Zvezdana Vuk-Pavlovic

Pneumocystis Cell Wall β-Glucans Induce Dendritic Cell Costimulatory Molecule Expression and Inflammatory Activation through a Fas-Fas Ligand Mechanism

Respiratory failure during Pneumocystis pneumonia is mainly a consequence of exaggerated inflammatory responses to the organism. Dendritic cells (DCs) are the most potent APCs in the lung and are key to the regulation of innate and adaptive immune responses. However, their participation in the inflammatory response directed against Pneumocystis infection has not been fully elucidated. Therefore, we studied the role of Pneumocystis carinii, as well as Saccharomyces cerevisiae, cell wall-derived β-glucans, in DC costimulatory molecule expression. We further studied the impact of β-glucans on subsequent T cell activation. Because cytokine secretion by DCs has recently been shown to be regulated by Fas ligand (FasL), its role in β-glucan activation of DCs was also investigated. β-Glucan-induced DC activation occurred in part through dectin-1 receptors. We demonstrated that DC activation by β-glucans elicits T cell activation and polarization into a Th1 patterned response, but with the conspicuous absence of IL-12. These observations differed from LPS-driven T cell polarization, suggesting that β-glucans and LPS signal DC activation through different mechanisms. We additionally determined that IL-1β and TNF-α secretion by β-glucan-stimulated DCs was partially regulated by Fas-FasL. This suggests that dysregulation of FasL could further enhance exuberant and prolonged cytokine production by DCs following DC-T cell interactions, further promoting lung inflammation typical of Pneumocystis pneumonia.

Surfactant protein d-mediated aggregation of Pneumocystis carinii impairs phagocytosis by alveolar macrophages

ABSTRACT Pneumocystis carinii remains an important and potentially fatal cause of opportunistic pneumonia. Animal studies reveal that substantial quantities of surfactant protein D (SP-D) accumulate in the airspaces during P. carinii pneumonia and are particularly abundant in aggregates of organisms. Due to the multimeric structure of SP-D, we hypothesized that SP-D mediates aggregation of the organism. From previous clinical studies it is known that aggregated organisms are conspicuous in sections of lung tissue and bronchoalveolar lavage (BAL) fluids of humans with active P. carinii pneumonia. Herein, we observe that SP-D levels increased at least fourfold in BAL fluids of patients with P. carinii pneumonia. Next, a spectrophotometric sedimentation assay was developed to assess the aggregation of P. carinii in vitro by SP-D. P. carinii organisms were first stripped with glutathione to remove bound SP-D and subsequently incubated in the presence of SP-D and 2 mM calcium. P. carinii incubated with natural SP-D (10 μg/ml) containing dodecamers and higher-order forms exhibited aggregation and enhanced sedimentation compared to that of glutathione-stripped P. carinii. Aggregation was also enhanced by the concentrated supernatant of rat BAL fluid, and this effect was abolished by the selective removal of SP-D from the lavage fluid. P. carinii aggregation was reduced by maltose, mannose, and EDTA, consistent with the role of the SP-D C-type lectin domain (CRD) in the aggregation event. Comparisons of different molecular forms of SP-D showed that dodecamers—but not trimeric subunits—mediate optimal aggregation of P. carinii. Aggregation of P. carinii by SP-D was shown to be responsible for the impaired phagocytosis of the organisms by alveolar macrophages. Thus, SP-D-mediated aggregation of P. carinii may represent one means by which the organism avoids elimination by the host.

Fitness of Cell-Mediated Immunity Independent of Repertoire Diversity

Fitness of cell-mediated immunity is thought to depend on TCR diversity; however, this concept has not been tested formally. We tested the concept using JH−/− mice that lack B cells and have TCR Vβ diversity <1% that of wild-type mice and quasimonoclonal (QM) mice with oligoclonal B cells and TCR Vβ diversity 7% that of wild-type mice. Despite having a TCR repertoire contracted >99% and defective lymphoid organogenesis, JH−/− mice rejected H-Y-incompatible skin grafts as rapidly as wild-type mice. JH−/− mice exhibited T cell priming by peptide and delayed-type hypersensitivity, although these responses were less than normal owing either to TCR repertoire contraction or defective lymphoid organogenesis. QM mice with TCR diversity contracted >90%, and normal lymphoid organs rejected H-Y incompatible skin grafts as rapidly as wild type mice and exhibited normal T cell priming and normal delayed-type hypersensitivity reactions. QM mice also resisted Pneumocystis murina like wild-type mice. Thus, cell-mediated immunity can function normally despite contractions of TCR diversity >90% and possibly >99%.

Characteristics of Monocyte Angiotensin‐Converting Enzyme (ACE) Induction by Dexamethasone

Monocyte maturation to macrophages and transformation into epithelioid granuloma cells in some granulomatous diseases are accompanied by the induction of membrane‐bound angiotensin‐converting enzyme (ACE). The physiologic and pathophysiologic roles of ACE generated in these processes are not known. The pattern and the mechanism of ACE induction in human monocytes are also not well understood. Dexamethasone is one of the agents reported to induce elevated ACE activity in human monocytes, and therefore a suitable tool for studying the phenomenon. This study shows that dexamethasone augments monocyte ACE in a Diphasic dose‐dependent manner with maximum effect at 10‐5 M concentration. Although it enhances the level of ACE activity, dexamethasone doss not alter the time course for ACE induction from that found in unstimulated monocytes. The ACE activity of monocytes cultivated in 10 nM dexamethasone and then exposed to 10‐3 M diazosulfanilic acid (DASA) is reduced approximately by 80% in comparison with cells not treated with DASA, demonstrating that dexamethasone‐induced ACE is an ectoenzyme. Dexamethasone does not increase the activity of other monocyte ectoenzymes: γ‐glutamyltransferase, alkaline phosphodiesterase‐I, and leucine aminopeptktase, showing that dexamethasone induction of ACE is a specific, rather than generalized, effect on plasma membrane enzymes. It is suggested that the increase in ACE activity is due to the increased rate of enzyme synthesis.

Cotton Condensed Tannin: A Potent Modulator of Alveolar Macrophage Host-Defense Function

Alveolar macrophages are the resident airway cells primarily responsible for the protection of the lungs against inhaled toxins and other biologically active material. A number of functional capabilities constitute their host-defense function. They can phagocytize and inactivate foreign material by production of reactive oxygen intermediates or the action of hydrolytic enzymes. In the absence of phagocytosis, macrophages can secrete reactive oxygen intermediates or enzymes that inactivate extracellular biologically active material. They also can secrete metabolites of arachidonic acid and other cytokines that contribute to the inflammatory response of the lungs. Macrophages also secrete a variety of peptide and lipid chemotactic factors that lead to the recruitment of other inflammatory cells into the airways. The condensed tannins, which constitute a significant percentage of the water soluble compounds present in respirable cotton mill dust, dramatically alter the host-defense function of alveolar macrophages in vitro. Tannin inhibits both phagocytosis and production of reactive oxidants in a dose-dependent manner with EC50s of 16 micrograms/mL and 3 micrograms/mL, respectively. This inhibition dramatically decreases the ability of resident alveolar macrophages to clear and detoxify potentially harmful inhaled particles. However, at similar concentrations, tannin stimulates the dose-dependent secretion (EC50 = 15 micrograms/mL) of a low molecular weight lipid neutrophil chemotactic factor that could result in an inflammatory reaction with the recruitment of neutrophils into the lungs. At slightly higher concentrations, tannin promotes the dose-dependent release of arachidonic acid from the macrophage membranes (EC50 = 65 micrograms/mL), which could also contribute to the local inflammatory reaction. Finally, tannin also causes secretion of the cytokine, interleukin-1, from the monocyte precursors of macrophages with an EC50 of 32 micrograms/mL. Interleukin-1 has been implicated as one of the causative agents in the development of fever.

Induction of Interleukin-1-Beta Release from Human Monocytes by Cotton Bract Tannin

The human T lymphocyte proliferative response to cotton bract tannin was shown to be dependent upon the presence of monocytes. Since monocytes support the T cell mitogenic response by interleukin-1 (IL-1) production, it was anticipated that tannin has IL-1-inducing ability. To examine this possibility, human monocytes were cultured alone or with peripheral blood T lymphocytes, and stimulated with tannin. Control cultures included unstimulated cells, and cells challenged with other IL-1 inducers: concanavalin A (Con A) and lipopolysaccharide from Escherichia coli or Enterobacter agglomerans. IL-1 beta was measured in culture supernatants 24 h after initiation of the culture by the use of an ELISA or an RIA. The results showed that tannin stimulated monocytes to secrete IL-1 beta in a manner similar to Con A, i.e. substantially more cytokine was measured in the supernatants of monocyte-T-lymphocyte co-cultures than in the cultures of monocyte alone. Endotoxin from E. coli was less effective than the endotoxin from E. agglomerans in IL-1 induction. Contaminating endotoxin present in the tannin preparation accounted for the majority of IL-1 beta released from monocytes alone stimulated with tannin, but only 20% of the IL-1 beta released from tannin-stimulated monocyte-T-lymphocyte co-cultures. These results show that tannin itself has IL-1-inducing ability. The dose-response studies show that the extent of IL-1 beta release is dependent on tannin dose and that increased levels of monocyte-produced IL-1 beta precede the increase in T lymphocyte proliferation.(ABSTRACT TRUNCATED AT 250 WORDS)