Hemoglobin Enhances the Biological Activity of Synthetic and Natural Bacterial (Endotoxic) Virulence Factors: A General Principle

Abstract

Although hemoglobin (Hb) is mainly present in the cytoplasm of erythrocytes (red blood cells), lower concentrations of pure, cell-free Hb are released permanently into the circulation due to an inherent intravascular hemolytic disruption of erythrocytes. Previously it was shown that the interaction of Hb with bacterial endotoxins (lipopolysaccharides, LPS) results in a significant increase of the biological activity of LPS. There is clear evidence that the enhancement of the biological activity of LPS by Hb is connected with a disaggregation of LPS. From these findings one questions whether the property to enhance the biological activity of endotoxin, in most cases proven by the ability to increase the cytokine (tumor-necrosis-factor-, interleukins) production in human mononuclear cells, is restricted to bacterial endotoxin or is a more general principle in nature. To elucidate this question, we investigated the interaction of various synthetic and natural virulence (pathogenicity) factors with hemoglobin of human or sheep origin. In addition to enterobacterial R-type LPS a synthetic bacterial lipopeptide and synthetic phospholipid-like structures mimicking the lipid A portion of LPS were analysed. Furthermore, we also tested endotoxically inactive LPS and lipid A compounds such as those from Chlamydia trachomatis. We found that the observations made for endotoxically active form of LPS can be generalized for the other synthetic and natural virulence factors: In every case, the cytokine-production induced by them is increased by the addition of Hb. This biological property of Hb is connected with its physical property to convert the aggregate structures of the virulence factors into one with cubic symmetry, accompanied with a considerable reduction of the size and number of the original aggregates.

Morphology, size distribution, and aggregate structure of lipopolysaccharide and lipid A dispersions from enterobacterial origin

“…This interpretation is strengthened by recent observations that haemoglobin and part structures are enhancing the biological activity of LPS, concomitant with a clear expression of a cubic SAXS pattern for LPS Rc as well as Ra. 24,25…”

Identification of Hemopexin as an Anti-Inflammatory Factor That Inhibits Synergy of Hemoglobin with HMGB1 in Sterile and Infectious Inflammation

“…A series of early studies revealed that hemoglobin synergistically increases the production of TNF and other cytokines by macrophages exposed to LPS and other activators(1-3). It has previously been proposed that hemoglobin enhances the presentation of LPS to immune cells, resulting in a synergistic increase in cytokine release (3-5). Other studies, however, implicated a critical role of the heme moiety of hemoglobin in the mechanism that enhances macrophage TNF release during endotoxemia (6).…”

Usefulness of ELISA Methods for Assessing LPS Interactions with Proteins and Peptides

“…However, HB, which promotes LPS toxicity [ 11 ], produced an increase in OD signals in the competitive ELISAs tested. Although there is still some controversy as to whether the active forms of LPS are monomers or multimers with a particular conformation [ 52 ], most of the available data attribute the enhanced LPS biological activity to the ability of certain proteins to disaggregate LPS (decrease in the size of aggregates) and/or to promote transition from multilamellar structures to cubic structures [ 16 , 25 , 53 ]. Our results are consistent with this type of interaction, as the enhanced OD values obtained in the competitive ELISAs are probably due to disaggregation of LPS.…”