Lipopolysaccharide binding protein potentiates airway reactivity in a murine model of allergic asthma.

The development of allergic asthma is influenced by both genetic and environmental factors. Epidemiologic data often show no clear relationship between the levels of allergen and clinical symptoms. Recent data suggest that bacterial LPS may be a risk factor related to asthma severity. Airborne LPS is typically present at levels that are insufficient to activate alveolar macrophages in the absence of the accessory molecule LPS binding protein (LBP). LBP levels are markedly elevated in bronchoalveolar lavage fluids obtained from asthmatic subjects compared with those in normal controls. We hypothesized that LBP present in the lung could augment the pulmonary inflammation and airway reactivity associated with allergic asthma by sensitizing alveolar macrophages to LPS or other bacterial products and triggering them to release proinflammatory mediators. We compared wild-type (WT) and LBP-deficient mice using a defined Ag immunization and aerosol challenge model of allergic asthma. Immunized LBP-deficient mice did not develop substantial Ag-induced airway reactivity, whereas WT mice developed marked bronchoconstriction following aerosol Ag sensitization and challenge with methacholine. Similarly, production of NO synthase 2 protein and the NO catabolite peroxynitrite was dramatically higher in the lungs of WT mice following challenge compared with that in LBP-deficient mice. Thus, NO production appears to correlate with airway reactivity. In contrast, both mice developed similar pulmonary inflammatory cell infiltrates and elevated mucin production. Thus, LBP appears to participate in the development of Ag-induced airway reactivity and peroxynitrite production, but does not seem to be required for the development of pulmonary inflammation.

Identification of a sea urchin Na(+)/K(+)/2Cl(-) cotransporter (NKCC): microfilament-dependent surface expression is mediated by hypotonic shock and cyclic AMP.

We report the identification of an invertebrate Na(+)/K(+)/2Cl(-) cotransporter, NKCC. As a model system, we used the immune cells (coelomocytes) of the Mediterranean sea urchin Paracentrotus lividus. These cells are particularly interesting because they can be activated to undergo a rapid and dynamic change in cell shape. We demonstrate that forskolin, a cyclic AMP agonist known to regulate NKCC, induced coelomocyte transformation at doses of 10 micromol l(-)(1) and greater. Using two distinct monoclonal antibodies (T4 and T9) raised against the human intestinal epithelial NKCC, we have identified a high-molecular-mass (195 kDa) protein in coelomocyte extracts. We propose a novel method for the isolation of NKCC in one step by using bumetanide-Sepharose affinity chromatography under low-[Cl(-)] conditions. This method was successful in isolating coelomocyte 195 kDa NKCC. The T4 monoclonal antibody was used in immunocytochemical experiments to localize NKCC in resting and activated coelomocytes. In petalloid coelomocytes, a punctate, cytoplasmic distribution was observed in close proximity to actin filament bundles; in transformed coelomocytes, the immunofluorescence was distributed along the length of the filopodia and uniformly throughout the perinuclear region. The change in subcellular distribution of NKCC between the resting and the activated state was further investigated by using cell surface biotinylation followed by immunoprecipitation. These studies revealed an upregulation of NKCC at the plasma membrane upon activation, a process that was blocked by the F-actin-stabilizing drug phalloidin. These studies identify a novel model system in which to investigate a newly identified invertebrate Na(+)/K(+)/2Cl(-) cotransporter.

Polarity of A2b adenosine receptor expression determines characteristics of receptor desensitization.

UNLABELLED: It is not known if, in polarized cells, desensitization events can be influenced by the domain on which the receptor resides. Desensitization was induced by 5'-(N-ethylcarboxamido)adenosine (NECA) and was quantitated by measurement of short-circuit current (I(sc)) in response to adenosine. NECA added to either the apical or basolateral compartments rapidly desensitized receptors on these respective domains. Although apical NECA had no effect on the basolateral receptor stimulation, basolateral NECA induced a complete desensitization of the apical receptor. We hypothesized that desensitization of apical receptor by basolateral desensitization could relate to a trafficking step in which A2b receptor is first targeted basolaterally upon synthesis and transported to the apical surface via vesicular transport/microtubules. Because desensitization is associated with downregulation of receptors, apical adenosine receptor can thus be affected by basolateral desensitization. Both low temperature and nocodazole inhibited I(sc) induced by apical and not basolateral adenosine. IN CONCLUSION: 1) a single receptor subtype, here modeled by the A2b receptor, differentially desensitizes based on the membrane domain on which it is expressed, 2) agonist exposure on one domain can result in desensitization of receptors on the opposite domain, 3) cross-domain desensitization can display strict polarity, and 4) receptor trafficking may play a role in the cross-desensitization process.

Roles of lipoarabinomannan in the pathogenesis of tuberculosis.

Tuberculosis is a worldwide public health threat caused by Mycobacterium tuberculosis. All mycobacteria express a unique cell envelope glycolipid, lipoarabinomannan, which can be released at sites of infection. Lipoarabinomannan is a potential virulence factor which can bind to leukocytes and modulate immune responses. Here, we provide an overview of the interactions of mycobacteria and lipoarabinomannan with immune cells.

Distinct Ca2+- and cAMP-dependent anion conductances in the apical membrane of polarized T84 cells.

Monolayers of the human colonic epithelial cell line T84 exhibit electrogenic Cl- secretion in response to the Ca2+ agonist thapsigargin and to the cAMP agonist forskolin. To evaluate directly the regulation of apical Cl- conductance by these two agonists, we have utilized amphotericin B to permeabilize selectively the basolateral membranes of T84 cell monolayers. We find that apical anion conductance is stimulated by both forskolin and thapsigargin but that these conductances are differentially sensitive to the anion channel blocker DIDS. DIDS inhibits thapsigargin-stimulated responses completely but forskolin responses only partially. Furthermore, the apical membrane anion conductances elicited by these two agonists differ in anion selectivity (for thapsigargin, I- > Cl-; for forskolin, Cl- > I-). However, the DIDS-sensitive component of the forskolin-induced conductance response exhibits anion selectivity similar to that induced by thapsigargin (I- > Cl-). Thus forskolin-induced apical anion conductance comprises at least two components, one of which has features in common with that elicited by thapsigargin.

Induction of epithelial chloride secretion by channel-forming cryptdins 2 and 3.

Salt and water secretion from intestinal epithelia requires enhancement of anion permeability across the apical membrane of Cl- secreting cells lining the crypt, the secretory gland of the intestine. Paneth cells located at the base of the small intestinal crypt release enteric defensins (cryptdins) apically into the lumen. Because cryptdins are homologs of molecules known to form anion conductive pores in phospholipid bilayers, we tested whether these endogenous antimicrobial peptides could act as soluble inducers of channel-like activity when applied to apical membranes of intestinal Cl- secreting epithelial cells in culture. Of the six peptides tested, cryptdins 2 and 3 stimulated Cl- secretion from polarized monolayers of human intestinal T84 cells. The response was reversible and dose dependent. In contrast, cryptdins 1, 4, 5, and 6 lacked this activity, demonstrating that Paneth cell defensins with very similar primary structures may exhibit a high degree of specificity in their capacity to elicit Cl- secretion. The secretory response was not inhibited by pretreatment with 8-phenyltheophyline (1 microM), or dependent on a concomitant rise in intracellular cAMP or cGMP, indicating that the apically located adenosine and guanylin receptors were not involved. On the other hand, cryptdin 3 elicited a secretory response that correlated with the establishment of an apically located anion conductive channel permeable to carboxyfluorescein. Thus cryptdins 2 and 3 can selectively permeabilize the apical cell membrane of epithelial cells in culture to elicit a physiologic Cl- secretory response. These data define the capability of cryptdins 2 and 3 to function as novel intestinal secretagogues, and suggest a previously undescribed mechanism of paracrine signaling that in vivo may involve the reversible formation of ion conductive channels by peptides released into the crypt microenvironment.

Surface expression, polarization, and functional significance of CD73 in human intestinal epithelia.

During active intestinal inflammation polymorphonuclear leukocytes (PMN) transmigrate into the lumen and release 5'-AMP (J. Clin. Invest. 1993. 91:2320-2325). 5'-AMP is converted to adenosine by the apical epithelial surface with subsequent activation of electrogenic Cl- secretion (the basis of secretory diarrhea) via apical A2b adenosine receptors (J. Biol. Chem. 1995. 270:2387-2394). Using a polarized human intestinal epithelial monolayer (T84), we now characterize the basis of the observed conversion of 5'-AMP to adenosine required for this paracrine signaling pathway. An inhibitor of the ecto-5'-nucleotidase CD73, alpha, beta-methylene ADP (AOPCP), inhibited epithelial Cl- secretory responses to 5'-AMP, but not to authentic adenosine. Confocal immunofluorescent microscopy revealed CD73 to be surface expressed on both model and natural human intestinal epithelia. Expression was about sixfold greater on the apical cell surface as assessed biochemically by selective cell surface biotinylation, and morphologically by immunofluorescence. Treatment with phosphotidylinositol specific-phospholipase C (PI-PLC) released 95% of apical CD73, indicating that the intestinal CD73 possesses a glycosylphosphatidylinositol (GPI) anchor. Neither adenosine nor 5'-AMP stimulation induced intact T84 cells to shed surface CD73. The bulk of apical CD73 ( approximately 60%) was released from the cell surface by treatment with 1% Triton X-100 (TX-100) at 4 degrees C, but such release was not affected by pretreatment with ligand or by prior, antibody-mediated cross-linking of CD73. Subsequent analyses showed that the subpool of CD73 released by TX-100 at 4 degrees C was not truly solubilized, but rather represented TX-100-induced release of CD73-containing membrane fragments. These membrane fragments displayed light density on sucrose gradients characteristic of detergent insoluble glycosphingolipid-rich membrane domains (DIGs)/ caveolae, were solubilized by n-octyl glucoside (NOG, 1%) at 4 degrees C, and contained caveolin. These data indicate that human intestinal epithelia express CD73, which is apically polarized and targeted to microdomains with DIGs/caveolae characteristics. CD73 likely participates in translating paracrine, PMN-derived 5'-AMP signals to the authentic effector adenosine. These studies define CD73 as central to PMN-mediated intestinal Cl- secretion, the major directacting mechanism by which PMN induce intestinal epithelial Cl- secretion.

Neutrophil functional responses depend on immune complex valency.

Ligand-induced cross-linking of Fc gamma receptors (Fc gamma R) on neutrophils plays a significant role in their stimulation, shown here by contrasting the responses induced by low valency immune complexes (LICs) and high valency immune complexes (HICs) and by cross-linking LICs in situ (L/Ab) after their addition to the cells. Multiparameter flow cytometry was used to measure immune complex (IC)-elicited changes in cytoplasmic Ca2+ concentration and initiation of the oxidative burst simultaneously in the same cell and to correlate these with Fc gamma R occupancy. We have previously shown that subpopulations of neutrophils respond maximally to subsaturating concentrations of HIC; saturating dosages stimulate the entire population. This discrepancy was not due to differences in receptor occupancy. The magnitude of the transient Ca2+ increase was independent of the dose of HIC but depended on the dose when an LIC was used. As shown here, L/Ab cross-linking elicited Ca2+ responses similar to those observed in HIC-stimulated cells. In contrast, LIC elicited only minimal intracellular delta pH and no oxidative burst or membrane potential changes at all unless Fc gamma R was cross-linked, accomplished by HIC or by L/Ab. However, azurophilic degranulation, as determined by elastase release, was not observed in cells stimulated by the in situ cross-linking method, whereas the HIC preparation triggered azurophilic degranulation. Thus, some Fc gamma R-mediated neutrophil effector functions such as azurophilic degranulation and oxidative burst initiation have an absolute requirement for Fc gamma R cross-linking, whereas signaling functions such as changes in membrane potential, intracellular pH, and intracellular Ca2+ concentration can occur, albeit more slowly and to a lesser extent, if single Fc gamma R are occupied.

Role of the Fc gamma R subclasses Fc gamma RII and Fc gamma RIII in the activation of human neutrophils by low and high valency immune complexes.

Two Fc gamma receptor (Fc gamma R) subclasses on human neutrophils, Fc gamma RII and Fc gamma RIII, activate different cellular functions. To examine the involvement of each receptor subtype in polymorphonuclear leukocyte activation, Fab and F(ab')2 fragments of subclass-specific monoclonal antibodies ([mAbs] mAb IV.3 against Fc gamma RII and mAb 3G8 against Fc gamma RIII, respectively) were used to block the binding of low valency immune complexes (LICs) and high valency immune complexes (HICs). Flow cytometry then permitted the simultaneous quantitation of antibody and ligand binding, the elicited intracellular Ca2+ concentration (delta[Ca2+]int), initiation of the oxidative burst, and/or the phospholipase A activation in the same cell. We have previously demonstrated that subsaturating dosages of HIC bind uniformly to all the cells but elicit an "all-or-none" (i.e., dose independent) maximal delta[Ca2+]int in a dose-dependent subpopulation of the cells. In contrast, both the proportion of cells responding and the magnitude of the delta[Ca2+]int transient depend on the subsaturating dose of LIC, even though it too binds uniformly to all the cells, nonresponding as well as responding. These earlier findings have here been extended by single cell flow cytometric analysis to demonstrate that F(ab')2 Fc gamma RIII is the major Fc gamma R involved in HIC binding (and [Ca2+]int mobilization), as well as in oxidative burst and phospholipase A activation. In contrast, both receptor subclasses must be available for LIC-elicited delta[Ca2+]int, as blockage by either of the mAb Fab or F(ab')2 fragments abrogates this response, even though LIC binding to the receptors is not decreased. Furthermore, LIC elicited little oxidative burst activity and failed to activate phospholipase A but cross-linking to achieve multivalency, previously shown to induce [Ca2+]int and oxidative burst responses, elicited phospholipase A activity via Fc gamma RIII. Fc gamma RII's role appears to be modulation of the small, late Ca2+ influx observed at > 1 min, whereas Fc gamma RIII modulates all the earlier larger events. Thus, simultaneous observation of receptor identity, receptor occupancy, and consequent activation parameters in the same cell by flow cytometry permits use to demonstrate that Fc gamma RII is necessary for the small signal transduction elicited by LIC; it plays a relatively small role in polymorphonuclear leukocyte stimulation by HIC. Fc gamma RIII is the main receptor responsible for immune complex-elicited polymorphonuclear leukocyte responses; its efficacy is greatly enhanced when the receptors are cross-linked, either by preequilibrated multivalent complexes or by in situ cross-linking of bound LIC with excess antibody.

The A2b adenosine receptor mediates cAMP responses to adenosine receptor agonists in human intestinal epithelia.

Adenosine is thought to be a major effector in immunological stimulation of Cl- secretion in intestinal epithelia. Previous studies indicate that both apical and basolateral domains of intestinal epithelial cells possess functionally defined adenosine receptors. However, it is unclear whether the same receptor subclass is expressed, what the receptor subclass(es) is, or how the receptors signal the Cl- secretory response. We now characterize the intestinal epithelial adenosine receptor subtype using the model epithelium, T84. Both apical and basolateral adenosine receptor agonist response profiles revealed a hierarchy (ED50) of 5'-(N-ethylcarboxamido)adenosine > adenosine > CGS-21680. Similarly inhibition studies revealed identical ID50 hierarchies for apical and basolateral antagonism by xanthine amine congener > 1,3-diethyl-8-phenylxanthine > aminophylline. Analyses of both agonist and antagonist pharmacological hierarchies in Chinese hamster ovary cells stably expressing the A2b receptor revealed these same hierarchies. Northern blots performed on RNA extracted from polarized T84 monolayers demonstrated no detectable message for A1 or A2a adenosine receptor, but strong hybridization was detected for the A2b adenosine receptor. Subsequent Northern blots of RNA prepared from human alimentary tract revealed that A2b adenosine receptor message was heavily expressed throughout the colon, in the appendix, and more modestly expressed in the small intestine (ileum). Analyses of cAMP generation in T84 cells in response to adenosine indicated that the basolateral A2b receptor elicits Cl- secretion through this signaling pathway. Stimulation of Cl- secretion through the apical A2b receptor exhibited relatively small but significant increases in cAMP compared with basolateral stimulation. The protein kinase A inhibitor H-89, used at concentrations that did not affect short circuit current responses to the Ca(2+)-mediated agonist carbachol, effectively inhibited short circuit current elicited by either apical or basolateral adenosine. These data suggest that the major intestinal epithelial adenosine receptor is the A2b subclass, which is positively coupled to adenylate cyclase. Such observations have potentially important implications for the treatment of diarrheal diseases.