IL-6
Description
IL-6 is an interleukin that acts as both a pro-inflammatory cytokine and an anti-inflammatory myokine. IL-6 is an important mediator of fever and of the acute phase response, is capable of crossing the blood-brain barrier and initiating synthesis of PGE2 in the hypothalamus, thereby changing the body's temperature setpoint. In muscle and fatty tissue, IL-6 stimulates energy mobilization that leads to increased body temperature. IL-6 can be secreted by macrophages in response to pathogen-associated molecular patterns (PAMPs) antigens. IL-6 can be modulated by chemopreventive drugs, small molecules, monoclonal antibodies, and immune checkpoint inhibitors (Unver and McAllister 2018). IL-6 is an important biomarker used in infectious and autoimmune diseases, cancer, neurodegenerative and cardiovascular diseases, and aging (Erol 2007). There is just one commercially available mAb specific for marmoset IL-6 (www.Ucytech.com), which so far has not been employed in published studies.
Alignment
Protein alignment for human, rhesus macaque and marmoset IL-6:
Protein alignment for marmoset, owl monkey, and squirrel monkey IL-6:
References
- Erol, A. (2007). "Interleukin-6 (IL-6) is still the leading biomarker of the metabolic and aging related disorders." Med Hypotheses 69(3): 708.
- Unver, N. and F. McAllister (2018). "IL-6 family cytokines: Key inflammatory mediators as biomarkers and potential therapeutic targets." Cytokine Growth Factor Rev 41: 10-17.
Status
Recombinant marmoset (Mst) IL-6 was produced in HEK293 cells, tagged with either C-tag or C-tag plus immunogenicity tag. Forty-six hybridomas recognizing MAR-IL-6 were identified by ELISA and further evaluated in Octet showing that a subset of nine monoclonal antibodies (mAbs) recognized Mst-IL-6 with high affinity and bound to different epitopes.
The nine selected mAbs were produced as pure or biotinylated forms and tested in ELISA assays that crisscrossed all possible capture/detection possibilities, using supernatant of marmoset PBMC stimulated with LPS, a known TLR4 ligand that induces expression of IL-6 in mammalian cells. There were several mAb combinations that detected the recombinant Mst IL-6 protein used for immunization, but none of them recognized IL-6 produced by Mst PBMCs stimulated with LPS.
Thus, the next step was to perform ELISA assays using a commercially available anti-IL-6 rabbit polyclonal antibody for detection (Figure 1). The results showed that a few of the mAbs (BU6 and BU7) were able to capture MAR IL-6, for detection by the polyclonal antibody. This reactivity was specific for marmoset (Mst) IL-6 since no reactivity was seen for human (Hu), baboon (Bn), or rhesus macaque (Rh) PBMC stimulated with LPS (Figure 1).
We hyperimmunized two rabbits with the recombinant marmoset IL-6 protein and new ELISA assays were run. Using either BU6 or BU7 mAbs as capture antibodies, we were able to confirm that the two anti-MAR IL-6 rabbit polyclonal antibodies detected IL-6 produced by LPS-stimulated PBMCs from marmoset, squirrel monkeys, and owl monkeys (Figure 2).
The BU6 and BU7 mAbs target the same IL-6 epitope; however, BU6 seems to have higher affinity for IL-6, since ELISA optical density (OD) values were higher for BU6 compared with BU7 (Figure 2). Also, polyclonal antibody (pAb) from rabbit 1 produced higher ODs than rabbit 2. Finally, some of the mAbs used for detection generated OD values 2-3 times higher than background or unstimulated supernatants, which was a promising result.
We covalently bound BU1, BU6 or BU7 to LMX beads and performed assays with diluted supernatants of marmoset PBMC unstimulated or stimulated with LPS. To our surprise, the highest fluorescence values were obtained with the mAbs pairs BU6/BU20 and BU7/BU13, instead of with the rabbit pAb (Figure 3). The combination of BU6 capture and BU20 detection also provided the best dose response curve using recombinant marmoset IL-6 (Figure 4).
Having identified the combination of BU6 capture/BU20 detection as the best mAb pair in a LMX assay, we sought to demonstrate that this pair was also able to recognize IL-6 from squirrel monkeys (SQM) and owl monkeys (OLM), using the in vivo samples obtained from animals exposed to a low level intravenous (IV) LPS challenge (section 1b). Figure 5 shows that IL-6 in plasma peaks at 1-2 hours post-LPS challenge in marmosets and squirrel monkeys. For owl monkeys, however, changes are not that significant. There are two possible explanations for this lack of IL-6 detection: a) the anti-marmoset IL-6 antibody pair does not crossreact with OLM IL-6; or b) the in vivo LPS challenge was not done properly. The argument that perhaps OLM do not respond to LPS is not valid, since we were able to detect OLM production in the ELISA assay using the polyclonal antibody (Figure 2).
In summary, we developed BU6 and BU20 mAbs, which recognize marmoset and squirrel monkey IL-6 in both ELISA and LMX assays. We also identified owl monkey IL-6 in ELISA assays using a combination of BU6 or BU7 mAb capture and pAb detection. We will continue to test mAb/mAb and mAb/pAb combinations trying to identify pairs that work for all NWM species. Production of BU6 and biotin BU20 for distribution to investigators in underway.