Gemma Rigter

Rat C-reactive protein activates the autologous complement system

Activation of complement is a biological function of human C‐reactive protein (hCRP), whereas rat CRP (rCRP) has been claimed to be unable to activate complement. As important biological functions of proteins are probably conserved among species, we re‐evaluated, using various ligands, the capability of rCRP to activate complement. The activation of complement by hCRP and rCRP was investigated in solid‐ and fluid‐phase systems. In the solid‐phase system, purified CRP was fixed to enzyme‐linked immunosorbent assay (ELISA) plates and incubated with human or rat recalcified plasma. Dose‐dependent binding of human and rat C3 and C4 was observed to human and rat CRP, respectively. In the fluid‐phase system, recalcified rat plasma, which contains about 500 mg/l of CRP, or human plasma supplemented with hCRP, were incubated with lyso‐phosphatidylcholine. A dose‐dependent activation of complement was observed upon incubation with this ligand, as reflected by the generation of activated C4 as well as of CRP–complement complexes. This activation was, in both cases, inhibited by preincubation of plasma with p‐aminophosphorylcholine, a specific inhibitor of the interaction of CRP with its ligands, or by chelation of calcium ions. We conclude that rat CRP, similarly to human CRP, can activate autologous complement. These results support the notion that opsonization of ligands with complement is an important biological function of CRP.

The potentiation of human C1-inhibitor by dextran sulphate is transient in vivo: studies in a rat model.

C1-inhibitor (C1-Inh) is an important regulator of inflammatory reactions because it is a potent inhibitor of the contact and complement system. C1-Inh application in inflammatory disease is, however, restricted because of the high doses required. The glycosaminoglycan-like molecule dextran sulphate (DXS) enhances C1-Inh function in vitro. Hence, we investigated whether co-administration with dextran sulphate reduces the amount of C1-Inh required, through enhancement in vivo. C1-Inh potentiation was measured in a newly developed C1s-inactivation assay that is based on activation of C4 by purified C1s. Activated C4 in rat plasma was quantified with a newly developed ELISA. Human C1-Inh (2.5 microM) inhibited C1s in rat plasma 55-fold faster in the presence of dextran sulphate (15 kDa, 5 microM). To study the stability of the complex in vivo, rats were given a mixture of C1-Inh (10 mg/kg) and dextran sulphate (3 mg/kg). C1-Inh activity during 5 h was analyzed ex vivo with the C1s inactivation assay. The noncovalent C1-Inh-dextran sulphate complex resulted in a transient enhancement of the inhibitory capacity of C1-Inh, lasting for 60-90 min. Dextran sulphate did not affect plasma clearance of C1-Inh. We conclude that the enhanced inhibitory capacity of C1-Inh complexed to dextran sulphate is transient in vivo. Hence, co-administration of these compounds seems a feasible approach to achieve short-term inhibition of complement in vivo.

Intravenous immunoglobulin preparations induce mild activation of neutrophils in vivo via triggering of macrophages studies in a rat model

Despite widespread use in various immune disorders, the in vivo mechanisms of action of intravenous immunoglobulin (IVIG) preparations are not well known. We previously reported that human neutrophils degranulate after incubation with IVIG in vitro as a result of interaction with FcγRII. The purpose of this study was to determine whether IVIG might stimulate neutrophils in vivo. Anaesthetized rats received a bolus intravenous injection of IVIG preparations, containing either high (aged IVIG) or low (fresh IVIG) amounts or IgG dimers at a dose of 250 mg/kg. Administration of aged IVIG induced neutrophil activation in vivo, whereas no effect was observed after infusion of fresh IVIG. Histological examination of lung tissue demonstrated mild influx of neutrophils into the pulmonary tissue after aged IVIG administration, though gross damage did not occur. Macrophage‐depleted rats no longer showed activation of neutrophils after infusion of aged IVIG, suggesting that neutrophils become activated via an indirect macrophage dependent way. We conclude that IVIG induces a mild activation of neutrophils in vivo via triggering of macrophages depending on the amount of IgG dimers. For this reason, IVIG preparations with a high content of dimers may not always be as harmless as generally believed and may be responsible for some of the side‐effects observed during IVIG infusions.

Intravascular Reduction of Methemoglobin in Plasma of the Rat in Vivo.

The formation and reduction of extracellular methemoglobin (metHb) in plasma was studied in vivo in conscious rats after isovolemic exchange transfusions with polymerized hemoglobin solutions. After exchange transfusions of 40 and 70% of the blood volume with hemoglobin solutions, containing less than 6% methemoglobin, the methemoglobin level remained below 15%, whereas exchange transfusions of greater than 90% resulted in an increase in the metHb level to about 30% after 24 hours. The reduction of metHb was studied after exchange transfusions with fully oxidized hemoglobin. A gradual decrease in the metHb level to 20% was observed after exchanges of 5 or 40%. A higher exchange transfusion (70%) resulted also in a decrease in the metHb level but only to approximately 40% in 24 hours. In another series of experiments the reduction of metHb was studied in vitro with isolated human erythrocytes. Incubation of the erythrocytes in the presence of oxidized polymerized hemoglobin (3 g%) resulted in a decrease in the percentage of metHb from 91% to 64%. In the presence of 0.2 mM ascorbic acid the metHb level declined to 22%, suggesting a synergistic effect. These results indicate (1) that there is a potent reducing mechanism present in blood that can reduce extracellular oxidized polymerized hemoglobin and (2) that isolated erythrocytes have a large capacity to reduce extracellular metHb, and may also play an important role in the reduction of extracellular metHb in vivo.

Brief Report: Tumor Necrosis Factor and Interleukin-6 Differentially Regulate Dkk-1 in the Inflamed Arthritic Joint

Tumor necrosis factor (TNF) drives bone destruction, but it also inhibits new bone formation by inducing Dkk‐1, an inhibitor of the Wnt pathway. Accordingly, blocking of Dkk‐1 reverses the phenotype in experimental arthritis from a pattern of bone destruction to a pattern of bone formation. To delineate the potential role of Dkk‐1 in the structural phenotype of human arthritis, we analyzed the expression of Dkk‐1 and its regulation by proinflammatory cytokines in the inflamed peripheral joints of patients with spondyloarthritis (SpA) and rheumatoid arthritis (RA).

Clearance of human factor XIa–inhibitor complexes in rats

The serpins C1 esterase inhibitor (C1Inh), antithrombin (AT), α1‐antitrypsin (α1AT) and α2‐antiplasmin (α2AP) are known inhibitors of coagulation factor XIa (FXIa). Although initial studies suggested α1AT to be the main inhibitor of FXIa, we recently demonstrated C1Inh to be a predominant inhibitor of FXIa in vitro in human plasma. The present study was performed to investigate the plasma elimination kinetics of preformed human FXIa–FXIa inhibitor complexes injected in rats. The amounts of complexes remaining in circulation were measured using enzyme‐linked immunosorbent assays. The plasma half‐life time of clearance (t1/2) was 98 min for FXIa–α1AT complexes, whereas it was considerably shorter, i.e. 19, 18 and 15 min for FXIa–C1Inh, FXIa–α2AP and FXIa–AT complexes, respectively. Thus, due to this different plasma t1/2, preferentially FXIa–α1AT complexes may be detected in clinical samples. Furthermore, measuring FXIa–FXIa inhibitor complexes in patient samples may not help to clarify the relative contribution of the individual serpins to inactivation of FXIa in vivo

Effect of Polymerization on Clearance and Degradation of Free Hemoglobin

In the present study we investigated the mechanism of prolongation of the plasma retention of free hemoglobin by polymerization. Polymerization of intramolecularly crosslinked hemoglobin with glutaraldehyde yields a mixture of large polymers, small polymers and monomers. In exchange transfusion experiments in rats we analyzed plasma samples by gel filtration to determine the clearance of polymers of different size. A positive correlation was found between polymer size and vascular retention. Furthermore, the clearance of large polymers appeared to be highly dose-dependent: after 20% and 70% exchange transfusions, we observed for large polymers a plasma half-life of 12 and 26 hours, respectively, whereas the half-life for 64 kD monomers was 4 hours in both cases. The degradation of hemoglobin was followed by measuring the bilirubin excretion. The infused heme was recovered as bilirubin within 72 hours. The delay between the disappearance of free hemoglobin from the plasma and the recovery as bilirubin was about six hours and was not affected by polymerization or dose. We conclude that polymerization prevents the operation of certain clearance mechanisms, while still allowing a route of clearance that is easily saturated. The intracellular degradation of heme into bilirubin is not affected by the modifications of hemoglobin and is not easily saturated.

Hemorrhagic Disorders After Administration of Glutaraldehyde-polymerized Hemoglobin

Glutaraldehyde is frequently used for the chemical modification of hemoglobin to improve the characteristics for use as a blood substitute. Polymerization of hemoglobin prolongs the vascular retention by increasing the molecular size. There are different ways to perform the cross-linking reaction, and this determines the stability and the oxygen affinity of the final product. In our laboratory we developed a glutaraldehyde-polymerized hemoglobin solution (PolyHbXI) with a high degree of intramolecular cross-linking and lowered oxygen affinity. In preclinical safety studies, PolyHbXI was generally well tolerated. However, in three animal models (rats, rabbits and rhesus monkeys), we observed transient hemorrhagic disorders. We further analyzed this side effect in a histopathological study with rats. Hemorrhagic lesions were observed in several tissues (with predilection for the intestinal wall) two days after administration of PolyHbXI, and the lesions were resolved after one week. Microscopically, the bleedings were characterized as “small vessel vasculitis” with a neutrophil infiltration. Platelet numbers, bleeding time and APTT values were normal. This leads to the hypothesis that endothelial cell injury plays a central role in the pathophysiology. By comparing different modified hemoglobin preparations we found evidence that the toxic factor is the result of the cross-linking procedure with glutaraldehyde.

Tumor necrosis factor and interleukin-6 differentially regulate Dkk-1 in the inflamed arthritic joint.

Tumor necrosis factor (TNF) drives bone destruction, but it also inhibits new bone formation by inducing Dkk‐1, an inhibitor of the Wnt pathway. Accordingly, blocking of Dkk‐1 reverses the phenotype in experimental arthritis from a pattern of bone destruction to a pattern of bone formation. To delineate the potential role of Dkk‐1 in the structural phenotype of human arthritis, we analyzed the expression of Dkk‐1 and its regulation by proinflammatory cytokines in the inflamed peripheral joints of patients with spondyloarthritis (SpA) and rheumatoid arthritis (RA).

EVALUATION OF THE IMMUNOGENICITY OF POLYMERIZED HEMOGLOBIN SOLUTIONS IN A RABBIT MODEL

Chemical modification of proteins carries the risk that neo-antigens are introduced. To investigate the potential immunogenicity of human glutaraldehyde-polymerized hemoglobin (polyHbX1), we analyzed the antibody responses of rabbits after hyperimmunization with complete Freunds adjuvant. In view of the species difference, we also tested rabbit hemoglobin that was modified in the same way as human polyHbX1. Thereafter, we studied the antibody response after weekly intravenous infusion of clinically relevant doses of polyHbX1 to evaluate whether an immune response is likely to occur when modified hemoglobin is used as blood substitute. The occurrence of an antibody response was tested by using an enzyme immunoassay (ELISA). To find out whether antibodies were directed against neo-epitopes on human polyHbX1 we used a competitive ELISA. The results showed that polymerized hemoglobin may weakly activate the immune system in special conditions, but is unlikely to do so when it is used as blood substitute.