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. 2025 Oct 1;10(40):47544–47558. doi: 10.1021/acsomega.5c07484

Developing a Human Immune-Endothelial Coculture Model for Inflammatory Vasculitis and Drug Screening

Mi-lang Kyun †, Ji-In Kwon †,‡, Jeongha Kim †,‡, Minseong Jo †,§, Hyewon Jung †, Yu Bin Lee †,∥,*, Kyoung-Sik Moon †,∥,*
PMCID: PMC12529162  PMID: 41114267

Abstract

Vasculitis is an inflammatory condition characterized by immune cell activation and vascular endothelial dysfunction leading to severe clinical complications, including cytokine release syndrome. Despite existing treatment strategies such as corticosteroids and NSAIDs, severe cases remain difficult to manage, necessitating improved in vitro models for mechanistic studies and drug evaluation. This study established a direct coculture model of human umbilical vein endothelial cells (HUVECs) and peripheral blood mononuclear cells to investigate immune-endothelial cell interactions under lipopolysaccharide (LPS)-induced inflammatory conditions. Upon LPS stimulation, the cocultured HUVECs exhibited increased expression of inflammatory cytokines (IL-6 and IL-8) and adhesion molecules (VCAM-1 and ICAM-1), along with reduced CD31 level, indicating endothelial barrier disruption. Vascular permeability assays confirmed that the coculture model exacerbated permeability changes beyond those observed in monocultures, highlighting the synergistic inflammatory effects mediated by immune-endothelial cell interactions. Furthermore, cytokine secretion analysis demonstrated that coculture conditions significantly amplified IL-6, IL-8, and TNF-α expression, mimicking the inflammatory milieu observed in vasculitis. Treatment with tocilizumab, an IL-6 receptor antagonist, effectively suppressed cytokine release and restored endothelial integrity, highlighting the potential of this model for therapeutic screening. This study provides a physiologically relevant in vitro platform for investigating the mechanisms of drug-induced and immune-mediated vasculitis with potential applications in preclinical safety assessments of biotherapeutics.

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1. Introduction

The increasing use of biopharmaceuticals, including monoclonal antibodies and cytokine therapies, has led to a rise in immune-mediated adverse effects, including drug-induced vasculitis. However, current preclinical models lack the complexity required to accurately predict these immune responses, which limits effective drug safety assessments. Vasculitis is an inflammatory vascular disease characterized by immune-endothelial cell interactions that drive cytokine release and endothelial dysfunction. In severe cases, this can lead to cytokine release syndrome (CRS), causing multiorgan failure. Despite advances in immunotherapy and drug development, there remains a critical gap in evaluating the risk of vasculitis and associated inflammatory vascular complications induced by pharmaceutical agents.

Vasculitis pathogenesis involves complex interactions between immune and endothelial cells. Activated immune cells release pro-inflammatory cytokines such as IL-1β and TNF-α, which upregulate cell adhesion molecules including intracellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1), promoting immune cell adhesion to the endothelium. Simultaneously, tight junction proteins such as ZO-1 and endothelial adhesion molecules such as CD31 are downregulated, leading to increased vascular permeability. This increased permeability facilitates immune cell infiltration, further exacerbating inflammation within the vessel walls. Drug-induced vasculitis, a subtype of this condition, is an adverse effect of certain pharmaceuticals, particularly biopharmaceuticals, that can inadvertently activate the immune system. The resulting excessive immune activation can trigger CRS, a severe hyper-inflammatory response that leads to vascular damage and multiorgan dysfunction. Given the growing reliance on biotherapeutics, it is crucial to develop robust preclinical models that enable accurate assessment of the potential vasculitis-inducing effects of drugs and provide insights into their underlying mechanisms.

Existing in vitro models of vasculitis are primarily based on monocultures of endothelial cells. , These models assess drug-induced effects by measuring the changes in cell viability, expression of endothelial junction proteins, and alterations in permeability. Some studies have incorporated immune cells such as macrophages to evaluate the adhesion dynamics and cytokine secretion. , However, these approaches fail to capture the full spectrum of immune-endothelial cell interactions that characterize vasculitis. Current methodologies for predicting CRS and drug-induced vasculitis rely on whole-blood assays or peripheral blood mononuclear cell (PBMC)-based cytokine release tests. Although informative, these models do not sufficiently replicate the direct cellular crosstalk between immune and vascular endothelial cells, limiting their translational relevance. Therefore, there is an urgent need for physiologically relevant coculture systems that mimic immune-endothelial cell interactions and allow for a more accurate assessment of drug-induced inflammatory vascular responses.

To address this gap, we developed a direct coculture model of human umbilical vein endothelial cells (HUVECs) and peripheral blood mononuclear cells (PBMCs) using a transwell system. Unlike conventional transwell assays that physically separate cell types, our model enables direct interaction between immune and endothelial cells, thus better replicating the dynamic interplay observed in vasculitis. Inflammatory conditions were induced using lipopolysaccharide (LPS), a well-established immune stimulant, to mimic the inflammatory environment of vasculitis. , We hypothesize that this coculture model will more accurately reflect key vasculitis-related mechanisms, including the upregulation of adhesion molecules (VCAM-1and ICAM-1), increased inflammatory cytokine secretion (IL-6, IL-8, IL-17, IL-2, IFN-γ, and TNF-α), and endothelial barrier dysfunction, compared to monoculture conditions. Additionally, we investigated the potential of adhesion molecule knockdown , and immunosuppressive treatment (tocilizumab) to mitigate inflammatory responses, thereby providing insights into therapeutic strategies for controlling immune-mediated vascular damage.

Beyond drug-induced vasculitis, this model could serve as a platform for studying autoimmune vasculitis, endothelial dysfunction in sepsis, and therapeutic screening for novel immunomodulators that target vascular inflammation. By establishing a more physiologically relevant in vitro system, we aimed to bridge the gap between preclinical drug evaluation and clinical outcomes, thereby facilitating the development of safer and more effective biotherapeutics.

2. Materials and Methods

2.1. Cell Culture

Human umbilical vein endothelial cells (HUVECs) purchased from Lonza (Basel, Switzerland), at passages 4–7 or 12–14, were cultured in EGM-2 medium (CC-3156, Lonza) supplemented with EGM-2 Endothelial SingleQuots (CC-4176, Lonza), 1% penicillin–streptomycin (P/S) (15140-122, Sigma–Aldrich, St. Louis, MO, USA), and 2% fetal bovine serum (FBS) (F0900-050, Gibco, Carlsbad, CA, USA). HUVECs at passages 4–7 were used for initial experiments to ensure high viability and endothelial phenotype stability, while passages 12–14 were selected for validation experiments to confirm model robustness across extended cultures, avoiding potential variability in intermediate passages (8–11) due to senescence or dedifferentiation. PBMCs were separated by density centrifugation (Ficoll-Paque) from the blood provided by the Korean Red Cross Blood Services and cultured in RPMI 1640 medium (BR-E011, BRC, Anyang, Korea) supplemented with 10% FBS (F0900-050, Gibco) and 1% P/S (15140-122, Sigma–Aldrich). The medium was changed every 2–3 days. The cells were subcultured in 0.05% trypsin–EDTA (Cat. 25300-054; Gibco) when they reached 80–90% confluency. All cell cultures were maintained in a humidified incubator at 37 °C with 5% CO2.

2.2. Coculture of HUVECs and PBMCs

A direct coculture system was established using HUVECs and PBMCs to examine the interactions between endothelial and immune cells. HUVECs 1.0 × 105 cells/well) were seeded into a 12-well transwell insert (Cat# 3401, Sigma–Aldrich) and allowed to adhere for 24 h. Subsequently, the culture medium was replaced, and PBMCs (1.0 × 105 cells/well) were added to the lower chamber containing RPMI 1640 medium supplemented with 10% FBS and 1% P/S. To induce an inflammatory response, LPS (O111:B4, Sigma–Aldrich) was simultaneously added to the lower chamber at a final concentration of 1 μg/mL.

To visualize the adherent interactions between the HUVEC layer and PBMCs, the cells were labeled with the live-cell-tagging fluorescent dyes, Paul Karl Horan 26 (PKH26) and Paul Karl Horan 67 (PKH67). Briefly, HUVECs seeded on a 12-well transwell insert were washed with PBS and incubated for 30 min with PKH26 diluted in RPMI 1640 medium to a final concentration of 2 × 10–6 M. PBMCs were labeled for 30 min with PKH67 diluted in RPMI 1640 medium to a final concentration of 2 × 10–6 M. After labeling, 1.0 × 105 PBMCs were cocultured with the HUVEC layer for 24 h. Upon treatment with LPS according to the experimental conditions, the cells were washed with PBS 3 times to remove nonadherent PBMCs and observed by confocal microscopy (Olympus IX-51, Olympus, Tokyo, Japan).

2.3. Reverse Transcription Quantitative PCR

After washing with phosphate-buffered saline (PBS), RNA was extracted using the TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA). Cell lysates (0.5 mL) were vigorously mixed with chloroform (100 μL) and centrifuged for 15 min at 12000 rpm. The supernatants collected after centrifugation were mixed with isopropanol and centrifuged (10 min for 12,000 rpm). The collected RNA samples were washed with 75% ethanol by repeated centrifugation (10 min, 7500×g), mixed, and reconstituted in diethylpyrocarbonate-treated water. RNA was quantified using a NanoDrop spectrophotometer, and cDNA synthesis was performed. Reverse transcription quantitative PCR using SYBR Green premix (RK21219; ABclonal, Woburn, MA, USA) was performed for 45 cycles using QuantStudio5 (Thermo Fisher Scientific). Each cycle involved melting at 95 °C for 15 s, followed by annealing at 55 °C and extension at 75 °C. CT values were used for data analysis. The relative expression levels of the target genes were normalized to those of the control group using β-actin. The experiment was conducted in triplicate (n = 3). Primers (Bioneer, Daejeon, Korea) used in these experiments are listed in Supplementary Table 1.

2.4. Cell Viability Assessment via DNA Quantification and Cell Counting Kit-8 (CCK-8) Assays

To determine the effects of LPS on cell viability, DNA quantification and CCK-8 assays were performed. HUVECs were seeded into 96-well plates at a density of 2.0 × 104 cells/well and incubated for 24 h. Subsequently, cells were treated with LPS at concentrations of 10 ng/mL, 1, and 10 μg/mL. For DNA quantification using the PicoGreen dsDNA Assay Kit (Life Technologies Corp., Grand Island, NY, USA), cells were incubated for 48 h and then dissolved in RIPA lysis buffer (Thermo Fisher Scientific). The lysates were centrifuged (10 min, 15,000 rpm), and the supernatants were analyzed according to the manufacturer’s protocol.

For the CCK-8 assay, cells were incubated for 48 h (n = 3), and then the culture medium was replaced with fresh medium containing 10% CCK-8 solution (Lot. TS502, DOJINDO Laboratories, Kumamoto, Japan). The plates were incubated for 1 h at 37 °C and the absorbance was measured at 450 nm using a microplate reader (iD3, Molecular Devices, San Jose, CA, USA). Cell viability was expressed as a percentage of the untreated control.

2.5. Immunofluorescence Staining

Immunocytochemistry was performed to visualize the presence and localization of specific proteins or antigens within the cells using antibodies and fluorescent dyes. HUVECs were seeded at a density of 1.0 × 105 cells/well onto sterile coverslips placed in a 12-well culture plate. After 24 h, the same amount of PBMCs were seeded and treated with 1 μg/mL LPS. Following treatment, the culture medium was removed and the cells were washed three times with PBS. The samples were then fixed with 4% paraformaldehyde solution (235885, Thermo Fisher Scientific) at room temperature for 10 min. After fixation, the samples were washed three times with PBS and permeabilized either by treating with cold methanol for 3 min or with PBST containing 0.5% Triton X-100 (Sigma–Aldrich) at 4 °C for 10 min.

After permeabilization, the samples were blocked with PBS containing 4% bovine serum albumin (BSA) overnight at 4 °C. Following blocking, the samples were washed three times with PBS and then incubated for 24 h with primary antibodies targeting VCAM-1 (13662S, Cell Singling Technology, Danvers, MA, USA) and CD31 (3528S, Cell Singling Technology) diluted at a ratio of 1:100 in 4% BSA at 4 °C. After washing three times with PBS, secondary antibodies conjugated with fluorescent dyes, appropriate for the primary antibody species, were diluted 1:100 in 4% BSA and incubated at room temperature in the dark for 1 h.

After additional washes with PBST (three times) and PBS (once), the samples were stained with 4′,6-diamidino-2-phenylindole (DAPI) diluted at a ratio of 1:1000 in 1% PBS at room temperature for 10 min. The samples were then washed once with PBS and three times with PBST. Finally, a drop of mounting medium (TA-030-FM, Thermo Fisher Scientific) was applied to the samples and fluorescent images were captured using a confocal microscope (Olympus IX-51).

2.6. Permeability Test

Increased vascular permeability resulting from the weakening of adhesion between endothelial cells was assessed. HUVECs were seeded at a density of 1 × 105 cells/well in 12-well transwell inserts. After 24 h, the same number of PBMCs was added, and the culture medium was treated with LPS at a concentration of 1 μg/mL. Permeability assays were conducted 24 and 48 h after LPS treatment. The medium in the lower wells of the transwell plate was replaced with fresh culture medium, and the inset wells were supplemented with FITC-dextran (CAS. 60842-46-8, Sigma–Aldrich) at a concentration of 1 mg/mL, with a total volume of 500 μL. The cells were then incubated at 37 °C for 1 h. To measure the fluorescence of FITC-dextran that permeated through the intercellular spaces, 100 μL of media was collected from the lower wells after 1 h, which was transferred to a black 96-well plate (Lot. 32919025, Corning, New York, NY, USA) and fluorescence was measured at an excitation wavelength of 485 nm and an emission wavelength of 535 nm (iD3, Molecular Devices, San Jose, CA, USA).

2.7. Knockdown of VCAM-1 with siRNA

To knockdown VCAM-1, HUVECs were seeded in a 6-well culture plate at a density of 4.0 × 105 cells/well. After 24 h, the cells were treated with either 20 μM of SC siRNA (scrambled-hm2 Duplex) or VCAM-1 siRNA (sc-29519, Santa Cruz Biotechnology, Dallas TX, USA) at concentrations of 10 and 20 μM. The siRNAs were mixed with the FuGENE 4 K Transfection Reagent (Lot. 0000566815; Promega, Madison, WI, USA) in Opti-MEM (Cat. 31985070; Gibco) without antibiotics. After 6 h of transfection, the original HUVECs culture medium was added back to the wells and the cells were incubated for an additional 24 h.

2.8. Western Blotting

Western blotting was performed to confirm the knockdown of VCAM-1. Cell lysates obtained by RIPA lysis buffer were used for Western blotting. Cell lysates were subjected to SDS–PAGE to separate α-tubulin and VCAM-1 protein bands and the proteins were then transferred onto a PVDF membrane. The membrane was incubated in blocking buffer for 1 h at 37 °C, and subsequently treated with primary antibody solution overnight at 4 °C for the detection of α-tubulin (sc-5286, Santa Cruz Biotechnology) and VCAM-1 (13662S, Cell Signaling Technology). After primary antibody incubation, the sections were incubated with horseradish peroxidase-conjugated secondary antibodies (Applied Biological Materials Inc., Richmond, BC, Canada) for 1 h at room temperature (25 °C). The immunoreactive signals were detected using an enhanced chemiluminescence (ECL) detection system. The protein bands were visualized using a Western blot detection system (Thermo Fisher Scientific, iBright CL750).

2.9. Animal Experiment

The ear skin was selected as the LPS injection site due to its thin, accessible structure, which facilitates high-resolution intravital imaging of vascular changes and immune cell infiltration with minimal trauma. This site offers advantages over deeper tissues (e.g., kidney or liver) by enabling clear visualization of CD31 and F4/80 dynamics, as established in models of cutaneous inflammation. We utilized six-week-old C57BL/6 (n = 4) mice to investigate the inflammatory response in ear skin. The mice were housed in a controlled environment with a 12-h light/dark cycle and had ad libitum access to food and water. Prior to the experiment, the mice were acclimatized for 1 week. Under anesthesia (isoflurane), three punctures were made on the ear skin of each mouse, avoiding the major blood vessels to minimize trauma and bleeding. The left ear was treated with 3 μL of PBS as a control, while the right ear received 3 μL of LPS at a concentration of 50 mg/mL to induce inflammation. Following treatment, ear skin samples were collected at 0, 4, and 6 h postinjection for analysis. The expression levels of inflammatory cytokine genes, including IL-1β, IL-6, and TNF-α, were assessed using quantitative PCR. Additionally, to evaluate endothelial activation and macrophage infiltration, antibodies against CD31 (IVI-991-0003, IVIM Technology, Daejeon, Korea) and F4/80 (IVI-9991-0056, IVIM Technology; 123135, BioLegend, San Diego, CA, USA) were administered via intrapenile injections. The mice were monitored for any adverse reactions following antibody administration, and all procedures were conducted in accordance with the institutional animal care guidelines to ensure ethical treatment of the animals. Tissue samples were subsequently processed for histological analysis and immunofluorescence staining was performed to visualize the distribution of CD31 and F4/80 in the ear skin. All experimental procedures were conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee. The study protocol was approved by the institutional IACUC of the Korean Society of Toxicology (IAC-24-01-0280-0235). The procedures were designed to minimize discomfort and pain in the animals.

2.10. Statistical Analysis

The quantitative results presented in this paper are expressed as mean ± standard deviation. Statistical significance was determined using Student’s t-test and analysis of variance, with posthoc comparisons conducted using Tukey’s honest significant difference test (p < 0.05).

3. Results

3.1. Cellular Responses to LPS and Increased Expression of Inflammatory Cytokines and Adhesion Molecule Markers

To evaluate the cytotoxic response to LPS, cell viability was assessed using the CCK-8 assay and intracellular DNA content was measured using a DNA assay (Figure A–C). There were no significant changes in the viability of HUVECs or PBMCs across LPS concentrations. Similarly, there were no substantial changes in intracellular DNA content in response to varying LPS concentrations. Subsequently, the gene expression of the inflammatory cytokine IL-6 and the vascular cell adhesion molecule VCAM-1 in HUVECs was assessed following LPS treatment (Figure D). The results indicated a significant concentration-dependent upregulation of IL-6 and VCAM-1, particularly at 4 h postexposure to LPS.

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Cell viability and increased expression of inflammatory cytokines and cell adhesion molecule markers in response to LPS. (A) Assessment of HUVEC viability after treatment with different LPS concentrations. (B) Quantification of the DNA content in HUVECs treated with different LPS concentrations. (C) Evaluation of peripheral PBMCs viability at various LPS concentrations. (D) Expression levels of inflammatory cytokines and vascular cell adhesion molecules in HUVECs under monoculture conditions with different LPS concentrations. *p < 0.05; ****p < 0.0001. IL-6: interleukin-6, VCAM-1: vascular cell adhesion molecule-1.

Specifically, the fold increase in IL-6 expression levels was 1.9 ± 0.1 at 10 ng/mL, 4.3 ± 0.2 at 1 μg/mL, and 5.6 ± 0.5 at 10 μg/mL, compared to the control. For VCAM-1, the fold increase in expression levels were 6.6 ± 0.6 at 10 ng/mL, 15.6 ± 0.7 at 1 μg/mL, and 14.9 ± 0.4 at 10 μg/mL, compared to control. These results demonstrate a concentration-dependent increase in gene expression and suggests that LPS functions as a potent stimulus that induces inflammatory responses. Based on these results, a concentration of 1 μg/mL was selected as the optimal dose for inducing inflammatory responses.

3.2. Increased Expression of Inflammatory Cytokine Genes in HUVECS in the Presence of PBMCs and LPS

The LPS-induced inflammatory response was investigated in four experimental groups: HUVEC (monocultured HUVECs), HUVEC+LPS (HUVECs treated with LPS), Co_HUVEC (coculture of HUVECs and PBMCs), and Co_HUVEC+LPS (coculture of HUVECs and PBMCs followed by LPS treatment) (Figure A). Monocultured HUVECs exhibited a characteristic cobblestone-like morphology, which was maintained in both the HUVEC+LPS and Co_HUVEC groups. In contrast, the Co_HUVEC+LPS group exhibited a sharp and elongated morphology, indicating morphological changes in response to the inflammatory environment present under these conditions (Figure B). To evaluate the inflammatory response, gene expression levels of the inflammatory cytokines IL-6 and IL-8 were analyzed. The results showed that the fold increase of IL-6 expression level in the HUVEC+LPS group was 11.8 ± 3.1, whereas it was significantly elevated in the Co_HUVEC+LPS group to 76.5 ± 23.0. For IL-8 expression, fold increases in expression level in the HUVEC+LPS and Co_HUVEC+LPS groups were 161.5 ± 43.7 and 504.2 ± 80.6, respectively (Figure C). These findings suggest that the inflammatory response is significantly amplified in a coculture environment compared to monoculture conditions upon exposure to inflammatory stimuli, emphasizing the critical role of immune cells in modulating inflammation.

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Increased expression of inflammatory cytokine genes in the presence of PBMC and LPS. (A) Schematic representation of the experimental groups: HUVEC (monocultured HUVECs), HUVEC+LPS (HUVECs treated with LPS), Co_HUVEC (coculture of HUVECs and PBMCs), and Co_HUVEC+LPS (coculture of HUVECs and PBMCs followed by LPS treatment). (B) Phase-contrast images showing changes in cell morphology of HUVECs and cocultured PBMCs after LPS treatment (scale bar = 200 μm). (C) mRNA expression levels of the inflammatory cytokines, IL-6 and IL-8, released under coculture conditions after LPS treatment. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. IL-6: interleukin-6, IL-8: interleukin-8.

3.3. Protein Levels of LPS-Induced Inflammatory Cytokines in Coculture of HUVECs and PBMCs

The protein levels of inflammatory cytokines were analyzed in six experimental groups: HUVEC, HUVEC+LPS, Co_HUVEC, Co_HUVEC+LPS, PBMC, and PBMC+LPS (Figure A). Examination of representative cytokines associated with CRS revealed a significant increase in cytokine protein expression at 24 h compared to 4 h across all groups. In the case of IL-2, the amount of protein in the HUVEC+LPS and PBMC+LPS groups was 4.7 ± 0.2 and 3.4 ± 0.1 pg/mL, respectively. In the Co-HUVEC+LPS group, the IL-2 protein level was markedly increased to 11.6 ± 0.1 pg/mL, representing an approximately 2–3-fold increase. For IFN-γ, protein secretion levels in the HUVEC+LPS and PBMC+LPS groups were approximately 1.5 ± 0.1 and 1.2 ± 0.1 pg/mL, respectively, whereas in the Co-HUVEC+LPS group it was significantly higher, amounting to 3.9 ± 0.1 pg/mL. Similarly, for IL-17 expression levels in HUVEC+LPS and PBMC+LPS groups were 2.4 ± 0.1 and 1.6 ± 0.1 pg/mL, respectively, whereas in Co_HUVEC+LPS group it amounted to 6.7 ± 0.2 pg/mL. The IL-2, IFN-γ, and IL-17 were found to be secreted to some extent by both HUVECs and PBMCs. Additionally, in the Co_HUVEC+LPS group, the secretion levels were slightly higher than the combined levels obtained in the respective monocultured groups.

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Protein levels of LPS-induced inflammatory cytokines in coculture of HUVECs and PBMCs. (A) Schematic representation of the six experimental groups used for the protein secretion analysis. (B) Protein levels of LPS-induced inflammatory cytokines under various conditions. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. IL-6: interleukin-6, IL-8: interleukin-8, IL-2: interleukin-2, IL-17: interleukin-17, IFN-γ: interferon gamma, TNF-α: tumor necrosis factor-alpha.

Cytokines such as IL-6 and IL-8 play a crucial role in the onset and progression of vasculitis by activating endothelial cells and inducing inflammatory responses. IL-6 secretion levels in the HUVEC+LPS and PBMC+LPS groups amounted to 118.1 ± 2.6 and 219.9 ± 7.5 pg/mL, respectively. Notably, the Co_HUVEC+LPS group exhibited a dramatic elevation in secretion, surging to 5614.3 ± 208.0 pg/mL. Similarly, for IL-8, the cytokine expression in HUVEC+LPS and PBMC+LPS groups were 913.1 ± 24.9 and 576.8 ± 9.6 pg/mL, respectively. However, in the Co_HUVEC+LPS group, secretion level showed a striking increase, reaching 7716.8 ± 365.7 pg/mL. The markedly higher expression levels of IL-6 and IL-8, which are considered critical markers in the onset and progression of vasculitis, observed in the Co_HUVEC+LPS group compared to the monoculture groups suggest that employing coculture conditions can better simulate a disease-relevant inflammatory environment when studying vasculitis by enhancing inflammatory signaling pathways. TNF-α protein expression levels in the HUVEC+LPS and PBMC+LPS groups were 11.5 ± 0.5 and 66.7 ± 0.3 pg/mL, respectively, whereas in the Co-HUVEC+LPS group the level amounted to 82.3 ± 3.9 pg/mL. These results suggest that PBMCs demonstrate a greater responsiveness to LPS than HUVECs in terms of TNF-α secretion (Figure B). The significant increase in cytokine secretion associated with CRS observed in the coculture environment following LPS stimulation compared to that in monoculture conditions indicates that interactions between immune and endothelial cells significantly amplify the inflammatory response. PBMCs predominantly drive cytokine production, with HUVECs facilitating amplification through direct interactions. These findings suggest that cellular interactions play a critical role in driving inflammation within the vasculature.

3.4. Endothelial Junction Damage Induced by LPS in the Coculture Model of HUVECs and PBMCs

We investigated the damage to the endothelial junctions induced by LPS treatment. CD31, a cell adhesion molecule expressed at endothelial junctions and responsible for connecting adjacent endothelial cells, was examined by immunostaining. In the control group, CD31 expression was prominently observed, indicating the presence of intact endothelial junctions. However, in the HUVEC+LPS group, in which an inflammatory response was induced by LPS treatment, a noticeable reduction in CD31 expression was detected, suggesting that endothelial cell damage was caused by LPS. Similarly, in the coculture of PBMCs and HUVECs, a decrease in CD31 expression was observed following LPS treatment. This reduction was significantly more pronounced than in the monoculture group, with CD31 expression becoming nearly undetectable (Figure A). These findings suggested that the interaction between HUVECs and PBMCs amplified the effects of LPS, leading to a more severe reduction in CD31 expression. Under inflammatory conditions, activated endothelial cells undergo structural changes that destabilize the intercellular junctions. These alterations weaken the adhesion between endothelial cells, significantly contributing to increased vascular permeability.

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Endothelial junction damage induced by LPS in the coculture model of HUVECs and PBMCs. (A) Immunofluorescence staining images of the endothelial junction molecule CD31 (red) and nuclei (DAPI, blue) (scale bar = 50 μm). (B) LPS-induced changes in permeability were assessed under various conditions using fluorescein isothiocyanate-dextran. **p < 0.01; ****p < 0.0001. CD31­(PECAM-1): platelet endothelial cell adhesion molecule-1.

In the permeability assay, LPS treatment of HUVECs resulted in an increase in permeability to 110.5 ± 0.1 compared to the control group (set at 100). This increase was significantly elevated to 137.4 ± 3.2 following LPS addition to cocultures of HUVECS and PBMCs. After 48 h, LPS treatment also increased permeability in HUVECs to 120.3 ± 3.1-fold compared to the control group, with significantly greater increase to 166.4 ± 2.4 observed in the Co_HUVEC+LPS group (Figure B). These results confirm that LPS weakens the adhesive properties of endothelial cells, creating gaps between weakened cells, thereby increasing permeability. Notably, the interaction between immune and vascular cells in the coculture model significantly amplified the LPS-induced increase in cell permeability. Moreover, a time-dependent increase in permeability was observed, with the values at 48 h being notably higher than those at 24 h.

3.5. Adhesion Capacity of Activated Immune Cells in Coculture Model of HUVECs and PBMCs in the Presence or Absence of LPS

To investigate whether the hyper-inflammatory response in the LPS-induced coculture model was associated with increased interactions between endothelial and immune cells, live cells were tagged with the fluorescent dyes PKH26 and PKH67. Fluorescent dye staining was performed prior to cell seeding. The expression of adhesion molecules was not activated in the Co_HUVEC group, and there was minimal contact between PKH67-tagged PBMCs and HUVECs, which appeared to be separated. In contrast, in the Co_HUVEC+LPS group, LPS stimulation activated adhesion molecules, allowing PBMCs to adhere to HUVECs. This was confirmed at 6-h post coculture; after 24 h, a greater number of aggregated PBMCs adhered to endothelial cells (Figure A). Furthermore, after washing to remove medium containing the suspended PBMCs, a higher number of PBMCs remained attached to the endothelial cells in the Co_HUVEC+LPS group than in the Co_HUVEC group (arrows, Figure B). The observation that PBMC remained attached despite media washout indicated that the activation of adhesion molecules facilitated the adhesion of these suspended immune cells to HUVECs. Consequently, even after removing the media in which the PBMCs were cultured, the immune cells still adhered to the endothelial cells. Quantitative analysis using fluorescence measurements further confirmed that the number of adherent cells was significantly higher in the Co_HUVEC+LPS group than in the Co_HUVEC group (Figure C).

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Adhesion capacity of activated immune cells in the coculture model of HUVECs and PBMCs in the presence or absence of LPS. (A) Live cell tagging imaging using the fluorescent dyes PKH26 and PKH67 to assess the effects of LPS on PBMCs adherence to HUVECs. Scale bars in the images represent 25 μm (6 h, 80× magnification), 50 μm (24 h, 40× magnification). (B) Immunofluorescence staining images of adherent PBMCs after medium washout (scale bar = 50 μm). (C) Quantification of the fluorescence intensity of adherent immune cells in the HUVEC layer. ****p < 0.0001. PKH26: Paul Karl Horan 26, PKH67: Paul Karl Horan 67, CD31­(PECAM-1): platelet endothelial cell adhesion molecule-1, HO-1: heme oxygenase-1.

To investigate whether inflammatory cytokine levels were elevated through direct contact between PBMCs and HUVECs, we conducted both indirect and direct coculture experiments using transwell systems (Supplementary Figure 1A). The same number of cells and LPS concentration were maintained under both experimental conditions. Notably, in the direct coculture setting, the mRNA expression levels of IL-1β, IL-6, and IL-8 were significantly higher compared to the indirect coculture without direct contact between PBMCs and HUVECs (Supplementary Figure 1B). These findings confirm that, even when triggered by the same LPS stimulus, immune cells can enhance inflammatory responses through direct interactions with vascular endothelial cells.

These results demonstrate that LPS stimulation leads to an increased expression of cell adhesion molecules on endothelial cells, thereby promoting the adhesion of immune cells to endothelial cells.

3.6. Knockdown Response of Adhesion Factors

Building on previous findings that TLR4, a receptor for LPS, is expressed in vascular endothelial cells and that LPS enhances inflammatory cytokine production via NF-kB and AP-1-dependent pathways, we hypothesized that LPS would increase PBMC adhesion to HUVECs by upregulating VCAM expression. To test this, we knocked down VCAM to evaluate its effect on PBMC adhesion and predicted that reduced VCAM levels would lead to decreased PBMC attachment and, consequently, reduced inflammatory cytokine release in the coculture model. We confirmed that the mRNA expression levels of the adhesion molecules VCAM-1 and ICAM-1 were elevated in the Co_HUVEC+LPS group (Figure A). This was further validated by immunocytochemistry, which demonstrated increased VCAM-1 protein expression (Figure B). Based on these findings, we identified VCAM-1 as a key adhesion molecule and performed its knockdown to examine whether the previously elevated cytokine expression would decrease. VCAM-1 knockdown was successfully achieved in endothelial cells at both 24 and 48 h (Figure C). However, in all culture conditions of Co_HUVEC and Co_HUVEC+LPS, VCAM-1 knockdown resulted in an unexpected increase in the mRNA expression levels of major inflammatory cytokines, as well as other adhesion molecules, including E-selectin and ICAM-1 (Figure D). These findings suggest that targeting VCAM-1 alone may not effectively regulate inflammatory responses.

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Knockdown response of adhesion factors. (A) mRNA expression levels of the adhesion factors VCAM-1 and ICAM-1. (B) Immunofluorescence staining of VCAM-1, an LPS-induced adhesion factor, in cocultures of HUVECs and PBMCs. (scale bar = 50 μm). (C) VCAM-1 knockdown in HUVECs at 24 and 48 h. (D) VCAM-1 knockdown in cocultured HUVECs and PBMCs following LPS treatment. *p < 0.05; **p < 0.01; ***p < 0.001; p < 0.0001. VCAM-1: vascular cell adhesion molecule-1, ICAM-1: intercellular adhesion molecule-1, IL-1β: interleukin-1 beta, IFN-γ: interferon gamma, E-selectin: endothelial leukocyte adhesion molecule-1.

3.7. Response of the HUVEC and PBMC Coculture Model to the Immunosuppressant Tocilizumab

To address the inability to verify the knockdown effect observed previously, two experimental hypotheses were proposed: (1) whether simultaneous knockdown of additional adhesion molecules alongside VCAM-1 could inhibit the elevated cytokine levels and binding responses between immune and endothelial cells, and (2) whether the immune response associated with the elevated cytokines could be suppressed by treatment with immunosuppressants. Since the knockdown of additional adhesion molecules was expected to cause unforeseen damage to HUVECs, we focused on validating our second hypothesis. Tocilizumab, a monoclonal antibody that binds to the IL-6 receptor and prevents IL-6 signaling, was administered at a concentration of 5 μg/mL to mitigate the vasculitis-like inflammatory responses in the Co_HUVEC+LPS group. Figure A showed that the fold increase in IL-6 gene expression was 59.6 ± 9.3 in the Co_HUVEC+LPS group, which was reduced significantly to 14.3 ± 0.2 following tocilizumab treatment. Similarly, the fold increase in IL-8 gene expression was 143.6 ± 18.2 in the Co_HUVEC+LPS group, and was decreased to 58.4 ± 13.3 following tocilizumab treatment. Additionally, the fold increase in MCP-1 gene expression rose to 45.8 ± 2.1 in the Co_HUVEC+LPS group but was reduced to 33.4 ± 3.1 following tocilizumab treatment. These findings confirmed the effectiveness of tocilizumab in suppressing inflammatory responses. MCP-1 decrease may involve indirect pathway inhibition beyond IL-6 signaling. We also report that the permeability increased to 187 ± 8.6 in the Co_HUVEC+LPS group, compared to the control group (set at 100), but decreased to 133 ± 8.8 following tocilizumab treatment (Figure B). Direct IL-6 inhibition likely drives CD31 restoration, as supported by temporal correlation and prior studies. These findings indicate that the immunosuppressive effects of tocilizumab reduce the intercellular disruptions caused by hyperimmune responses, thereby significantly decreasing vascular permeability.

7.

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Response of the immunosuppressant tocilizumab in the coculture model of HUVECs and PBMCs. (A) Expression of inflammatory cytokines (IL-6, IL-8, and MCP-1) after tocilizumab treatment. (B) Immunofluorescence staining images of the endothelial junction molecule CD31 (red) and nuclei (DAPI, blue) after treatment with tocilizumab. (scale bar = 50 μm). (C) Changes in cell permeability after tocilizumab treatment. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. (D) Immunofluorescence staining of VCAM-1 in cocultures of HUVECs and PBMCs treated with LPS and tocilizumab. (scale bar = 50 μm). IL-6, interleukin-6, IL-8: interleukin-8, MCP-1: monocyte chemoattractant protein-1, CD31­(PECAM-1): platelet endothelial cell adhesion molecule-1. VCAM-1: vascular cell adhesion molecule-1, Tocili, tocilizumab.

3.8. In Vivo Inflammatory Response and Vascular Remolding Induced by LPS

In our in vitro experiments, we confirmed an increase in inflammatory cytokines induced by LPS, with significantly elevated levels of cytokine gene expression and protein production observed in cocultures with peripheral PBMCs. Additionally, we observed a reduction in the expression of CD31, which correlated with increased vascular permeability and adhesion of immune cells to endothelial cells. To investigate whether the immune responses observed ex vivo can be replicated in vivo, we conducted experiments in mice (Figure A). The results showed significant inflammatory cytokine responses in the ear skin of LPS-treated mice at the 4-h mark (Figure B). Specifically, LPS treatment increases the mRNA levels for various cytokines. The fold increase in IL-6 expression was 1.2 ± 0.1 in the PBS group compared with 3.4 ± 0.8 in the LPS group. Similarly, the fold increase in expression of IL-8, IL-1β, CCL-2, and TNF-α went from 0.6 ± 0.1, 1.3 ± 0.2, 1.5 ± 0.2, and 1.4 ± 0.2, respectively, in the PBS group, to 3.3 ± 0.5, 3.1 ± 1.0, 4.7 ± 0.4, 2.2 ± 0.1, respectively, in the LPS group. These results indicated a significant increase in the mRNA levels of inflammatory cytokines, consistent with our in vitro findings, suggesting that LPS exerts a potent inflammatory effect in vivo. Subsequently, we administered PBS and LPS to the mouse ear skin and injected antibodies to CD31 and F4/80 via intrapenile injection (Figure C). Using CD31, an endothelial cell adhesion molecule, we assessed the vascular morphology in vivo, whereas F4/80, a specific macrophage marker, was used to observe macrophage migration. These observations mirrored the immune cell–endothelial cell adhesion responses observed in our in vitro experiments, validating the mechanism by which LPS induces inflammation and activates immune cells in vivo. One hour post-treatment images, captured using intravital confocal microscopy revealed that in PBS-treated mouse ear skin, the endothelial cells were distinctly organized and displayed clear vascular structures through CD31 staining. In contrast, in the LPS-treated mouse ear skin, F4/80 staining indicated that macrophages had migrated and adhered to blood vessels. After 4 h, mosaic imaging of the mouse ear skin showed that LPS treatment led to a significantly higher number of blood vessels than PBS treatment, with these vessels exhibiting irregular branching patterns. Imaging suggests early adhesion (1 h) evolving to migration/infiltration (4 h), reflecting activation progression. Overall, our findings demonstrate that LPS induces an inflammatory cytokine response, facilitates macrophage adhesion around blood vessels, and promotes abnormal angiogenesis in vivo. This study highlights the effect of LPS on inflammatory responses and vascular remodeling in biological systems.

8.

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Immune response assessment in mouse ear skin following LPS induction. (A) Schematic representation of the experimental design and analytical methods used to evaluate the immune response in mouse ear skin after LPS treatment. (B) mRNA expression levels of inflammatory cytokines in mouse ear skin following LPS induction, demonstrating the extent of the inflammatory response. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. (C) In vivo imaging of CD31 and F4/80 expression in mouse ear skin after LPS treatment, illustrating the adhesion of immune cells and associated vascular changes.

4. Discussion

In this study, we established a nonclinical evaluation model to investigate the potential induction of vasculitis owing to immunotoxicity stemming from pharmaceuticals or infections, with a particular focus on the effects of LPS derived from Escherichia coli. Vasculitis, a condition characterized by the inflammation of blood vessels, can have severe consequences, including ischemic tissue damage, which may lead to major health complications such as stroke and heart attack. Our model aimed to elucidate the mechanisms underlying immunotoxicity and associated vascular changes. By allowing direct cell–cell interactions, this model captures synergistic effects, such as amplified cytokine release and adhesion molecule upregulation, which are underestimated in indirect transwell systems (Supplementary Figure 1).

The difference in cytokine expression between the 1 and 10 μg/mL concentrations was minimal, while the 10 μg/mL concentration has been reported in previous studies to have cytotoxic effects. , Therefore, in this experiment, we selected the 1 μg/mL concentration as the appropriate inflammatory-inducing concentration for LPS treatment. Under normal culture conditions, HUVECs typically exhibit a rounded morphology, which is maintained when treated with LPS alone or when cocultured with PBMCs. However, in the Co_HUVEC+LPS group, the cells presented elongated, pointed, and disorganized morphologies, indicating damage to and activation of HUVECs in the presence of PBMCs and LPS. Such alterations may disrupt tight junctions and increase permeability, indicating a decline in the original function of HUVECs.

Among the experimental groups, the Co_HUVEC+LPS group showed the highest levels of cytokine secretion, suggesting that the interaction between HUVECs and PBMCs plays a crucial role in CRS. IL-2 plays a crucial role in immune responses by activating CD4+ and CD8+ T cells. IFN-γ is important for the activation of NK cells, T cells, and B cells, and the interaction with PBMCs can be interpreted as enhancing the expression of this cytokine. − IL-17 is associated with the activation of TH-17 T cells and plays a significant role in inflammatory diseases. − These three markers were secreted by both HUVECs and PBMCs, and coculture resulted in significantly higher secretion levels than the monocultured groups, confirming that the interaction between immune cells and endothelial cells contributes to the amplification of inflammatory responses. This amplification is driven by direct interactions, where PBMC-derived cytokines (e.g., TNF-α, IL-6) trigger HUVEC expression of adhesion molecules (VCAM-1, ICAM-1), fostering a feedback loop that enhances inflammation and vascular permeability (Figures –). IL-6 and IL-8, known as key markers of vasculitis, , play crucial roles in the onset and progression of the disease by activating vascular endothelial cells to induce inflammatory responses. Notably, these cytokines exhibited markedly increased expression in the Co_HUVEC+LPS group compared to the other groups, highlighting the distinct synergistic effects of the interactions between the two cell types on their secretions.

During the inflammatory response induced by LPS, activated endothelial cells undergo structural changes due to instability of intercellular junctions. These changes weaken the adhesion between endothelial cells, which is a major contributor to the increased vascular permeability. This cotreatment resulted in increased inflammatory cytokine expression and an endothelial-to-mesenchymal transition (EndoMT)-like phenotype in HUVECs. These changes suggest a transient EndoMT-like state contributing to permeability, rather than irreversible transition. EndoMT is characterized by the transition of endothelial cells into mesenchymal cells, which reduces intercellular adhesion and compromises vascular integrity, potentially leading to increased permeability and altered vascular function. This highlights the influence of hyperinflammation on vascular health, suggesting that the acquisition of an EndoMT-like phenotype may decrease vascular function.

The permeability assay confirmed that vascular permeability in the HUVEC+LPS group was significantly higher than that in the HUVEC control group. This finding is consistent with previous studies and supports the negative effects of LPS on endothelial cell function. , We also observed the most significant increase in permeability in the Co_HUVEC+LPS group, especially in the measurements taken 48 h post-treatment compared to those at 24 h. This suggests that coculture with PBMCs contributes to an increase in LPS-induced vascular permeability.

Previous studies have shown that the expression of adhesion molecules such as VCAM-1 and ICAM-1 increases in response to inflammation. This upregulation enhances intercellular adhesion and promotes cellular internalization. Although we achieved a strong knockdown of VCAM-1 under all culture conditions, we found that the mRNA expression of major inflammatory cytokines and other adhesion factors increased. This suggests a compensatory mechanism where inhibition of VCAM-1 induces upregulation of alternative adhesion molecules like ICAM-1 and E-selectin, potentially driven by NF-κB activation or increased cytokine release (e.g., IL-1β, IFN-γ) from unbound PBMCs, indicating that multitarget inhibition may be necessary to suppress inflammation effectively. Additionally, these results suggest that the interaction between HUVECs and PBMCs in an inflammatory environment is driven by the secretion of cytokines and chemokines as well as paracrine effects. However, as numerous reports have indicated that immune cell adhesion to the endothelium also plays a significant role, further studies are needed to validate this hypothesis.

Tocilizumab is a humanized monoclonal antibody that targets the IL-6 receptor, blocking the inflammatory effects of IL-6, thereby suppressing the immune response. We confirmed that the expression levels of IL-6 and IL-8, key cytokines in vasculitis, and MCP-1, an important cytokine in the inflammatory process, decreased following tocilizumab treatment. Furthermore, tocilizumab treatment reduced vascular permeability.

The administration of LPS to the ear significantly increased the expression of inflammatory cytokine genes and enhanced the adhesion of immune cells to the vascular endothelium, possibly mediated by ICAM-1 and VCAM-1, consistent with previously established phenomena associated with inflammatory environments. Our findings suggest that the mechanisms observed in our animal model can provide insights applicable not only to murine systems, but also to human pathophysiology. Though not target-specific, the model evaluates systemic effects; future enhancements could incorporate CRISPR for enzyme/protein targeting to refine screening. This platform could also be adapted for autoimmune vasculitis studies, e.g., by incorporating antineutrophil cytoplasmic autoantibodies (ANCA). A detailed examination of vascular permeability and immune cell interactions in animal models and human tissues could lead to a better understanding of the processes underlying conditions such as sepsis, autoimmune diseases, and chronic inflammatory disorders.

Despite the findings of the coculture model, our study has several limitations. We confirmed immune cell attachment to HUVECs upon LPS treatment, but lack a detailed analysis of the specific immune cell types involved in this interaction. In addition, although we employed VCAM knockdown to inhibit immune cell adhesion, compensatory increases in ICAM and E-selectin expression complicated this approach, indicating the need for a more comprehensive understanding of the dynamics of adhesion factors. Finally, in our mouse model, further investigations are necessary to assess immune cell leakage due to increased vascular permeability, and to analyze changes in blood vessel morphology over various time points.

5. Conclusions

This study provides important insights into the immunotoxicity-induced vasculitis, particularly in the context of pharmaceutical compounds and specific viral infections. Our nonclinical evaluation method enhances the potential for advanced safety assessments of immunotoxicity, addressing a critical gap in current biopharmaceutical drug development practices. In pipelines, it bridges in vitro and in vivo testing for safer biotherapeutics. Future research should focus on testing our model against drugs known to cause vascular toxicity, such as tyrosine kinase inhibitors, to further validate its applicability and relevance in assessing immunotoxicity and vascular health.

Supplementary Material

ao5c07484_si_001.pdf (168.1KB, pdf)

Acknowledgments

This study was supported by the Korea Institute of Toxicology, Republic of Korea (Grant No. 2710086920), Korea Health Industry Development Institute (RS-2025-25454991), and the Ministry of SMEs and Startups (MSS) under the Scale-up TIPS Program (RS-2024-00509945).

The data sets supporting the conclusions of this article are fully available within the manuscript and the Supporting Information files.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.5c07484.

  • Differences in inflammatory cytokine expression between indirect and direct coculture conditions; and primer sequences used for PCR in this study (PDF)

M.l.K. and J.I.K. contributed equally to this study as corresponding authors. Y.B.L. and K.-S.M. contributed equally to this study as the first authors. M.l.K. and J.I.K.: Data curation, writingoriginal draft, investigation, methodology, formal analysis. J.K., M.J., and H.J.: Methodology and formal analysis. Y.B.L.: Supervision, data curation, investigation, writingreview and editing. K.S.M.: Supervision, funding acquisition, project administration. All the authors have read and approved the manuscript. M.l.K. and J.I.K. contributed equally to this study as the first authors. Y.B.L. and K.S.M. contributed equally to this study as corresponding authors.

This study was conducted in accordance with the principles of the ‘3Rs’ (Replacement, Reduction, Refinement) and approved by the Institutional Animal Care and Use Committee (IACUC) of the Korea Institute of Toxicology (IAC-24-01-0280-0235). All procedures were designed to minimize pain and distress in the animals, including the use of appropriate anesthesia and analgesia where necessary. The study was conducted with utmost consideration for animal welfare.

The authors declare no competing financial interest.

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Associated Data

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Supplementary Materials

ao5c07484_si_001.pdf (168.1KB, pdf)

Data Availability Statement

The data sets supporting the conclusions of this article are fully available within the manuscript and the Supporting Information files.

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