleted animals. This decreased bacterial clearance was not due to an impaired PMN recruitment; on the contrary, the K. pneumoniae-induced PMN recruitment in AM-depleted lungs was sevenfold greater 48 h postinfection than that in control infected lungs. Together with an increased PMN infiltration, 3- and 10-fold increases in lung homogenate tumor necrosis factor alpha (TNF-alpha) and macrophage inflammatory protein 2 (MIP-2) levels, respectively, were measured. Neutralization of TNF-alpha or MIP-2, 2 h before infection, reduced the numbers of infiltrating PMN by 41.6 and 64.2%, respectively, indicating that these cytokines mediate PMN influx in infected lungs, rather then just being produced by the recruited PMN themselves. Our studies demonstrate, for the first time, the relative importance of the AM in the containment and clearance of bacteria in the setting of Klebsiella pneumonia.

In vivo study on the immunomodulating effects of OM-85 BV on survival, inflammatory cell recruitment and bacterial clearance in Klebsiella pneumonia.

Oral administration of the bacterial extract OM-85 BV has been shown to prime alveolar macrophages (AM) in such a way that they secrete significantly more nitric oxide, tumor necrosis factor-alpha and interleukin-1beta upon in vitro stimulation with lipopolysaccharide (LPS). As increased cytokine secretion by AM may account for the therapeutic effect of OM-85 BV in respiratory tract infections, we studied the effect of orally administered OM-85 BV on the outcome of Klebsiella pneumoniae-induced pneumonia. Mice received a daily oral dose of OM-85 BV (350 mg/kg body weight) for 5 days and were intratracheally infected with 333, 1000 or 3333 CFU K. pneumoniae on day 8. It was shown that OM-85 BV pretreatment of mice has no effect on bacterial clearance, neutrophil recruitment and survival in this acute respiratory tract infection. Also, OM-85 BV treatment had no protective effect in a recurrent infection with K. pneumoniae. It is concluded that AM activation by oral treatment with OM-85 BV is not sufficient to play a protective role in respiratory tract infection with K. pneumoniae.

Dose- and time-dependent activation of rat alveolar macrophages by glucocorticoids.

Effects of glucocorticoids on immune functions are generally thought to be suppressive and anti-inflammatory. However, most reports dealing with this issue describe effects of long-term treatment with high doses of glucocorticoids on immune functions. In the present study we have investigated both dose and timing effects of exposure of alveolar macrophages with dexamethasone on lipopolysaccharide (LPS)-induced IL-1 beta and nitric oxide secretion. For this purpose, alveolar macrophages were preincubated with various doses of dexamethasone during varying intervals, followed by stimulation of the cells with endotoxin, either in the absence or presence of dexamethasone. Subsequently, the effects of this treatment of IL-1 beta and nitric oxide secretion were measured. It was shown that both short-term incubation of alveolar macrophages with high doses of dexamethasone and long-term incubation with low doses of dexamethasone lead to enhanced nitric oxide and enhanced IL-1 beta secretion upon subsequent stimulation of the cells with LPS. In contrast, long-term incubation of alveolar macrophages with high-dose dexamethasone leads to decreased IL-1 beta and nitric oxide secretion upon subsequent stimulation. Thus, it is concluded that the effects of dexamethasone on rat alveolar macrophages are both time- and dose-dependent. It is therefore argued that effects of glucocorticoids on immune functions are not a priori suppressive, but that both dose and timing effects should be taken into account.

Phagocytosis and intracellular killing of serum-opsonized Staphylococcus aureus by mouse fibroblasts expressing human Fcgamma receptor type IIa (CD32).

Phagocytes bear more than one class of receptors for the Fc domain of IgG (FcgammaR). In addition the same ligand can interact with different classes of FcgammaR. This complexity makes it difficult to study the contribution of the various classes of FcgammaR to antimicrobial functions. To circumvent this difficulty, in the present study mouse 3T6 fibroblasts transfected with cDNA encoding for human FcgammaR type IIa (FcgammaRIIa-expressing cells) were used to determine the role of this receptor in phagocytosis and intracellular killing of serum-opsonized Staphylococcus aureus. Experiments using microbiological and fluorescent techniques to discriminate between cell-adherent and intracellular bacteria revealed that serum-opsonized bacteria are phagocytized by FcgammaRIIa-expressing cells, but not by parental fibroblasts. Non-opsonized bacteria were poorly internalized by FcgammaRIIa-expressing as well as parental fibroblasts. Furthermore, incubation of FcgammaRIIa-expressing cells with opsonized bacteria at 4oC and incubation of FcgammaRIIa-expressing cells with cytochalasin E prior to addition of opsonized bacteria inhibited the phagocytosis of these bacteria almost completely. Phagocytosis of opsonized bacteria by FcgammaRIIa-expressing cells was partly inhibited by selective inhibition of protein tyrosine kinases (PTK). FcgammaRIIa cross-linking initiated transient tyrosine phosphorylation of various proteins in FcgammaRIIa-expressing cells. These data indicate that activation of PTK is involved in the FcgammaRIIa-mediated phagocytosis of opsonized S. aureus by transfected fibroblasts. Human serum from normal individuals and agammaglobulinemic patients triggered the intracellular killing of S. aureus by FcgammaRIIa-expressing fibroblasts. Surprisingly, heat-inactivated human serum, IgG and incubation with anti-FcgammaRII antibodies followed by a bridging secondary antibody did not stimulate the killing process. The possibility that these ligands did not interact with FcgammaRIIa on the cells can be excluded since they induced tyrosine phosphorylation of cellular proteins. The serum factor that stimulates the intracellular killing of bacteria by FcgammaRIIa-expressing cells is not yet identified. Oxygen-independent mechanisms are thought to be responsible for the killing of intracellular bacteria by these cells since the NADPH oxidase inhibitor diphenylene iodonium did not affect the serum-stimulated intracellular killing of S. aureus and no reactive oxygen and nitrogen intermediates were produced by FcgammaRIIa-expressing cells after appropiate stimulation. Taken together, these data show that phagocytosis but not intracellular killing of S. aureus is mediated via FcgammaRIIa on cells expressing this receptor.

Changes in cytokine and nitric oxide secretion by rat alveolar macrophages after oral administration of bacterial extracts.

Oral administration of the bacterial immunomodulator Broncho-Vaxom (OM-85), a lysate of eight bacteria strains commonly causing respiratory disease, has been shown to enhance the host defence of the respiratory tract. In this study we examined the effect of orally administered (in vivo) OM-85 on stimulus-induced cytokine and nitric oxide secretion by rat alveolar macrophages in vitro. The results show that alveolar macrophages isolated from OM-85-treated rats secreted significantly more nitric oxide, tumour necrosis factor-alpha (TNF-alpha) and IL-1 beta upon in vitro stimulation with lipopolysaccharide (LPS), whereas, in contrast, LPS-induced IL-6 secretion was significantly lower. The observed effects of in vivo OM-85 treatment on stimulus-induced cytokine secretion in vitro are not due to a direct effect of OM-85 on the cells, because in vitro incubation of alveolar macrophages with OM-85 did not result in altered activity, nor did direct intratracheal instillation of OM-85 in the lungs of rats result in altered alveolar macrophage activity in vitro. It is hypothesized that oral administration of OM-85 leads to priming of alveolar macrophages in such a way that immune responses are non-specifically enhanced upon stimulation. The therapeutic action of OM-85 may therefore result from an enhanced clearance of infectious bacteria from the respiratory tract due to increased alveolar macrophage activity.