New Research Points the Way Towards Mechanism of Action, Receptor for MS Copolymer Drugs

Multiple_sclerosis_T_cellsNeurological damage in multiple sclerosis (MS) is caused by autoreactive immune cells, which attack myelin sheathing on axons of the brain and spinal cord, leading to inflammation and myelin loss.  The MS drug Copaxone is a copolymer of glutamic acid, lysine, alanine, and tyrosine (YEAK) that is thought to impair myelin attack by inhibiting MBP self antigen presentation to autoreactive T cells; a related copolymer in which phenylalanine replaces glutamic acid (YFAK) has been developed based on MBP binding to class II MHC.  However, little is known about these drugs’ molecular targets or mechanisms of actionIn vitro studies suggest IL-10 secretion by B cells or regulatory and Th2-CD4+ T cells is involved.  Recent data demonstrate YEAK and YFAK also have MHC-independent effects on macrophages and dendritic cells, although the receptor which mediates these effects is unknown.  In a recent article in The Journal of Immunology, Koenig and colleagues isolated YEAK- and YFAK-interacting proteins from macrophage lysates and identified structures required for copolymer interaction with cells.

Following incubation with RAW264.7 macrophage lysate, biotinylated copolymers were recovered using avidin-coated beads and the associated cellular proteins were identified using mass spectroscopy.  One high-frequency hit with known surface expression and involvement in immune signaling was gp96.  Cell surface gp96 directly activates innate immune cell cytokine production, acts as a class I MHC antigen chaperone, and has been proposed as a Th2-specific co-stimulatory molecule.  CD91 has been implicated in gp96 stimulation of antigen-presenting cells (APC) and is involved in signaling and endocytosis of several ligands.  App was also identified as a surface protein that interacts with YEAK and YFAK.  A β-amyloid species precursor in Alzheimer’s disease, its function on myeloid cells is not well understood.

Macrophages secrete CCL22, a chemoattractant for regulatory and Th2 T cells, in response to YEAK or YFAK.  Koenig et al. studied this response in wild-type versus gp96-, CD91-, and App-deficient cells and found no impairment in any of the knock-out cell lines, indicating that despite their interactions with YEAK and YFAK, neither gp96, CD91, nor App are involved in cell signaling by these copolymers.

Lysine confers a positive charge on these copolymers, leading Koenig et al. to propose that cellular binding may be mediated by electrostatic interaction rather than conformation.  Indeed, increasing salt concentration reduced protein interactions with biotinylated copolymer.  Importantly, using cell lines lacking specific sulfation enzymes, the authors demonstrated that YEAK and YFAK bind to negatively charged heparan sulfate proteoglycans (HSPG).  This interaction is functional: RAW264.7 cells stimulated with YFAK in the presence of heparin sulfate, a structurally similar competitor of HSPG, did not produce CCL22.

HSPG are glycoproteins which contain one or more covalently attached heparin sulfate (HS) chains.  Membrane HSPG are known to act as co-receptors for many growth factors and could therefore play a role in the cellular effects of YEAK by activating cell signaling through an associated receptor or preventing signaling by that receptor’s natural ligand.  For example, YEAK binding to HS, a co-receptor for gp96 binding to CD91, may alter cellular uptake of gp96-peptide complexes via CD91, affecting self antigen cross-presentation and T cell activation by APC.

Alternatively, HSPG also function as receptors for constitutive as well as ligand-induced endocytosis.  YEAK interaction with HSPG may promote its fluid-phase uptake and delivery to intracellular target(s) in a manner similar to that of cationic cell-penetrating peptides.  In fact, gene ontology term enrichment analysis highlights “RNA binding” as a molecular function of YEAK- and YFAK-interacting proteins identified in this study, supporting a potential cytosolic or nuclear site of action.

Significant work remains to define the receptors and molecular mechanisms of action for these copolymers and aid rational design of future immune-modulating drugs.  The authors’ list of 222 copolymer-interacting proteins and characterization of sulfated glycosaminoglycans as the moieties responsible for functional interaction of these copolymers with innate immune cells serve as a solid foundation for further research in this area.

Further Reading:

Amino acid copolymers that alleviate experimental autoimmune encephalomyelitis in vivo interact with heparan sulfates and glycoprotein 96 in APCs.  Koenig PA, Spooner E, Kawamoto N, Strominger JL, Ploegh HL.  J Immunol.  2013 Jul 1; 191(1):XXX.  Epub ahead of print 2013 June 5.

Heparan sulphate proteoglycans fine-tune mammalian physiology.  Bishop JR, Schuksz M, Esko JD.  Nature. 2007 Apr 26; 446(7139):1030-7.

Interactions between heparan sulfate and proteins – design and functional implications.  Lindahl U, Li JP.  Int Rev Cell Mol Biol. 2009; 276:105-59.

Cell surface heparan sulfate proteoglycans influence MHC class II-restricted antigen presentation.  Léonetti M, Gadzinski A, Moine G.  J Immunol. 2010 Oct 1; 185(7):3847-56.



Considerations for measuring cytokine levels in serum or plasma

Changes in circulating cytokine and chemokine levels have been associated with many human diseases, and thus understanding the relationships between these changes and disease is an important area of medical research.  Circulating levels of these proteins or other chemistries are measured from plasma or serum collected from peripheral blood draws.  It is important to note that the methods of sampling and storage of plasma or serum are critical for accurate measurements.  Here are some important considerations when planning to measure the levels of cytokines and chemokines in serum or plasma.

Blood collection tubes are available in a choice of factors and should blood tubesbe selected based on the analysis being done, as different anti-coagulants support different chemistries.  Plasma is collected from blood drawn into tubes containing anticoagulants, including sodium or lithium heparin which act to inhibit thrombin from blood clotting, or sodium citrate or EDTA which chelate calcium ions to prevent coagulation.  Serum collection tubes contain clot activators, however this method does not allow collection of peripheral blood mononuclear cells (PBMCs) from the same vial, which means that oftentimes, plasma will be the product of choice to maximize the value of blood drawn in a minimal number of tubes from study participants and healthy donors.

luminex service 2Following collection, plasma or serum should be cryopreserved at -80º C.  Cytokines and chemokine levels can be measured by Enzyme-linked immunosorbent assay (ELISA).  However, this method is time consuming and allows measurement of only one factor at a time.  Luminex, a bead-based multiplex assay, can measure up to 100 cytokines, chemokines, or other soluble proteins at a time. Thus, for a given disease cohort, multitudes of measurements can be made from a single small sample of serum or plasma.  Notably, many cytokines and chemokines exist in very low levels in peripheral blood, thus for each cytokine or chemokine to be measured it is important to determine if the detection range of the assay used is sufficient for the known range of circulating levels of that protein.  Also, levels of these proteins may differ depending on whether they were measured in serum or plasma collected in various anticoagulants, so determinations should be done using the most similar methodologies as comparisons.

A methodology paper by de Jager et. al, discusses several important considerations for analyzing cytokine levels from serum or plasma by Luminex.  In this paper, due to unmeasurably low levels of many cytokines, to allow for more dynamic determinations, whole blood was spiked with recombinant cytokines, or treated with LPS for a time period to upregulate expression of cytokines, prior to plasma collection, cryopreservation, and Luminex assays.

One comparison made was the difference in profiles of 15 cytokines in serum, versus plasma from the same donors collected in sodium heparin, EDTA, or citrate.  Overall, cytokine levels were similar with a few exceptions, including IL-6 having the lowest values in serum compared with plasma, while CXCL8 was significantly higher in serum.  The authors concluded that plasma collected in sodium heparin allowed the best measurements overall for the cytokines assessed.  The time it takes to process and store samples after blood collection may also influence cytokine levels and should be done as consistently as possible for the most robust comparisons.

Another hugely important factor is sample storage time.  As with all assays, experimental variation should be minimized, and thus it is common to store plasma or serum samples until the entire cohort has been collected and then analyze all of the samples simultaneously.  This also comes into play when changes in cytokine profiles over time are to be measured from serial samples from an individual. The authors measured cytokine levels from sodium heparin plasma stored at -80º C over time, for up to four years.  Several cytokines including IL-13, IL-15, IL-17 and CXCL8 began to be degraded within one year of storage, while levels of IL-1α, IL-1β, IL-5, IL-6, and IL-10 were degraded by over 50% in 2-3 yearsIL-2, IL-4, IL-12 and IL-18 were much more stable, maintaining their initial levels out to 3 years post initial storage.  Thus, depending on the cytokines being analyzed it is critical to keep these issues in mind.  These are the same issues that are faced with storage of recombinant proteins that are used to generate the ELISA or Luminex standard curves or in other cytokine assays.

Stability of cytokines following several rounds of freeze-thawing were also assessed.  Almost all of the cytokines analyzed with the exception of IL-6 and IL-10 were affected by freeze thawing the samples.  Thus, when storing plasma or serum samples, it is important to freeze the samples in multiple aliquots such that additional assays can be performed while avoiding this issue.

In conclusion, handling and storage of serum and plasma samples as well as the choice of serum versus plasma collected in different anti-coagulants are all important factors to consider when planning for studies that will include measurement of circulating cytokines and chemokines.

Further Reading:

Prerequisites for cytokine measurements in clinical trials with multiplex immunoassays.  de Jager W, Bourcier K, Rijkers GT, Prakken BJ, Seyfert-Margolis V. BMC Immunol. 2009 Sep 28;10:52. doi: 10.1186/1471-2172-10-52.