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- Materials and methods
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.
Although intravenous immunoglobulin (IVIG) preparations were initially introduced as replacement therapy in primary antibody deficiency disorders, they were found to have beneficial effects in patients with autoimmune thrombocytopenic purpura and other autoimmune diseases (Dwyer, 1992; Wolf, 1996). Nowadays, IVIG is used in a broad range of autoimmune and systemic inflammatory disorders. Despite its widespread use, the precise mechanisms of action of IVIG are still largely unknown. Different mechanisms have been proposed, such as Fcγ-receptor blockade, inhibition of complement deposition, neutralization of superantigens, neutralization of cytokines and manipulation of the idiotypic network (Imbach et al, 1981; Sultan et al, 1984; Aukrust et al, 1994; 1997; Mouton et al, 1994; Andersson et al, 1996; Basta, 1996; Skansén-Saphir et al, 1997).
We previously reported that IVIG interacts with neutrophils in vitro through binding of IgG dimers and polymers to their Fcγ-receptors (Teeling et al, 1998). In particular, binding of IVIG to FcγRIIa was shown to induce activation and, subsequently, degranulation of neutrophils.
Neutrophils are endowed with potent mechanisms to kill microbes and thus contribute to the host's defence against microbicidal infections. However, they have also long been recognized as important effectors of the adverse effects of inflammatory reactions because their excessive accumulation in tissues may contribute to injury of the host as a result of release of a variety of toxic products. The lungs are particularly susceptible to such insults and constitute a primary site of damage induced by host-defence mechanisms (Chignard & Renesto, 1995; Kubo et al, 1998). Hence, interaction of IVIG with neutrophils in vivo may, among others, contribute to the development of clinical (side) effects of IVIG.
Because IVIG is able to directly activate neutrophils in vitro, an animal model was developed to study if this also occurs in vivo. IVIG preparations, differing in their IgG dimer content, were administered and activation of neutrophils was determined in the circulation as well as at tissue level. As we considered a bolus infusion of IVIG, rather than continuous infusion, to be more robust and hence more suitable to detect potential effects, IVIG was administered as a bolus infusion in most experiments. However, to mimic the clinical situation, we also evaluated the effect of continuous infusion. Independent of the mode of infusion, our results showed that IVIG activates neutrophils in vivo, albeit not directly. Instead, macrophages are involved, because macrophage depletion completely abolished all parameters for neutrophil activation.
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- Materials and methods
The present study demonstrates a mild activation of neutrophils after infusion of IVIG in a rat model. For several reasons, we tested two IVIG preparations, i.e. fresh IVIG and aged IVIG, which only differed in their IgG dimer content. First, polymers and dimers present in the preparations were previously found to be responsible for the activation of human neutrophils in vitro (Teeling et al, 1998). Second, in some commercially available IVIG preparations we found high amounts of IgG dimers, up to 15% (Bleeker et al, 2000). During the production of the different IVIG preparations, IgG dimers can be formed, depending on the temperature, ionic strength, pH, additions such as glucose and duration of the whole manufacturing process.
Rat neutrophils were activated to the same extent as human neutrophils in vitro, indicating that these cells are able to interact with human IVIG preparations as was confirmed in direct binding studies.
In general, systemic activation of neutrophils in vivo is associated with a rapid disappearance of neutrophils from the circulation followed by sequestration in the different tissues, especially the lungs (Carlos & Harlan, 1990;Butcher, 1991; Worthen et al, 1991; van Zee et al, 1992; Borregaard et al, 1994). This sequestration of neutrophils is caused by changes in the cytoskeleton, transient cell-stiffening and changes in expression of adhesion molecules (Worthen et al, 1991; Borregaard et al, 1994). Administration of LPS, a well-known agonist for neutrophils, caused a marked neutropenia within 5 min of infusion. Aged IVIG also caused a decrease in circulating neutrophils whereas fresh IVIG did not, suggesting that activation of neutrophils is caused by IgG dimers present in the IVIG preparation and not by antibodies against constituents on rat cells. Continuous infusion of aged IVIG further demonstrated that the effects observed were not caused by the rapid administration of IVIG.
Activation of neutrophils was phenotypically studied by assessment of the expression of CD11b and L-selectin on the surface of circulating neutrophils. Administration of IVIG (either aged or fresh) did not clearly exhibit signs of activation as was observed in the in vitro experiments. One reason for this discrepancy between in vitro and in vivo findings could be that the activated neutrophils became under represented in the blood samples by the coinciding granulocytosis, with neutrophils freshly released from the bone marrow exhibiting a higher expression of L-selectin (van Eeden et al, 1995). Administration of LPS did not induce a coinciding granulocytosis, thus explaining the marked changes in CD11b and L-selectin induced by this agonist. Alternatively, histological observations and the MPO contents of the lungs support the idea that activated neutrophils may leave the circulation.
Conversely, L-selectin expression on neutrophils increased after infusion of fresh IVIG. As explained above, recruitment of neutrophils from the bone marrow may explain the increase in mean fluorescence intensities of L-selectin on circulating neutrophils after infusion of fresh IVIG. The histological results suggest that despite minimal changes observed in peripheral blood, administration of fresh IVIG as a bolus infusion also leads to mild activation of neutrophils in vivo. Interestingly, continuous infusion of aged IVIG showed less sequestration of neutrophils into lung tissue, suggesting that the rate of infusion plays a role in the IVIG-induced neutrophil activation. This finding is very recrudescent of clinical practice, in which adverse reactions are associated with rapid infusion rates (Duhem et al, 1994; Bagdasarian et al, 1998). Our observations suggest possible involvement of neutrophil activation in the induction of these clinical side-effects.
Mobilization of the marginated pool, as occurs during a stress reaction (Gordon, 1955), cannot explain the marked granulocytosis observed after infusion of aged IVIG. This is because demargination of neutrophils after administration of adrenaline, resulted in a maximum two- to threefold increase in the circulating numbers of neutrophils. Several inflammatory mediators are able to induce granulocytosis, such as C5a, cytokines, the growth factors, granulocyte colony-stimulating factor (G-CSF), granulocye macrophage CSF (GM-CSF), eicosanoids and glucocorticosteroids (van Zee et al, 1992; Jagels & Hugli, 1994; 1995; Ulich et al, 1987; Kubo et al, 1998; Opdenakker et al, 1998). In the present study, we tested whether TNF-α, one of the early response cytokines, was released and hence constitutes a mechanism by which IVIG induced granulocytosis. LPS caused a rise in TNF-α plasma levels, whereas levels of TNF-α remained undetectable after infusion of IVIG, making the involvement of TNF-α less probable. Another mediator, previously shown to be involved in IVIG-induced changes, may be PAF that is released from macrophages (Bleeker et al, 1989). Although rPAF-AH protected rats from hypotension, this inhibition could not prevent the quantitative changes in neutrophil counts. Yet, macrophages were major contributors herein: the biphasic effect of aged IVIG is completely abrogated in macrophage-depleted rats. Notably, the experiments with IVIG were carried out 2 d after depletion of macrophages by Cl2MBP, by which time an inflammatory reaction induced by the depletion would have been unlikely to blunt the phenomena studied (van Rooijen et al, 1989; van Rooijen & Sanders, 1990; Qian et al, 1994). At the moment, complement activation and subsequent formation of C5a as a cause of the granulocytosis cannot be ruled out but it seems less apparent knowing that macrophages play such an important role.
A more likely explanation for the discrepancy between in vivo and in vitro findings would be indirect activation of neutrophils via macrophages because macrophage-depleted rats no longer show activation of circulating neutrophils after infusion of a bolus injection of aged IVIG. IgG dimers were present in the circulation during the entire 4-h study period, making it less probable that direct activation of neutrophils by IgG dimers occurs in vivo. Furthermore, a second dose of aged IVIG did not induce neutropenia. However, the macrophage mediators involved in the activation of neutrophils still remains to be elucidated because potential mediators like TNF-α and PAF were not found to play a major role in our model.
The present study demonstrates that a mild activation of neutrophils occurs after infusion of IVIG in a rat model. To what extent this occurs in patients receiving IVIG remains to be established, although we have found increasing plasma levels of elastase and lactoferrin in a few patients experiencing clinical side-effects after IVIG infusion (unpublished observations). In addition, a recent clinical study in volunteers was performed in which different experimental IVIG-preparations were compared (Spycher et al, 1999). In this study, a correlation was found between IgG dimer content in the preparations and the occurrence of clinical adverse reactions in humans. These clinical side-effects in the humans were accompanied by a transient decrease in neutrophil and monocyte numbers in the peripheral blood and an increase in TNF serum concentrations.
Although, in general, the neutrophil activation will be mild and hence well tolerated by most patients, IVIG infusion may have serious consequences in patients with pre-activated neutrophils in their circulation. Recently, a healthy volunteer was described who developed adult respiratory distress syndrome (ARDS) after administration of IVIG (Dooren et al, 1998).
The beneficial effect of IVIG therapy in the treatment of several autoimmune diseases is thought to be mediated by autoantibody idiotypes present in the IVIG preparations. Formation of such idiotype–anti-idiotype dimers may therefore prevent autoantibodies from binding to antigens (Roux & Tankersley, 1990; Mouton et al, 1994; Vassilev et al, 1995). In this study, we have shown that the dimers present in IVIG preparations may also constitute the less favourable effects in the treatment of patients. For this reason, IVIG with a high content of dimers may be harmful in patients with pre-activated neutrophils in the circulation.