Removal of bronchoalveolar cells augments the late eosinophilic response to segmental allergen challenge.

BACKGROUND: In patients with quiescent asthma, macrophages are the most prevalent cells recovered by bronchoalveolar lavage (BAL). Through activation via their FcepsilonRII receptors or by acting as antigen-presenting cells, macrophages could, in theory, promote the late airway response to allergen. OBJECTIVE: In order to investigate the importance of macrophages and other airway luminal cells in inducing the late airway response, a novel washout experiment was designed. METHODS: Five patients with ragweed-allergic asthma underwent bronchoscopy and segmental bronchial challenge with either normal saline or short ragweed extract in two segments of one lung. In a third segment of the opposite lung, 12 successive BALs (25 mL each) were performed, followed by challenge with an identical dose of short ragweed (washed-challenged segment). After 24 h, all three challenged segments underwent BAL. RESULTS: Initially, in the washed-challenged segment, over 80% (mean 80.4%, range 68-88%) of the recoverable airway dwelling cells were removed. Unexpectedly, 24 h later these same washed-challenged segments contained more eosinophils in the BAL than the challenged segments from the opposite lung (P = 0.033). CONCLUSIONS: Removing the majority of airway luminal cells followed by allergen bronchoprovocation increased the number of eosinophils recovered 24 h after challenge. Our results suggest that in quiescent allergic asthma, the airway luminal cells are protective and attenuate the late eosinophilic response to allergen challenge.

Carbohydrate recognition domain of surfactant protein D mediates interactions with Pneumocystis carinii glycoprotein A.

Pneumocystis carinii continues to cause severe pneumonia in immunocompromised patients. Surfactant protein D (SP-D), a lung collectin, markedly accumulates during P. carinii pneumonia and binds to glycoprotein A (gpA) on the surface of P. carinii, thereby enhancing interactions with alveolar macrophages. Herein, we report the structural basis of the interaction of SP-D with gpA. We demonstrate that natural SP-D binds to purified gpA in the presence of 2 mM calcium in a saturable, concentration-dependent manner, which is abolished by 10 mM ethylenediaminetetraacetic acid. Increasing concentrations of calcium under otherwise cation-free conditions significantly enhance SP-D binding to gpA, whereas manganese and magnesium cations have minimal effect. Maximal SP-D binding occurs at pH 7.4, with significant inhibition at pH 4. SP-D binding to gpA is also competitively inhibited by maltose>glucose>mannose>N-acetyl-glucosamine. Comparison of the binding of various natural and recombinant forms of SP-D to gpA reveals that the number of carbohydrate recognition domains (CRDs) in a given SP-D form determines the relative extent of binding to gpA. Maximal binding is observed with natural SP-D (dodecamers and higher order SP-D complexes) followed by recombinant dodecamers. In contrast, recombinant full-length trimers exhibit substantially less binding, which is similar to that observed with a recombinant truncated molecule consisting of the CRD and neck regions, and containing trimers of this portion of the molecule. Taken together, these findings strongly indicate that the CRD of SP-D mediates interaction with P. carinii gpA through its attached oligosaccharides and that the extent of SP-D binding to P. carinii is greatest with dodecamers and higher order forms of SP-D.

Mechanisms of defence in the lung: lessons from Pneumocystis carinii pneumonia.

Pneumocystis carinii continues to represent an important complication of individuals with compromised immunity. P. carinii interacts with immune and non-immune cells in the lung and mediates lung injury through a variety of mechanisms. CD4+ T lymphocytes are the cornerstone in defence against P. carinii. Recent studies indicate that alveolar macrophages provide essential functions that significantly enhance clearance of P. carinii infection. P. carinii also attaches to alveolar epithelial cells, causing inhibition of epithelial growth and replication. In addition to cellular interactions, P. carinii organisms bind to a variety of host adhesive proteins present in the lower respiratory tract. Binding of these proteins to P. carinii modulates host cell recognition and immune responses to the parasite. During the course of P. carinii pneumonia, several inflammatory mediators are produced in the lung. Although necessary for control of infection, exuberant inflammatory responses also predispose the host to the development of acute lung injury. Thus, host defences against P. carinii depend on complex interactions between immune and non-immune cells as well as several mediators that facilitate host recognition and eventual elimination of infection. Understanding these complex processes may enable development of novel therapeutic approaches for management of this important infection.

Calcium influx is required for tannin-mediated arachidonic acid release from alveolar macrophages.

The role of Ca2+ was investigated in the response of alveolar macrophages to cotton tannin, an agent implicated in the lung disease byssinosis in textile mill workers. A physiological concentration of extracellular Ca2+ was found to be required for tannin-mediated release of radiolabeled arachidonic acid (AA). Flow cytometry using indo 1 indicated that tannin caused a rapid and dose-dependent Ca2+ increase in macrophages that also required extracellular Ca2+. Ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid virtually abolished the Ca2+ influx mediated by tannin but had little effect on intracellular Ca2+ release induced by thapsigargin, N-formylmethionyl-leucylphenylalanine, or thimerosal. A mechanism for extracellular Ca2+ influx was demonstrated by rapid Mn2+ quenching of indo 1 by tannin. Verapamil inhibited tannin-mediated Ca2+ influx and AA release, but the effective concentration was 100 microM. 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid chelated all Ca2+ in the cells and effectively abolished the tannin response. Exposure to tannin was not associated with cytotoxicity, as judged by 51Cr release. The data suggest that tannin induces Ca2+ influx in alveolar macrophages, which represents an important prerequisite for a cell-signaling pathway resulting in the accumulation of free AA.

Condensed tannin promotes the release of arachidonic acid from rabbit resident alveolar macrophages.

The condensed tannin present in cotton mill dust profoundly alters the functional capabilities of resident alveolar macrophages. Previous studies from this laboratory have shown that in vitro exposure of rabbit resident alveolar macrophages to condensed tannin significantly inhibits the ability of these cells to produce reactive oxygen intermediates or to ingest particles. In the present study, we demonstrate that condensed tannin also alters arachidonic acid (C20:4) metabolism in these cells. Exposure of rabbit resident alveolar macrophages to condensed tannin results in the time- and dose-dependent release of C20:4 from the membrane phospholipids. The release of C20:4 occurred only at tannin concentrations greater than 25 micrograms/ml and was maximal 90 min after the onset of exposure. The EC50 for release was 75 micrograms/ml. Exposure to 100 micrograms/ml tannin resulted in the release of 20 +/- 3% of the [14C]C20:4 incorporated in the cell membrane. In comparison, exposure to 160 micrograms/ml zymosan resulted in the release of 14 +/- 4% of the [14C]C20:4. For both tannin and zymosan, phosphatidylcholine and phosphatidylinositol were the principal sources of the released C20:4. Approximately 63% of the C20:4 released after zymosan stimulation was further metabolized, mainly via the cyclooxygenase pathway. The major metabolites were 6-keto-prostaglandin F1 alpha, prostaglandin F2 alpha, and prostaglandin E2. In contrast, only 24% of the C20:4 released by tannin was subsequently further metabolized. The metabolites formed were essentially evenly distributed between products of the cyclooxygenase pathway and the lipoxygenase pathway. Exposure of alveolar macrophages to 50 micrograms/ml tannin for 30 min reduced the ability of the cells to subsequently incorporate C20:4 by 50 to 70%. In contrast, exposure of the cells to 160 mg/ml zymosan for 30 min had only a minimal effect on the subsequent ability of these cells to incorporate C20:4. These results indicate that tannin promotes C20:4 release, at least in part, by inhibiting its reacylation back into phospholipids, a mechanism that differs from that of zymosan.

Monocyte heterogeneity in angiotensin-converting enzyme induction mediated by autologous T lymphocytes.

The ability of monocyte subpopulations to be induced selectively by T lymphocytes to synthesize enhanced levels of angiotensin-converting enzyme (ACE) was examined using an in vitro model employing normal peripheral blood monocytes and T lymphocytes. Separation of monocytes into subpopulations on the basis of buoyant density indicated no difference in the ability of the resulting monocyte subpopulations to produce basal levels of ACE when cultured in the absence of T lymphocytes. However, the subpopulations differed significantly in their ability to synthesize enhanced levels of ACE in response to the presence of autologous T lymphocytes; low-density monocytes were induced by T lymphocytes to synthesize three-fold more ACE than were high-density monocytes. Surface antigen labelling using MoAbs demonstrated that the low-density monocyte subpopulations also had a significantly higher percentage of Leu-M2+ monocytes compared with the high-density monocyte subpopulations. When monocytes were separated on the basis of the presence of the Leu-M2 antigen using an immune rosetting technique, T lymphocytes were able to induce significantly elevated levels of ACE in the Leu-M2+ enriched monocyte subpopulation but were unable to induce ACE beyond basal levels in the Leu-M2(+)-depleted monocyte subpopulation. These results demonstrate that monocytes are heterogeneous with respect to their ability to be induced by T lymphocytes to synthesize ACE. This raises the possibility that selective accumulation of a monocyte subpopulation in the granulomatous inflammation of sarcoidosis may be one of the factors required for elevated ACE synthesis in the resulting granuloma epithelioid cells.

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 EC50's 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.

Modulation of inflammatory cell function by cotton bract tannin: changes in the capacity of alveolar macrophages and neutrophils to produce hydrogen peroxide.

Inhalation of cotton mill dust leads to the development of the occupational lung disease byssinosis in a portion of the exposed workers. Condensed tannins present in the dust have biologic activities consistent with the hypothesis that they are one of the etiologic agents for the disease. Inhalation of either cotton dust or tannin provokes an acute inflammatory response characterized by the influx of neutrophils into the airways. The secretion of a low-molecular-weight, lipid neutrophil chemotactic factor from the alveolar macrophages in response to tannin stimulation appears to be important in this inflammatory process. In these studies, the effect of tannin the ability of alveolar macrophages and neutrophils to produce hydrogen peroxide was examined. Low concentrations of tannin itself induced a modest production of hydrogen peroxide from conditioned rabbit alveolar macrophages, while higher concentrations failed to induce peroxide production. In the presence of an independent stimulator of peroxide production (concanavalin A), tannin inhibited peroxide production at all concentrations examined. Aqueous extracts of cotton mill dust (CDE) had an identical effect on peroxide production in a manner that indicated that the tannin present in the dust was responsible for the effect. Like its direct effect on macrophage peroxide production, tannin induced modest peroxide production in human neutrophils. However, unlike its effect on macrophages, tannin enhanced the peroxide production induced by the presence of an independent stimulator (phorbol myristate acetate). CDE had a similar effect on peroxide production, but the dose-response curves suggested that only the high-molecular-weight polymers of tannin present in the CDE were able to enhance peroxide production.(ABSTRACT TRUNCATED AT 250 WORDS)

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)

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 biphasic dose-dependent manner with maximum effect at 10(-8) M concentration. Although it enhances the level of ACE activity, dexamethasone does 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: gamma-glutamyltransferase, alkaline phosphodiesterase-I, and leucine aminopeptidase, 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.

An in vitro model for the induction of angiotensin-converting enzyme in sarcoidosis: possible parallels to the immune response.

The T lymphocyte-dependent induction of angiotensin-converting enzyme (ACE) in cultured normal peripheral blood monocytes is a useful experimental model of the in vivo induction of ACE in monocyte-derived sarcoid granuloma epithelioid cells. Because of the possible parallels between ACE induction and immune activation in this model system, the ability of T lymphocytes to induce ACE was compared under a variety of conditions to their immune activation, as measured by 3H-thymidine incorporation. The ability of T cells to induce monocyte ACE was compared in the presence or absence of proliferation. The on-going autologous mixed lymphocyte reaction did not stimulate a further increase in ACE activity compared to the cultures where no proliferation occurred, showing that ACE induction does not depend on full immune T cell activation. Recognition of self though is crucial since in allogeneic monocyte/T lymphocyte co-cultures ACE was not induced above the levels found in monocytes alone. It was also shown that the immune cooperation between monocytes and T cells in reaction against monocyte-presented soluble antigen prevented T lymphocyte stimulated monocyte ACE induction, suggesting the possibility that self class II antigen recognition rather than foreign antigen recognition may be involved in the pathogenesis of sarcoidosis. Reaction to T mitogens (PHA, ConA), known to stimulate the production of interleukins, decreased monocyte ACE synthesis, indicating that soluble ACE-inducing factor probably does not belong to the group of immune interleukins.

Induction of angiotensin converting enzyme in cultured human monocytes by a factor present in the bronchoalveolar lavage fluid of sarcoidosis patients.

Human peripheral blood monocytes synthesize a low level of angiotensin converting enzyme (ACE) when cultured in vitro for six days. If cell-free bronchoalveolar lavage fluid from sarcoidosis patients was included with the monocytes during culture, the monocyte ACE level increased significantly in a dose dependent manner. In contrast, the cell-free bronchoalveolar lavage fluid from control subjects and patients with idiopathic pulmonary fibrosis or hypersensitivity pneumonitis raised basal monocyte levels little, if at all. These results suggested that sarcoid lavage fluid contains a soluble ACE-inducing factor (AIF) that was absent or present in much lower concentrations in the bronchoalveolar lavage fluid of the other patients studied. Initial attempts to characterize this AIF indicated an apparent molecular weight of greater than 5000 daltons as determined by Amicon ultrafiltration. The AIF was stable to heating at 56 degrees for 1 hour. This finding demonstrated that lavage ACE was not responsible for the AIF activity since lavage ACE is inactivated under these conditions. Gamma-interferon (macrophage-activating factor) was also eliminated as the agent responsible for AIF activity since gamma-interferon decreased rather than increased monocyte ACE. In summary, bronchoalveolar lavage fluid from sarcoid patients contains a soluble factor (AIF) that induces ACE in cultured monocytes. Action of this factor in vivo may be responsible for the increased ACE levels seen in the monocyte-derived epithelioid granuloma cells of sarcoidosis.

Cotton bract tannin: a novel human T-lymphocyte mitogen and a possible causative agent of byssinosis.

Cotton bract tannin, a major organic component of cotton dust, was tested for mitogenic activity upon human T lymphocytes. Tannin caused polyclonal activation of human T lymphocytes in vitro. The pattern of T-cell response to tannin was similar to the pattern seen in lymphocytes stimulated with the well-known plant lectin T-cell mitogens, PHA and ConA. The response to tannin was shown to be dependent upon tannin dose and presence of monocytes. The maximum response occurred after 3-4 days in culture with the magnitude comparable to the one achieved by ConA. Lipopolysaccharide from Enterobacter agglomerans, a major contaminant of cotton dust, was shown not to be a mediator of tannin mitogenicity in vitro. A role for tannin as a polyclonal cell activator and, therefore, a possible etiologic agent of byssinosis is suggested by these observations.

Modulation of in vitro growth of murine myeloid leukemia by an autologous substance immunochemically cross-reactive with insulin and antiinsulin serum.

Murine myeloid leukemia secretes a substance immunochemically cross-reactive with insulin (SICRI) both in vivo and in serum-free media. High SICRI concentrations in peripheral blood of tumorous animals do not affect circulating glucose levels. In culture, DNA synthesis rate per leukemic cell is proportional to cell density and is reduced by antiinsulin serum. Culture medium conditioned by leukemia cells as well as SICRI affinity purified from this medium stimulate DNA synthesis in cultured leukemia cells. It appears that autocrine stimulation of murine myeloid leukemia can be mediated in part by an insulin-related growth factor.