Differential roles of Toll-like receptors in the elicitation of proinflammatory responses by macrophages.

BACKGROUND: Mammalian Toll-like receptor (TLR) proteins are pattern recognition receptors for a diverse array of bacterial and viral products. Gram negative bacterial lipopolysaccharide (LPS) activates cells through TLR4, whereas the mycobacterial cell wall glycolipids, lipoarabinomannan (LAM) and mannosylated phosphatidylinositol (PIM), activate cells through TLR2. Furthermore, short term culture filtrates of M. tuberculosis bacilli contain a TLR2 agonist activity, termed soluble tuberculosis factor (STF), that appears to be PIM. It was recently shown that stimulation of RAW264.7 murine macrophages by LPS, LAM, STF, and PIM rapidly activated NF-kappaB, AP1, and MAP kinases. RESULTS: This study shows that signalling by TLR2 and TLR4 also activates the protein kinase Akt, a downstream target of phosphatidylinositol-3'-kinase (PI-3-K). This finding suggests that activation of PI-3-K represents an additional signalling pathway induced by engagement of TLR2 and TLR4. Subsequently, the functional responses induced by the different TLR agonists were compared. LPS, the mycobacterial glycolipids, and the OspC lipoprotein (a TLR2 agonist) all induced macrophages to secrete tumour necrosis factor alpha (TNFalpha), whereas only LPS could induce nitric oxide (NO) secretion. Human alveolar macrophages also exhibited a distinct pattern of cellular response after stimulation with TLR2 and TLR4 agonists. Specifically, LPS induced TNFalpha, MIP-1beta, and RANTES production in these cells, whereas the TLR2 agonists induced only MIP-1beta production. CONCLUSION: Together, these data show that different TLR proteins mediate the activation of distinct cellular responses, despite their shared ability to activate NF-kappaB, AP1, MAP kinases, and PI-3-K.

Costimulation of CD28- T cells through CD3 and beta1-integrins induces a limited Th1 cytokine response.

The costimulatory molecule CD28 regulates antigen-specific T-cell proliferation and the synthesis of multiple cytokines. The absence of CD28 on a subset of CD8bright+ T cells suggests that these cells may utilize alternative costimulatory pathways or have a limited cytokine response to presented antigen. We used fibronectin, a ligand for the beta1-integrins alpha4beta1 and alpha5beta1, as an alternate costimulatory ligand to assess the functional phenotype of CD8bright+CD28- T cells. CD25 expression was significantly up-regulated in CD8bright+CD28- T cells by immobilized anti-CD3i with fibronectin. Costimulation with fibronectin also significantly augmented anti-CD3i-induced IFN-gamma production only among CD8bright+CD28- T cells. The CD8bright+CD28- T cells did not produce significant IL-2 and IL-10 even in response to maximal stimulation with phorbol myristate acetate and ionomycin. These data support a costimulatory role for ss1-integrins in CD8bright+CD28- T cells and indicate that CD8bright+ CD28- T cells have a restricted Th1 cytokine repertoire.

Identification of IL-16 as the lymphocyte chemotactic activity in the bronchoalveolar lavage fluid of histamine-challenged asthmatic patients.

OBJECTIVE: We have previously demonstrated that the earliest lymphocyte chemotactic factors present in bronchoalveolar lavage fluid (BALF) of subjects with atopic asthma after subsegmental antigen challenge are IL-16 and MIP-1alpha, of which IL-16 appears to contribute a majority of the chemotactic activity. Because IL-16 is released in vitro after histamine stimulation of CD8+ T cells and epithelial cells, we evaluated the potential role of histamine in the release of IL-16 into the airways of allergic asthmatics in vivo. METHODS: Eight allergic asthmatic subjects, six normal subjects, and six atopic nonasthmatic subjects were challenged with saline in the lingula and with serial concentrations of histamine (1 x 10(-7) to 5 x 10(-5) mol/L) in the right middle lobe followed by bronchoalveolar lavage (BAL) 15 minutes and 6 hours later. RESULTS: The BALF from saline- and histamine-challenged lobes of normal subjects and atopic nonasthmatic subjects contained no significant lymphocyte chemoattractant activity. In six of the eight atopic asthmatic subjects, the histamine-challenged but not saline-challenged segment contained IL-16 chemotactic activity but no other identifiable lymphocyte chemoattractant activities at 6 hours. CONCLUSIONS: IL-16 appears in the airways after histamine challenge and therefore could contribute to the earliest infiltration of CD4+ T cells and eosinophils observed after antigen challenge due to histamine release from mast cells.

In vitro transendothelial migration of blood T lymphocytes from HIV-infected individuals.

OBJECTIVE: We hypothesized that differential extravasation of circulating CD4+ or CD8+ T lymphocytes contributes to HIV-associated CD8+ lymphocytic alveolitis. Differences in T-cell transendothelial migration may be intrinsic or emerge at sites where vascular endothelium is activated by overexpression of tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma. DESIGN: We used an in vitro model of lymphocyte extravasation to assess transendothelial migration of peripheral blood mononuclear cells (PBMC) from HIV-positive individuals. We assayed bronchoalveolar lavage (BAL) fluid from HIV-positive and normal individuals to determine if increased levels of TNF-alpha and IFN-gamma were present in the lungs of HIV-infected individuals. METHODS: Transendothelial migration was assessed by determining the number and flow cytometric phenotype of PBMC adherent to or migrating across unstimulated or TNF-alpha and IFN-gamma-activated endothelial cell monolayers. We measured BAL fluid cytokine concentrations using standard antigen-capture enzyme-linked immunosorbent assays for TNF-alpha and IFN-gamma. RESULTS: T cells migrating across unactivated endothelial cells were significantly enriched for CD4+ T cells. Cytokine activation of endothelial cells allowed significantly greater transendothelial migration of CD8+ T cells compared to unactivated endothelial cells. TNF-alpha was increased in BAL fluid from HIV-positive individuals relative to controls. CONCLUSIONS: These data suggest that, in HIV-positive individuals, CD4+ T cells are migration competent and blood CD8+ T cells do not have enhanced migration competence relative to CD4+ T cells. CD8+ T cell extravasation is aided by TNF-alpha and IFN-gamma-induced endothelial cells activation.

Erosion of the right mainstem bronchus by an esophageal stent.

Self-expanding metallic stents (SEMSs) are used to palliate malignant esophageal strictures. We describe a patient who had an extensive mediastinal tumor for which he was receiving irradiation therapy; chest pain, hemoptysis, and recurrent Gram-negative pneumonia developed in this patient after stent placement. Fiberoptic bronchoscopy revealed protrusion of the SEMS into the tracheobronchial tree, a novel complication for this new type of stent.

Migration of distinct subsets of CD8+ blood T cells through endothelial cell monolayers in vitro.

The immune response in many infections and to allografts is dependent on CD8+ cytotoxic T lymphocytes (CTL). Influx of CD8+ CTL from the blood has been documented during antigen challenge. We have previously found that a subset of CD8+ T cells from normal blood can migrate through endothelial cell monolayers in vitro. To further characterize migration-prone CD8+ T cells from normal blood, we examined the expression of CD28 and a restricted epitope of CD18/CD11a (S6F1), a CTL marker. Although normal blood CD8bright+ T cells were heterogeneous in their expression of CD28, three populations could be identified (CD28low, CD28moderate, and CD28high). CD8+ cells migrating across endothelial cell monolayers were enriched for CD8bright+ CD28high cells and a subset of CD8dim+ cells, which were CD28high. Both adherent and migrating CD8+ cells were exclusively (> 95%) S6F1high. There was also preferential adhesion and migration of CD8+ cells expressing the low-molecular-weight form of the leukocyte common antigen, CD45RO. Cytokine activation of the endothelium did not significantly alter preferential migration of these subsets. These data suggest that certain subsets of CD8+ memory T cells in normal human blood are prone to, adhere to, and migrate through allogeneic endothelial cells and would thus be likely to be recruited to sites of antigen challenge.

Expansion of a CD8+CD28- cell population in the blood and lung of HIV-positive patients.

CD8+ cytotoxic T lymphocytes (CTLs) in human immunodeficiency virus (HIV) patients have defects in cytolytic ability and proliferative potential. The surface receptor CD28 is important in regulating antigen-specific responses to T cells. We hypothesized that activated CD8+ CTLs in HIV patients would have altered expression of CD28. We examined surface expression of D44, a CD8+ CTL marker, and of CD28 on T cells from blood and bronchoalveolar lavage from HIV+ patients and normal volunteers. We found no significant difference between normal volunteers and HIV+ individuals in percentage of CD8+D44+ CTLs in blood or the lung. In contrast, CD8+CD28- T cells in the blood of HIV patients constituted 74% of CD8+ cells compared to 25% in normal subjects (p = 0.001), findings exaggerated in both normal and HIV+ lung. CD4+CD28- blood T cells were significantly increased in HIV+ patients compared to normal subjects (24 vs. 1.5%, p = 0.004). The HIV infection itself did not directly downmodulate CD28 expression, demonstrated in the CD28+ SUPT1 cell line. Increased numbers of CD28- T cells may be the result of immunologic activation or of expansion of a preexisting CD28- subset. These findings have immunologic consequences for the antigen-specific response of T cells in HIV+ patients.

Membrane permeability changes associated with DNA gyrase inhibitors in Escherichia coli.

Inhibition of DNA synthesis in Escherichia coli B/r by a DNA gyrase inhibitor results in cell death after a 50-min lag period. Examination of the cells under phase-contrast and electron microscopes revealed that they appeared to undergo plasmolysis coincident with the onset of cell death. The inhibited cells were also found to become susceptible to low levels of detergent at this time. With a fluorescent membrane probe, the level of membrane permeability was assessed and found to increase concurrently with the decrease in culture viability. Analysis of the cell envelope constituents revealed that, other than a shift in the protein/lipid ratio, the compositions of the cell membranes were unperturbed.

Characterization of polysaccharide accumulation in a cell division defective mutant of Escherichia coli 15T-.

Escherichia coli 15T-R1, a temperature-dependent cell division mutant, grows into filaments of various lengths (200 to 500 microgram) at 24 degrees C, but divides essentially normally at 37 degrees C. When grown to late-exponential phase at the restrictive temperature, the elongated cells showed discrete areas of increased density at polar regions and other sites in the cytoplasm, when viewed by phase contrast microscopy. Electron microscopy of preparations specifically stained for polysaccharide revealed clusters of granules with a similar distribution pattern to that of the dense areas seen by phase contrast microscopy. The granules were susceptible to alpha-amylase digestion, and chemical analysis of the extracted and purified polysaccharide showed that it consisted of polyglucose, including glycogen. At 24 degrees C the R1 cells contained about twice as much polyglucose and four times as much glucogen as at 37 degrees C.

Polysaccharide accumulation in the cell division defective mutant, Escherichia coli 15T-R1.

The low temperature dependent cell division defective mutant, Escherichia coli 15T-R1, displays a wide range of cell lengths (2 to 500 micrometer) in late logarithmic phase cultures grown at 24 degrees C. When these cells were stained with a periodic acid-Schiff technique and viewed under light microscopy, polysaccharide accumulations appeared as discretely stained areas at the poles, and at sites throughout the cytoplasm of elongated cells. A statistical analysis of the relationships between the size of stained areas and cell size, indicated that the total amount of polysaccharide increased with increasing cell length. This occurred both by an increase in size of existing polysaccharide-stained areas and by the creation of new area. Interestingly, over a wide range of cell size, the ratio of cell volume occupied by polysaccharide to total cell volume remained nearly constant with a mean of about 0.16. These data suggest the existence of a homeostatic mechanism for regulating polysaccharide concentration during elongation.

Importance of aliphatic amines in uremia.

Five main aspects were addressed: 1)The demonstration that creatinine is an endogenous precursor of dimethylamine (DMA) in chronic renal failure. 2) The size of the body amine pool measured in transplant patients suggests sequestration in some intracellular compartment. This illustrates the possible error in directly relating serum concentrations to neurological toxicity. 3) Bacterial overgrowth and increased generation of duodenal DMA in the small intestine becomes apparent at a serum creatinine above 8 mg/dl. Two cases show that bacterial overgrowth preceded the increased duodenal DMA. 4)Clinical toxicity is demonstrated by i) correlation of abnormal neurobehavioral parameters with serum amine levels, and ii) by improvement with administration of nonabsorbable broad spectrum antibiotics. Results with adsorption agents are inconclusive. 5) Preliminary tests of behavior modification in a rat model by direct instillation of amines into the brain are positive for TMA but negative for DMA, but no DMA entry into brain cells is demonstrated in the latter. The generation of aliphatic amines represents only one part of a spectrum of alteration induced by proximal intestinal bacterial enzyme action that occurs in renal failure. It is possible that some bacterial activity is beneficial and that the net clinical result is a balance between the "good" and the "evil" bacterial effects.

Bacterial populations of the small intestine in uremia.

The small intestinal bacterial flora of 15 patients with chronic renal insufficiency was compared with that of subjects with blind loop synDROME. 9 patients were on regular hemodialysis with high protein intake and 6 (serum creatinine 7.5 to 12.5 mg/dl) were maintained on low protein diet. The chronic renal patients harbored a greatly increased microbial flora of both anaerobes and aerobes in the duodenum and jejunum, quantitatively comparable to those in blind loop subjects. The composition did not differ significantly in the two groups. Some organisms may have the potential to metabolize substrates which reach the intestinal lumen from the diet and bile, and perhaps to generate toxic metabolites that could contribute to uremic toxicity or malabsorption.

Biochemical profile of uremic breath.

We attempted to define the substances that contribute to the characteristic "uremic breath" of patients with end-stage renal disease. Breath samples from nine patients underwent direct analysis before and after hemodialysis with use of gas chromatography and confirmation by mass spectrometry, and indirectly assessment by an organoleptic panel. Concentrations of secondary and tertiary amines, dimethylamine and trimethylamine were increased, with subsequent reduction after hemodialysis (dimethylamine from 2.00 +/- 0.19 [S.E.M.] to 0.88 +/- 0.12 microng per 30 minutes, P less than 0.001, and trimethylamine from 0.79 +/- 0.22 to 0.44 +/- 0.15 microng per 30 minutes, P less than 0.003). Treatment with nonabsorbable antibiotics in two patients reduced both serum and breath amine levels without dialysis. Loss of nitrogen via the breath was not quantitatively important. We conclude that uremic breath reflects the systemic accumulation of potentially toxic volatile metabolites, among which dimethylamine and trimethylamine have been positively identified and correlated with the classic fishy odor.

Amine metabolism and the small bowel in uraemia.

Intestinal intubation was carried out in 21 subjects: 9 with end-stage renal failure, 2 with early renal insufficiency, 7 untreated patients with blind-loop syndrome, and 3 normal volunteers. All 9 patients with uraemia had significantly raised duodenal dimethylamine (D.M.A.) concentrations compared with the other groups tested. Alteration of the intestinal bacterial flora with antibiotics markedly reduced serum D.M.A. and trimethylamine concentrations and modified the symptoms. Potentially toxic metabolites in the small bowel might have significant nutritional and toxic sequelae in uraemia and these findings suggest that current therapeutic approaches to the treatment of end-stage kidney disease should be re-examined in relation to bowel flora.