Detection of interleukin -1β from isolated human lymphocyte in response to lipopolysaccharide and lipoteichoic acid.

AIM: To detect the interleukin -1β levels from single and pooled isolated human lymphocytes in response to lipolysaccharide and lipoteichoic acid.
MATERIALS AND METHODS: Blood collected from healthy individuals (O +ve, A +ve, B +ve, and AB +ve) were subjected to gradient centrifugation to isolate lymphocytes. Different lymphocyte concentrations were used for in vitro pyrogen assay. Lymphocytes isolated were challenged with 5 EU of Gram negative (LPS) and 1 μg/μl of Gram positive (LTA) pyrogens in vitro and the inflammatory cytokine, Interleukin 1β (IL-1β) release was measured by Sandwich ELISA method.
RESULTS: The results indicated that the release of IL-1β increases immediately after the initiation of incubation and reaches a maximum at 4 to 6th hour and then stabilizes for both the pyrogens. Furthermore, IL-1β release by 5 EU of LPS and 1 μg/μl of LTA is dependent on lymphocytes concentration. It was also observed that the difference in blood group did not interfere with the IL-1β release.
CONCLUSION: The isolated lymphocyte system can be used as an alternative to the in vivo rabbit pyrogen assay.

Introduction

The measurement of pyrogens is an indispensable safety procedure for all parenterally administered drugs, as these contaminants are life threatening. The origin of pyrogenic substances varies and they may stem from bacteria, viruses, or certain protein components. A group of microbial products and other inorganic substances of varying origin can cause febrile response. Lipopolysaccharide (LPS), the cell wall components of Gram negative bacteria, is known to have various biological activities and it induces fever in minute amounts.[1] Moreover, lipoteichoic acids (LTA) from Gram positive bacteria are problematic in parenteral pharmaceuticals,[2] since many of the products are prone to contamination by the microbial remnants.[3]

The pharmacopoeia lists the in vivo rabbit pyrogen test, in vitro Limulus Amebocyte (LAL) test and monocyte-activation test for pyrogen testing. The rabbit pyrogen test measures the rise in body temperature after injection of the test substance.[4] However, it has several limitations and is not suitable for the detection of endotoxin limit. The LAL test cannot detect the LPS equivalents of Gram positive bacteria or fungi. To overcome these, an in vitro human whole blood assay has been developed for detecting pyrogenicity.[56]

Pyrogenic contaminants like LPS and LTA stimulate the mononuclear phagocytes in blood to release pro-inflammatory cytokines like IL-1, IL-6, and TNF-α, which then transmit the fever signal to thermoregulatory centers of the brain,[7] thereby inducing cyclooxygenase (COX)-2-dependent prostaglandin (PG) E2, the mediator of febrile response.[8] Interleukin-1β is one of the main pro-inflammatory cytokine produced by activated macrophages as a pro-protein, which is proteolically, processed into its active form by caspase 1. This cytokine is the principal mediator of the inflammatory process, which is immediately released upon exposure to minute concentration of pyrogens and can be readily measured by ELISA. A rapid, accurate, and cost effective ELISA method developed by Sree Chitra Tirunal Institute for Medical sciences and Technology (SCTIMST) measures the pyrogenic response of Gram negative, positive bacteria, chemical, and biological pyrogens on human whole blood.[9]

Human peripheral blood mononuclear cells (PBMC's) can be used as a measure of cytokine production.[10] The advantage of using isolated human lymphocytes is that they are the critical components in the human immune system to fight against infections and adapt to intruders. The present study makes use of the inflammatory response of isolated human lymphocytes in in vitro conditions toward LPS and LTA using the novel ELISA method developed by SCTIMST.

Materials and Methods

Heparin extra pure (Himedia, India), RPMI-1640 (Himedia, India), interleukin 1-β human recombinant expressed in Escherichia coli (Sigma, USA), albumin fraction V from bovine serum (BSA) for biochemistry (Merck, Germany), TMB plus liquid 1 component substrate (Amersco, India), hisep 1077 (Sigma, USA), lipoteichoic acid (Sigma, USA), lipopolysaccharide (Sigma, USA), and anti IL -1β antibody (collected from the rabbits immunized with IL-Iβ (Sigma, USA) at Toxicology Division, SCTIMST) were used. The reagents and materials used for the in vitro pyrogen assay were depyrogenized at 250°C for 30 min prior to the experiment.

Asys Expert plus ELISA plate reader (Grenier 96 well ELISA plate) with Digiread software (Austria), Eppendorf centrifuge 5810R (Germany), Fisher Scientific AB15+, pH meter (UK), ESCO Air stream vertical laminar flow cabinet (Singapore), Olympus CH-2 microscope (Japan), REMI Centrifuge (India), Heraeus Kelvitron T hot air oven (Germany), Deep freezer –20°C (Sanyo, Japan), Spectrophotometer (Schimadzu, Japan), and AKTA prime plus system (GE Healthcare, South Asia) were used.

Collection of Blood

Healthy, non allergic individuals who had no medication for at least 2 weeks prior to the blood collection served as donors. Informed consent was obtained from donors having different blood groups including O +ve, A +ve, B +ve, and AB +ve. Individual and pooled blood was used for the isolation of lymphocytes. Venous blood (8 ml) was collected from healthy volunteers and immediately transferred into sterile, pyrogen free tubes containing heparin (2 μg/ml).

Isolation of Lymphocytes

Lymphocytes were isolated using gradient centrifugation in Hisep LSM 1077 (Sigma) (Boyum, 1968). The cell viability was assessed with the Trypan blue exclusion method. The concentration of cells in the original mixture was calculated and the cells were subsequently diluted with RPMI 1640 (Himedia) to obtain different concentrations that were further used for the in vitro pyrogen assay. The number of cells in isolated lymphocyte sample were counted in a hemocytometer and the cells were diluted with appropriate volume of 1X PBS to obtain the concentrations of 4 × 105, 5 × 105 and 7 × 105 cells (O +ve and A +ve donors) and 5 × 105, 7 × 105 cells in individually pooled lymphocytes of A +ve, B +ve and AB +ve donors. This was used for the in vitro pyrogen assay.

Induction of IL-1b by LPS and LTA in Freshly Isolated Human Lymphocytes

Freshly isolated human lymphocytes (100 μl) from O +ve and A +ve donors were diluted separately in pyrogen stimulated RPMI-1640 (400 μl) to a final volume of 500 μl in 1.5 ml microfuge tubes. Lymphocytes were added in three different concentrations namely 4 × 105, 5 × 105 and 7 × 105 cells/100 μl. LPS from E. coli (5 EU) and LTA from Bacillus subtilis (1 μg/μl) were used in stimulating pyrogenic response in both the samples. Control experiments were also performed on non-stimulated lymphocytes. The reaction tubes were incubated at 37°C up to 8 h. At the end of each hour, the samples were harvested and centrifuged at 500 g for 2 min at 4°C. The supernatant was collected in a microfuge tube and immediately stored at –20°C until analysis.

Induction of IL-1b by LPS and LTA in Pooled Human Lymphocytes

The lymphocytes from multiple donors of A +ve blood group were pooled in two different concentrations such as 5 × 105 and 7 × 105 cells/100 μl. Similarly, lymphocytes from B +ve and AB +ve donors were pooled independently. Pyrogenic stimuli supplemented in RPMI 1640 (400 μl) were used to dilute pooled lymphocytes to final volume 500 μl in 1.5 ml microfuge tubes. LPS from E. coli (5EU) and LTA from Bacillus subtilis (1 μg/μl) were used in stimulating pyrogenic response in the three samples. The reaction tubes were incubated at 37°C up to 8 h. The samples were harvested at the end of each hour, and centrifuged at 500 g for 2 min at 4°C. The supernatant collected was immediately stored at –20°C until analysis.

Measurement of IL-1b by Sandwich ELISA Method

The microplate coated with anti-human IL-1β antibody in 50 mM carbonate bicarbonate buffer, pH 9.6 at 4°C overnight was blocked with 1% BSA for 1 h at room temperature. After washing with PBS, 50 μl of the lymphocyte samples was added per well and incubated for 2 h at room temperature. The wells were washed again and added with diluted HRP conjugated anti-IL1 β (prepared by in house method at Toxicology Division, Sree Chitra Tirunal Institute for Medical Sciences and Technology) and incubated at room temperature for 2 h. After washing, the wells were incubated with TMB substrate (Amersco) for 30 min and the reactions were terminated by adding 1M H2SO4 and incubated for 10 min in dark. The plates were read at 450 nm with the corrective filter at 620 nm using ELISA reader (ASYS Expert plus).

Results

Estimation and Detection of IL-1b Response

The IL-1β standards were assayed by the Sandwich ELISA method. The concentration of IL-1β released on stimulation with lymphocytes was calculated from the standard graph by plotting the IL-1β concentration (ng/μl) in X-axis and OD (450 nm) in Y-axis [Figure 1].

Figure 1

IL-1β ELISA Standard curve

Measurement of IL-1b in Response to LPS from O +ve and A +ve Donors

The time course IL-1β release in response to pyrogenic stimulation with 5 EU of LPS was evaluated for 8 h. Among the three concentrations viz.7 × 105, 5 × 105, and 4 × 105 cells, the maximum IL-1β (3 ng) level in O +ve donors was found to take place at 5th h of the in vitro reaction with 7 × 105 cell concentration [Figure 2a]. Similarly, lymphocytes from A +ve donors with 7 × 105 cell concentration displayed maximum release at 5th h (1.6 ng of IL-1β).

Figure 2

(a) represents Optical Density of control (open squares) and test (closed squares) of isolated human lymphocytes of O +ve donors (7 lakhs) stimulated with 5 EU of LPS. (b) represents Optical Density of control (open squares) and test (closed squares) of isolated human lymphocytes of O +ve donors (7 lakhs) stimulated with 1 μg/μl of LTA. (c) represents Optical Density of control (open squares) and test (closed squares) of isolated human lymphocytes of A +ve donors (7 lakhs) stimulated with 5 EU of LPS. (d) represents Optical Density of control (open squares) and test (closed squares) of isolated human lymphocytes of A +ve donors (7 lakhs) stimulated with 1μg/μl of LTA.

Measurement of IL-1b in Response to LTA from O +ve and A +ve Donors

In vitro pyrogen reaction stimulated with 1 μg/μl of LTA in O +ve donors with 7 × 105 of lymphocytes was monitored for 8 h and the maximum response with a release of 1.7 ng of IL-1β took place at 6th h [Figures 2b, c]. Compared to the O +ve donors, the A +ve donors displayed maximum release (5 ng) in 4th h of the reaction [Figure 2d].

Measurement of IL-1b in Response to LPS from Individually Pooled Lymphocytes of A +ve, B +ve, and AB +ve Donors

In pooled lymphocytes of A +ve donors, IL-1β release elicited by 5 EU of LPS was found maximum at 6 h post-stimulation with 7 × 105 lymphocyte concentration [Figure 3a]. Compared to the IL-1β concentration in 5 × 105 cells, the 7 × 105 cell population showed a release of 3 ng during the initial period of pyrogenic stimulation, which gets gradually increased to 20 ng and gets stabilized [Figure 1]. Further, B +ve donors displayed a maximum release (3.5 ng of IL-1β) at 6 h post stimulation [Figure 3c]. Also, pooled cells of AB +ve donors showed a maximum response of IL-1β (1.5 ng of IL-1β) in 6th h [Figure 3e].

Figure 3

(a):represents Optical Density of control (open squares) and test (closed squares) of pooled human lymphocytes of A +ve donors (7 lakhs) stimulated with 5 EU of LPS. (b) represents Optical Density of control (open squares) and test (closed squares) of pooled human lymphocytes of A +ve donors (7 lakhs) stimulated with 1 μg/μl of LTA. (c) represents Optical Density of control (open squares) and test (closed squares) of pooled human lymphocytes of B +ve donors (7 lakhs) stimulated with 5 EU of LPS. (d) represents Optical Density of control (open squares) and test (closed squares) of pooled human lymphocytes of B +ve donors (7 lakhs) stimulated with 1 μg/μl of LTA. (e) represents Optical Density of control (open squares) and test (closed squares) of pooled human lymphocytes of AB +ve donors (7 lakhs) stimulated with 5 EU of LPS. (f) represents Optical Density of control (open squares) and test (closed squares) of pooled human lymphocytes of AB +ve donors (7 lakhs) stimulated with 1 μg/μl of LTA

Measurement of IL-1b in Response to LTA from Individually Pooled Lymphocytes of A +ve, B +ve, and AB +ve Donors

The LTA (1 μg/μl) stimulated pyrogen reaction in pooled lymphocytes of A +ve donors showed maximum IL-1β release (2 ng) at 6th h when used in 7 × 105 cell concentration [Figure 3b]. The maximum release of IL-1β (5 ng) was found at 5th h when stimulated with 7 × 105 lymphocytes in B +ve donors [Figure 3d]. Moreover, the AB +ve donors yielded 2 ng of IL-1β at 5th h of the in vitro reaction in 7 × 105 cells [Figure 3f].

Discussion

Cytokines are involved in several aspects of inflammatory reactions. The increasing awareness of the key role of pro-inflammatory cytokines like IL-1β in inducing pyrogenic response necessitates the development of assays for cytokine quantification which have become a rapidly expanding part of the laboratory repertoire. The selection of a suitable cytokine assay depends to a large extent on the research objective to be achieved. In the present study, an attempt was made to isolate the lymphocyte cells from the human whole blood, to evaluate the release of IL-1β as a marker for pyrogenicity. The release of pro-inflammatory cytokine takes place when stimulated with 5 EU of LPS and 1 μg/μl of LTA.

IL-1β is a potent pro-inflammatory cytokine secreted by blood monocytes and tissue macrophages, when it comes in contact with exogenous pyrogens.[1112] It has an advantage as a read out parameter for Gram negative and Gram positive pyrogens because of the shorter incubation time required for its release.[13] Moreover, without pyrogenic (LPS and LTA) stimulation, no significant rise in IL-1β was observed. The release of IL-1β increases immediately after the initiation of incubation on challenge with exogenous pyrogens and reaches a maximum at 4 to 6th h and then stabilizes for both LPS and LTA. The data are supported by Jansky et al.[14] findings, where the net release of IL-1β in PBMCs increases immediately within the first few hours after the initiation of incubation with LPS and then remains stable during the 24-h test.

The result of the ELISA method in lymphocytes collected from O +ve donors indicates that the release of IL-1β was at a peak on 6th h when stimulated with 5 EU of LPS at a cell concentration of 5 × 105 and 4 × 105 [Table 1] of lymphocytes. The maximum release was noted at 5thh in 7 lakhs of cells which resulted in the production of 3 ng of IL-1β. The amount of endotoxin required to generate a pyrogenic reaction is 0.5 EU/ml which is considered as the threshold of fever induction. Further, it should be mentioned that the time course of IL-1β production after stimulation with LPS corresponds to the development of fever. Similarly, the IL-1β production in response to 1 μg/μl of LTA indicates a maximum of IL-1β level at 6th h of reaction when the concentration of lymphocyte was 7 × 105 [Figure 2b] and 4 × 105 [Table 1] cells. The cells yielded an IL-1β concentration of 1.7 ng, when challenged with 7 lakh cell concentrations.

Table 1

Optical density of control and test samples of isolated human lymphocytes from O +ve donors

The IL-1β level was found to be fluctuating in certain control samples which created minor technical variations in the Sandwich ELISA method. The reason for the inconsistency may be the inhibitory factors affecting the IL-1β release. This is supported by the finding[15] that steady state level of IL-1β increases with transient transcription and decreases due to the synthesis of a transcriptional repressor.

The results of the present study revealed a concentration dependent response of IL-1β toward lymphocytes with the pyrogenic stimulation of LPS and LTA. In O +ve donors, the concentration of IL-1β was increased from 1.5 ng to 3 ng when treated with LPS. Similarly, when treated with LTA, the IL-1β concentration increased from 1.5 ng to 1.7 ng compared to the LPS treated group. In another study conducted with A +ve donors, it was noticed that IL-1β formed a peak at 5th h of the reaction when treated with 5 EU of LPS at a cell concentration of 7 × 105 [Figure 2c] and 5 × 105 [Table 2]. The reaction indicates a production of 1.6 ng of IL-1β when stimulated with 7 lakhs of lymphocytes. A similar dose response relationship study was carried out with 1 μg/μl of LTA indicating a higher concentration of IL-1β (5 ng). This was found to be a much higher concentration (5 ng) than the LPS induced reaction. Furthermore, the peak elicited by 5 EU of LPS in inducing IL-1β release was found at 6th h of the reaction [Figure 3a] with 7 × 105 lymphocyte concentration (pooled lymphocytes of A +ve donors). The assay detected an IL-1β concentration of 1.5 ng during the initial period of pyrogenic stimulation which gradually increased to 20 ng. LTA also induced the highest IL-1β response in pooled sample (A +ve donors) on stimulation with 7 × 105 [Figure 3b] cell concentration.

Table 2

Optical density of control and test samples of isolated human lymphocytes from A +ve donors

In pooled lymphocytes (B +ve Donors), the IL-1β release elicited by 5 EU of LPS and 1 μg/μl of LTA took place at 6th [Figure 3c] and 5th h [Figure 3d] of the in vitro reaction in 7 × 105 of lymphocytes. Similarly, in pooled lymphocytes of AB +ve donors, LPS (5 EU) stimulated human lymphocytes exhibited IL-1β response at 6th h, when 5 × 105 [Table 3] and 7 ×105 [Figure 3e] of cell concentrations were used. Further, the IL-1β concentration was found as 2 ng in 5 lakhs cell concentration, whereas 1.5 ng was present in 7 lakh cell concentration. A similar dose–response relationship study was conducted with 1 μg/μl of LTA which yielded 2 ng of IL-1 β in both 7 × 105 and 5 × 105 lymphocyte concentrations [Table 4].

Table 3

Optical density of control and test samples of pooled human lymphocytes (5 lakh cell concentration)

Table 4

Optical density of control and test samples of pooled human lymphocytes (5 lakhs cell concentration)

The assay developed has a reduced incubation time and uses a different ELISA (Sandwich ELISA) detection strategy.[16] After validation, the lymphocyte test system can be used to detect Gram negative and Gram positive pyrogens since it measures the IL-1β release even with a small stimulation. The concentration of IL-1β and the threshold of fever induction can also be detected from the lymphocyte system. It was also observed that the difference in blood group did not interfere with the IL-1β release. Based on the results obtained, it can be concluded that the isolated lymphocytes system can be used as an alternative test system to the in vivo rabbit pyrogen assay. It is also concluded that the developed in vitro ELISA method is also an alternative to the LAL tests and is able to detect all types of pyrogens, irrespective of their chemical origin.