We describe a simple immunoassay to measure the production of pro-inflammatory cytokines, such as IL-1 beta production, in patients presenting with autoinflammatory phenotypes. By activating cells in whole blood cultures with pathogen-associated molecular patterns, specifically with lipopolysaccharide, cytokine secretion can be conveniently evaluated in whole blood supernatants.
Inflammatory processes resulting from the secretion of soluble mediators by immune cells, lead to various manifestations in skin, joints and other tissues as well as altered cytokine homeostasis. The innate immune system plays a crucial role in recognizing pathogens and other endogenous danger stimuli. One of the major cytokines released by innate immune cells is Interleukin (IL)-1. Therefore, we utilize a whole blood stimulation assay in order to measure the secretion of inflammatory cytokines and specifically of the pro-inflammatory cytokine IL-1β 1, 2, 3.
Patients with genetic dysfunctions of the innate immune system causing autoinflammatory syndromes show an exaggerated release of mature IL-1β upon stimulation with LPS alone. In order to evaluate the innate immune component of patients who present with inflammatory-associated pathologies, we use a specific immunoassay to detect cellular immune responses to pathogen-associated molecular patterns (PAMPs), such as the gram-negative bacterial endotoxin, lipopolysaccharide (LPS). These PAMPs are recognized by pathogen recognition receptors (PRRs), which are found on the cells of the innate immune system 4, 5, 6, 7. A primary signal, LPS, in conjunction with a secondary signal, ATP, is necessary for the activation of the inflammasome, a multiprotein complex that processes pro-IL-1β to its mature, bioactive form 4, 5, 6, 8, 9, 10.
The whole blood assay requires minimal sample manipulation to assess cytokine production when compared to other methods that require labor intensive isolation and culturing of specific cell populations. This method differs from other whole blood stimulation assays; rather than diluting samples with a ratio of RPMI media, we perform a white blood cell count directly from diluted whole blood and therefore, stimulate a known number of white blood cells in culture 2. The results of this particular whole blood assay demonstrate a novel technique useful in elucidating patient cohorts presenting with autoinflammatory pathophysiologies.
1. Collection of Whole Blood
2. Preparation of Whole Blood
3. Stimulation of Whole Blood
4. Cytokine Assay
5. Representative Results
Analysis of IL-1β production upon successful stimulation with LPS and ATP shows a dose response dependent on the number of cells being stimulated. We have found that 2.0 x 106 cells/mL is a sufficient concentration to produce an optimal and measurable concentration of IL-1β as well as other cytokines (Figure 2).
Whole blood assays are useful in studying patients that present with autoinflammatory manifestations. These patients may show a defect in the innate immune system, which can be characterized by overproduction of IL-1β (when compared to controls) upon stimulation with LPS alone (Figure 3).
Figure 1. Representative image of AO staining for nucleated white blood cells used in order to determine cell concentration in diluted whole blood samples.
Figure 2. IL-1β production measured in whole blood supernatants from a healthy donor upon stimulation with LPS at increasing cell concentrations. Samples were assayed in triplicate.
Figure 3. Representative results of IL-1β secretion measured in whole blood supernatants from two healthy normal controls as well as two patients presenting with IL-1β-associated autoinflammatory manifestations.
The whole blood assay described is a simple method that more closely mimics physiological conditions when compared to other techniques that would require cell isolation and extensive sample processing and manipulation. This method utilizes the Cellometer Vision with AO to count white blood cells in whole blood and therefore, provides a way to accurately standardize samples based on cell numbers.
It has to be noted that for this assay, whole blood should be drawn into sodium heparin collection tubes as other anticoagulants are known calcium chelators and can thus affect the results of the assay. Serum free media should also be used because this may affect cytokine concentrations. Also crucial is the immediate processing of samples once obtained. While the method described uses LPS and ATP to stimulate whole blood and measures IL-1βconcentration, other pathogens and stimuli can be used to measure various cytokine and chemokine responses upon stimulation; however, stimulation times and conditions may need to be adjusted accordingly. Finally, constant freeze-thaw cycles may affect cytokine concentrations.
The detection of IL-1β utilizing the whole blood stimulation assay described provides an alternative to other methods and is an effective immunoassay to study diseases with inflammatory-associated pathologies.
The authors have nothing to disclose.
This research was supported by the Intramural Research Program of the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health.
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
Sodium Heparin Blood Collection Tubes | BD | 366480 | ||
RPMI 1640 Media | GIBCO | 21870-100 | ||
Acridine Orange | Invitrogen | A3568 | ||
Cellometer Vision | Nexcelom Bioscience | Contact company for instrument information | ||
Cellometer Cell Counting Chambers | Nexcelom Bioscience | CHT4-SD100 | ||
24-well Tissue Culture Plate | Corning Costar | 3524 | ||
Ultra Pure E. coli K12 LPS | InvivoGen | tlrl-eklps | ||
Adenosine 5´-tripohasphate disodium salt hydrate | Sigma-Aldrich | A26209 | ||
Bio-plex 200 system | Bio-Rad | Contact company for instrument information | ||
Bio-Plex Pro human cytokine x-plex assay | Bio-Rad | x-plex assays are custom made according to specified cytokines of interest |