Insect tolerance to the crystal toxins Cry1Ac and Cry2Ab is mediated by the binding of monomeric toxin to lipophorin glycolipids causing oligomerization and sequestration reactions.

Endotoxins from the soil bacterium Bacillus thuringiensis are used worldwide to control insect pests and vectors of diseases. Despite extensive use of the toxins as sprays and in transgenic crops, their mode of action is still not completely known. Here we show that two crystal toxins binding to different glycoprotein receptors have similar glycolipid binding properties. The glycolipid binding domain was identified in a recombinant peptide representing the domain II of the crystal toxin Cry1Ac (M-peptide). The recombinant M-peptide was isolated from bacterial lysates as a mixture of monomers and dimers and formed tetramers upon binding to glycolipid microvesicles from gut tissues and lipid particles from hemolymph plasma. Likewise, when mature toxins and M-peptides where mixed with plasma, these peptides bind to lipid particles and can be separated with lipophorin particles on low-density gradients. When mature toxin and M-peptides are added to lipid particles in increasing amounts, the peptide-particle complexes form higher aggregates that are similar to aggregates formed in low-density gradients in the presence of the toxin. This could indicate that glycolipids on lipid particles are possible targets for toxin monomers in the gut lumen, which upon binding to the glycolipids form tetramers and aggregate particles and thereby sequester the toxin inside the gut lumen before it can interact with receptors on the brush border membrane. The implication is that domain II interacting with glycolipids mediate tolerance to the toxin that is separate from interaction of the toxin with glycoprotein receptors causing toxicity.

Developmental penalties associated with inducible tolerance in Helicoverpa armigera to insecticidal toxins from Bacillus thuringiensis.

Exposure of insect larvae to sublethal concentrations of crystal toxins from the soil bacterium Bacillus thuringiensis (Bt toxins) causes the induction of immune and metabolic responses that can be transmitted to offspring by epigenetic inheritance mechanisms. Given that the elevated immune status carries significant developmental penalties, we wanted to establish the relationships between immune induction, tolerance to the toxin and developmental penalties. A laboratory culture of Helicoverpa armigera was induced by a sublethal bacterial suspension containing crystal toxin Cry1Ac in one generation and maintained in the presence of toxin, acquiring significant levels of tolerance to the toxin within 12 generations of continuous exposure. Comparing tolerant and susceptible insects, we show that the induction of larval immune response and the coincident alteration of development-related metabolic activities by elicitors in the larval gut (larval induction) differs from the elevated immune status transmitted by epigenetic mechanisms (embryonic induction). Because the damaging effects of larval induction processes are higher compared to embryonic induction, it is likely that overall developmental penalties depend on the relative contribution of the two induction processes. When insects are kept with the same amount of toxin in the diet for subsequent generations, the embryonic induction process increases its contribution compared to the larval induction, resulting in reduced overall developmental penalty, while tolerance to the toxin is maintained.

Responses of endothelial cells from different vessels to inflammatory cytokines and shear stress: evidence for the pliability of endothelial phenotype.

BACKGROUND/AIMS: Local haemodynamic and stromal microenvironments may determine the phenotype of endothelial cells (EC) and regulate their inflammatory responses. METHODS: We compared neutrophil recruitment by EC from human umbilical veins (HUVEC) or arteries (HUAEC) or from human coronary arteries (HCAEC) after 'static' culture or exposure to shear stress (2 Pa for 24 h) and treatment with tumour necrosis factor-alpha (TNF-alpha) or interleukin-1beta (IL-1beta). RESULTS: Static cultures of each type of EC recruited flowing neutrophils efficiently after treatment with TNF-alpha or IL-1beta; differences in culture media caused minor variations. After shear conditioning, the response of HUVEC to TNF-alpha (but not IL-1beta) was much reduced, while the responses of HUAEC and HCAEC to both cytokines were reduced. However, swapping the culture media suggested that the differences in the shear response arose largely from medium constituents, particularly basic fibroblast growth factor. When gene expression profiles for HUVEC were examined immediately after isolation, after 5 days in static culture and after re-exposure to shear, variations in gene expression were only partially attributable to the effects of changes in shear stress. CONCLUSIONS: The behaviour of cultured EC may depend as much on the physico-chemical culture conditions as on their origins. The EC phenotype appears to be highly pliable, with environmental factors, such as shear stress and growth factors, modifying responses in an inter-linked manner.

Arsenic interactions with lipid particles containing iron.

While arsenic is toxic to all multicellular organisms, some organisms become tolerant by an unknown mechanism. We have recently uncovered an inducible tolerance mechanism in insects, which is based on a sequestration of toxins and pathogens by lipid particles. To examine whether arsenic interacts with lipid particles from mammals we compared binding of arsenic to lipid particles from insect and pig plasma after separation of lipid particles by low-density gradient centrifugation. Arsenic was found in both organisms in an area of the gradient, which corresponds to lipid-rich lipid particles. Since iron is known to affect arsenic toxicity in some organisms, we asked whether iron may be present in lipid particles. When low density cell (LDC) gradient fractions were analysed for the presence of iron we detected a peak in very low-density fractions similar to those that carried arsenic. This could indicate that arsenic interacts with lipid particles that contain iron and, if arsenic is removed from the plasma by lipid particles, that would also reduce iron-containing lipid particles at the time of arsenic emergence in the plasma. To test this assumption we measured the iron content in plasma at various time periods after the toxin ingestion. This time course revealed that iron is depleted in plasma fractions when arsenic shows a peak. Our data suggest that arsenic interacts with invertebrate and vertebrate lipid particles that are associated with proteins that may lead to detoxification by cell-free or cellular sequestration mechanisms.

Prolonged culture of endothelial cells and deposition of basement membrane modify the recruitment of neutrophils.

We tested whether endothelial cell conditioning during prolonged culture and deposition of basement membrane (BM) could modify neutrophil recruitment induced by the inflammatory cytokine, tumour necrosis factor-alpha (TNF). Confluent endothelial cells (EC) from human umbilical veins were cultured for 1 to 20 days and then stimulated with 1, 10 or 100 U/ml of TNF for 4 h. When isolated neutrophils were settled on EC stimulated with the lower doses of TNF, the levels of adhesion and the proportion of adherent cells that transmigrated increased markedly with time of culture. At 100 U/ml TNF, time of culture had little effect on recruitment, but the transmigrated neutrophils moved more slowly under the monolayer in longer-term cultures. The inhibitory effects of function-blocking antibodies against E-selectin and beta2-integrin, and studies in which neutrophils were perfused over short- or long-term cultures, suggested that increased adhesion and migration arose from increased efficiency of neutrophil activation by the EC. Prolonged culture was also associated with deposition of a distinct BM. When fresh EC were seeded on day 20 BM, transmigrated neutrophils moved more slowly under the EC than under control monolayers. Thus, EC change their pro-inflammatory phenotype during prolonged culture, and the deposited basement membrane influences neutrophil migration.

Differing mechanisms of leukocyte recruitment and sensitivity to conditioning by shear stress for endothelial cells treated with tumour necrosis factor-alpha or interleukin-1beta.

The cytokines tumour necrosis factor-alpha (TNFalpha) and interleukin-1beta (IL-1B) induce endothelial cells to recruit leukocytes. However, the exact adhesion and activation mechanisms induced by each cytokine, and their relative sensitivities to modulation by endothelial exposure to shear stress remain unclear. We cultured human umbilical vein endothelial cells (HUVEC) in glass capillaries at various shear stresses, with TNFalpha or IL-1B added for the last 4 h. Subsequently, human neutrophils were perfused over the HUVEC, and adhesion and migration were recorded. Both cytokines induced dose-dependent capture of neutrophils. However, while conditioning of HUVEC by increasing shear stress for 24 h diminished their response to TNFalpha, the response of HUVEC to IL-1B was similar at all shear stresses. The differing sensitivities were evident at levels of adhesive function and mRNA for adhesion molecules and chemokines. Analysis of nuclear factor kappaB (NF-kappaB)/Rel family of transcription factors showed that their expression and activation were modified by exposure to shear stress, but did not obviously explain differential responses to TNFalpha and IL-1B. Antibodies against selectins were effective against capture of neutrophils on TNFalpha-treated but not IL-1B-treated HUVEC. Stable adhesion was supported by beta2-integrins in each case. Activation of neutrophils occurred dominantly through CXC-chemokine receptor 2 (CXCR2) for TNFalpha-treated HUVEC, while blockade of CXCR1, CXCR2 and of platelet-activating factor receptors caused additive inhibition of migration on IL-1B-treated HUVEC. The mechanisms which underlie neutrophil recruitment, and their modulation by the haemodynamic environment, differ between cytokines. Interventions aimed against leukocyte recruitment may not operate equally in different inflammatory milieu.

Transmigrated neutrophils down-regulate the expression of VCAM-1 on endothelial cells and inhibit the adhesion of flowing lymphocytes.

As the first leukocytes recruited during inflammation, neutrophils are ideally situated to regulate the subsequent recruitment of mononuclear leukocytes. Here, we found that human neutrophils recruited by endothelial cells (EC), which had been stimulated with tumor necrosis factor alpha for 4 h, inhibited the adhesion of flowing, mixed mononuclear cells or purified lymphocytes over the subsequent 20 h but did not affect the adhesion of a secondary bolus of neutrophils. The degree of inhibition of lymphocyte adhesion increased with the duration of neutrophil-EC contact and with the number of recruited neutrophils. Antibody-blocking studies showed that lymphocyte adhesion was mediated predominantly by vascular cell adhesion molecule-1 (VCAM-1). Recruited neutrophils reduced the EC expression of VCAM-1 but not intercellular adhesion molecule-1 (ICAM-1) or E-selectin in a manner that mirrored the time- and number-dependent reduction in lymphocyte adhesion. VCAM-1 was not shed into the culture supernatant, and a panel of protease inhibitors was unable to reverse its down-regulation, indicating that it was not proteolytically degraded by neutrophils. In EC that had been in contact with neutrophils, the mRNA message for VCAM-1 but not ICAM-1 was down-regulated, indicating that alterations in transcriptional activity were responsible for the reduction in VCAM-1. Thus, under some inflammatory milieu, neutrophils may delay the recruitment of mononuclear leukocytes by regulating the expression of EC adhesion receptors.

Exposure to fluid shear stress modulates the ability of endothelial cells to recruit neutrophils in response to tumor necrosis factor-alpha: a basis for local variations in vascular sensitivity to inflammation.

Vascular endothelial cells are able to sense changes in the forces acting on them and respond, for instance, by modifying expression of a range of genes. However, there is little information on how such responses are integrated to modify homeostatic functions. We hypothesized that different shear stresses experienced in different regions of the circulation might influence endothelial sensitivity to inflammatory stimuli. We cultured human endothelial cells in tubes and exposed them for varying periods to shear stresses ranging from those typically found in postcapillary venules to those in arteries. When tumor necrosis factor-alpha was included in the flow cultures, we found startling differential effects of shear stress on the ability of endothelial cells to induce adhesion and migration of flowing neutrophils. Compared with static cultures, endothelial cells cultured at low shear stress (0.3 Pa) captured similar numbers of neutrophils but failed to induce their transendothelial migration. After exposure of endothelial cells to high shear stress (1.0 or 2.0 Pa), capture of neutrophils was largely ablated. The modification in response was detectable after 4 hours of exposure to flow but was much greater after 24 hours. From analysis of gene expression, loss of capture or migration was attributable to reduction in tumor necrosis factor-induced expression of selectins or CXC-chemokines, respectively. Thus, conditioning of endothelial cells by different flow environments may underlie variations in susceptibility to inflammation between different tissues or parts of the vascular tree.