Evidence for a dual role

MIF is a unique cytokine that functions both as an inflammatory chemokine and as an enzyme. The inflammatory activity of MIF is dependent on the enzymatic active site, thus these two functions are linked51_{. Swope et al. showed that by blocking the enzymatic active site, MIF}

had a reduced capacity to activate neutrophils51_{. Most studies that investigate the activity of}

MIF use protein that has been secreted from white blood cells. The study in this chapter investigated the activity of intracellular MIF. A key finding, that has relevance to the capacity of red blood cells to affect the immune response, was that intracellular MIF is enzymatically active and thus likely to be functional as a chemokine (Figure 3.6).

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3.4.3.1 Intracellular: Oxidoreductase activity

The less frequently discussed enzymatic function of MIF is its thiol-oxidoreductase activity and its ability to scavenge reactive oxygen species52,53_{. This study demonstrated that there is a}

high concentration of MIF in red blood cells, which may be explained by assuming that the primary function is intra-cellular oxidoreductase activity. The chemokine activity of MIF from red blood cells is likely to be observed by either release from intact cells or in much higher concentrations after red blood cell lysis. Nguyen et al. identified that the oxidoreductase activity is retained in a specific peptide fragment of MIF which is independent of its tautomerase activity52_{. This activity has since been demonstrated in a number of cell types.}

MIF deficient fibroblasts contained 2.3-fold more intracellular reactive oxygen species (ROS) than the MIF-positive controls53 _{and in neurons the increase in ROS after stimulation was}

decreased by intracellular administration of rMIF54_{. The effect of MIF on oxidative stress has}

also been demonstrated on whole organisms. Recently, MIF has been shown to play a crucial role in healthy lung development in neonates55_{. Hyperoxia-induced lung injury (HALI) is lung}

damage that can result from exposure to high levels of oxygen such as in a neonatal intensive care unit. In a recent article, Sun et al. identified that MIF plays an important regulatory role in dealing with high levels of oxygen56_{. When MIF was knocked down, there was a higher}

level of mortality among the mice. Similarly, altered expression of MIF in pre-term infants was associated with the incidence of bronchopulmonary dysplasia57_{. For premature mouse pups,}

only 8 % of MIF deficient mice survived compared to 75 % of the wild type mice55_{. The low}

mortality rate of MIF deficient mice was closely correlated with less mature lungs than the control group.

Red blood cells are burdened with substantial oxidative stress due to their primary oxygen- carrying function. To combat this, they contain a number of known mechanisms, such as the 20S proteasome, to deal with this stress and catabolise damaged proteins. Intracellular MIF is likely to be another part of the red blood cell stress mechanism. Investigation in the activity of MIF as an oxidoreductase in red blood cell lysates was complicated by the fact that red blood cells contain a variety of different oxidoreductase enzymes. Thus, investigation into this activity would require protein purification to isolate the red blood cell derived MIF in its native form. Quantifying the oxidoreductase activity of red blood cell derived MIF would be interesting, but was not in the scope of this study.

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3.4.3.2 Extracellular: Cytokine activity

MIF has also been described to have a number of roles in inflammation. It was originally identified as the factor that inhibited macrophages from migrating away from the site of inflammation8_{. Since then, it has been identified as a chemotactic attractant for monocytes, a}

modulator of glucocorticoid activity, and involved in neutrophil activation17,19,51_{. Since MIF}

has multiple enzymatic and chemotactic functions, studying the cytokine activity in isolation is not straightforward. Glucocorticoid regulation by MIF is associated with its oxidoreductase activity and the leukocyte activation is associated with its tautomerase activity51,52_{. The}

migration assay used to measure MIF activity involves the quantification of cell migration in the presence of MIF compared to controls. Numerous problems have been reported with the assay due to cell aggregation, surface adhesion, and passive migration, which lead to high standard deviations and only semi-quantitative results58_{. In addition, there have been reports of}

difficulty in purifying active MIF, which have led to suggestions that tertiary structural changes or an unknown co-factor affects the activity58_.

The data presented in this chapter indicate that MIF is present at a concentration of approximately 30 µg/mL in whole blood, which is one million times higher than the pg/mL levels usually reported for cytokines in plasma. Given that acute injury events, such as those reported by Pohl et al.22 _{can cause the release of high quantifies of MIF locally and}

systemically, it seems likely that a mechanism for the attenuation of MIF chemokine activity would exist.

3.4.3.3 Other: Tautomerase activity

Substantial research has been done on investigating the tautomerase activity of MIF with a particular emphasis on the development of MIF inhibitors7,59_{. In spite of this, its biological}

substrate is yet to be identified and the reason for MIF having this activity remains unknown. A number of chemical substrates for MIF tautomerase activity have been identified (such as D-dopachrome, L/D-dopachrome methyl ester, or p-hydroxyphenyl-pyruvate) and are used to quantify the level of activity in vitro. D-dopachrome tautomerase (DDT), or MIF-2, catalyses the same substrates in vitro, but its activity is not inhibited by the same chemical, ISO-135_.

This study identified that red blood cell lysates and cytosols are enzymatically active and are active at a rate that is comparable to recombinant MIF. Considering that the majority of MIF is present in the cytosolic fraction of red blood cells (Figure 3.1) it is not surprising that there was not a significant difference in the rate of substrate degradation between the whole lysate

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and the cytosol (Figure 3.6). Inhibition of recombinant MIF, lysate derived MIF, and cytosolic MIF using ISO-1 resulted in a significant attenuation of the breakdown of the substrate (Figure 3.7). However, it was difficult to quantify how effective this inhibition was in red blood cells samples. For ISO-1 to act effectively as an inhibitor of MIF activity, it required a high concentration (~50 %) of organic solvent to be present. The most widely reported solvent for ISO-1 is DMSO. Although the presence of DMSO did not affect recombinant MIF, it interfered with the red blood cell samples and the addition of the DMSO vehicle alone turned the solutions opaque. This interference has been documented in the literature60_{, so to circumvent this, the}

effects of a range of other organic solvents were tested and methanol was chosen as the optimal solvent. Methanol did not impede the inhibitory effect of ISO-1 and it had the lowest background interference for red blood cell samples. The background effect of methanol as a vehicle on the samples was less for the cytosolic fraction (Figure 3.7c) than for the lysates (Figure 3.7b) as some precipitation was observed in the lysate samples at the conclusion of the incubation with the sample.

Although MIF inhibition did occur, it was not possible to quantify the level of inhibition with the current assay and the complex nature of the red blood cell lysate. It is not clear if the tautomerase activity was a result of MIF enzymatic activity alone, or if it was a combination of MIF and D-dopachrome tautomerase enzymatic activity. Future studies should target the identification of MIF inhibitors that are compatible with complex protein solutions such as cell lysates.