Enhanced CD95 and interleukin 18 signalling accompany T cell receptor Vß21.3+ activation in multi-inflammatory syndrome in children.

Multisystem inflammatory syndrome in children is a post-infectious presentation SARS-CoV-2 associated with expansion of the T cell receptor Vß21.3+ T-cell subgroup. Here we apply muti-single cell omics to compare the inflammatory process in children with acute respiratory COVID-19 and those presenting with non SARS-CoV-2 infections in children. Here we show that in Multi-Inflammatory Syndrome in Children (MIS-C), the natural killer cell and monocyte population demonstrate heightened CD95 (Fas) and Interleuking 18 receptor expression. Additionally, TCR Vß21.3+ CD4+ T-cells exhibit skewed differentiation towards T helper 1, 17 and regulatory T cells, with increased expression of the co-stimulation receptors ICOS, CD28 and interleukin 18 receptor. We observe no functional evidence for NLRP3 inflammasome pathway overactivation, though MIS-C monocytes show elevated active caspase 8. This, coupled with raised IL18 mRNA expression in CD16- NK cells on single cell RNA sequencing analysis, suggests interleukin 18 and CD95 signalling may trigger activation of TCR Vß21.3+ T-cells in MIS-C, driven by increased IL-18 production from activated monocytes and CD16- Natural Killer cells.

Allergens induce upregulated IL-18 and IL-18Rα expression in blood Th2 and Th17 cells of patients with allergic asthma.

Allergic asthma (AA) is closely associated with the polarization of T helper (Th)2 and Th17 cells. Interleukin (IL)-18 acts as an inducer of Th2 and Th17 cell responses. However, expressions of IL-18 and IL-18 receptor alpha (IL-18Rα) in blood Th2 and Th17 cells of patients with AA remain unclear. We therefore investigated their expressions in Th2 and Th17 cells using flow cytometric analysis, quantitative real-time PCR (qPCR), and murine AA model. We observed increased proportions of Th2, Th17, IL-18+, IL-18+ Th2, and IL-18+ Th17 cells in blood CD4+ T cells of patients with AA. Additionally, house dust mite seemed to upregulate further IL-18 expression in Th2 and Th17, and upregulate IL-18Rα expression in CD4+ T, Th2, and Th17 cells of AA patients. It was also found that the plasma levels of IL-4, IL-17A, and IL-18 in AA patients were elevated, and they were correlated between each other. In ovalbumin (OVA)-induced asthma mouse (AM), we observed that the percentages of blood CD4+ T, Th2, and Th17 cells were increased. Moreover, OVA-induced AM expressed higher level of IL-18Rα in blood Th2 cells, which was downregulated by IL-18. Increased IL-18Rα expression was also observed in blood Th2 cells of OVA-induced FcεRIα-/- mice. Collectively, our findings suggest the involvement of Th2 cells in AA by expressing excessive IL-18 and IL-18Rα in response to allergen, and that IL-18 and IL-18Rα expressing Th2 cells are likely to be the potential targets for AA therapy.

A novel anti-human IL-1R7 antibody reduces IL-18-mediated inflammatory signaling.

Unchecked inflammation can result in severe diseases with high mortality, such as macrophage activation syndrome (MAS). MAS and associated cytokine storms have been observed in COVID-19 patients exhibiting systemic hyperinflammation. Interleukin-18 (IL-18), a proinflammatory cytokine belonging to the IL-1 family, is elevated in both MAS and COVID-19 patients, and its level is known to correlate with the severity of COVID-19 symptoms. IL-18 binds its specific receptor IL-1 receptor 5 (IL-1R5, also known as IL-18 receptor alpha chain), leading to the recruitment of the coreceptor, IL-1 receptor 7 (IL-1R7, also known as IL-18 receptor beta chain). This heterotrimeric complex then initiates downstream signaling, resulting in systemic and local inflammation. Here, we developed a novel humanized monoclonal anti-IL-1R7 antibody to specifically block the activity of IL-18 and its inflammatory signaling. We characterized the function of this antibody in human cell lines, in freshly obtained peripheral blood mononuclear cells (PBMCs) and in human whole blood cultures. We found that the anti-IL-1R7 antibody significantly suppressed IL-18-mediated NFκB activation, reduced IL-18-stimulated IFNγ and IL-6 production in human cell lines, and reduced IL-18-induced IFNγ, IL-6, and TNFα production in PBMCs. Moreover, the anti-IL-1R7 antibody significantly inhibited LPS- and Candida albicans-induced IFNγ production in PBMCs, as well as LPS-induced IFNγ production in whole blood cultures. Our data suggest that blocking IL-1R7 could represent a potential therapeutic strategy to specifically modulate IL-18 signaling and may warrant further investigation into its clinical potential for treating IL-18-mediated diseases, including MAS and COVID-19.

Elevated frequency of IL-37- and IL-18Rα-positive T cells in the peripheral blood of rheumatoid arthritis patients.

OBJECTIVE: To detect the expression of IL-37 and its receptors IL-18Rα and IL-1R8 in CD4+ T cells and total lymphocytes in rheumatoid arthritis (RA) patients and the relationship between autoantibodies and disease activity. To investigate the mechanism of IL-37 and its receptors involved in the pathogenesis of RA. To evaluate the effects of different concentrations of rhIL-37 on peripheral blood mononuclear cells (PBMCs) in RA patients with TNF-α, and IL-6. METHODS: The expression of IL-37 and its receptor IL-18Rα and IL-1R8 in peripheral blood CD4+ T cells and total lymphocytes in RA patients and healthy controls were measured by flow cytometry. The levels of TNF-α and IL-6 in the supernatant were measured by ELISA after rhIL-37 stimulation with PBMCs. RESULTS: The expression of IL-37 and IL-18Rα in the total lymphocytes, especially in CD4+ T cells in RA patients, was significantly higher than in the healthy control group. There was a positive correlation between the frequency of IL-37- or IL-18Rα-positive CD4+ T cells and ESR, CRP, and DAS28 values. Additionally, rhIL-37 significantly down-regulated TNF-α and IL-6 production in RA patients' PBMCs. CONCLUSIONS: IL-37 plays an important role in the regulation of inflammation in RA. IL-37 and its receptors may play an immunoregulatory role in the activation of lymphocytes, especially CD4+ T cells, in RA patients. IL-37 may represent a therapeutic target for rheumatoid arthritis.

Adenoviral vaccine induction of CD8+ T cell memory inflation: Impact of co-infection and infection order.

The efficacies of many new T cell vaccines rely on generating large populations of long-lived pathogen-specific effector memory CD8 T cells. However, it is now increasingly recognized that prior infection history impacts on the host immune response. Additionally, the order in which these infections are acquired could have a major effect. Exploiting the ability to generate large sustained effector memory (i.e. inflationary) T cell populations from murine cytomegalovirus (MCMV) and human Adenovirus-subtype (AdHu5) 5-beta-galactosidase (Ad-lacZ) vector, the impact of new infections on pre-existing memory and the capacity of the host's memory compartment to accommodate multiple inflationary populations from unrelated pathogens was investigated in a murine model. Simultaneous and sequential infections, first with MCMV followed by Ad-lacZ, generated inflationary populations towards both viruses with similar kinetics and magnitude to mono-infected groups. However, in Ad-lacZ immune mice, subsequent acute MCMV infection led to a rapid decline of the pre-existing Ad-LacZ-specific inflating population, associated with bystander activation of Fas-dependent apoptotic pathways. However, responses were maintained long-term and boosting with Ad-lacZ led to rapid re-expansion of the inflating population. These data indicate firstly that multiple specificities of inflating memory cells can be acquired at different times and stably co-exist. Some acute infections may also deplete pre-existing memory populations, thus revealing the importance of the order of infection acquisition. Importantly, immunization with an AdHu5 vector did not alter the size of the pre-existing memory. These phenomena are relevant to the development of adenoviral vectors as novel vaccination strategies for diverse infections and cancers. (241 words).

T cell expression of IL-18R and DR3 is essential for non-cognate stimulation of Th1 cells and optimal clearance of intracellular bacteria.

Th1 cells can be activated by TCR-independent stimuli, but the importance of this pathway in vivo and the precise mechanisms involved require further investigation. Here, we used a simple model of non-cognate Th1 cell stimulation in Salmonella-infected mice to examine these issues. CD4 Th1 cell expression of both IL-18R and DR3 was required for optimal IFN-γ induction in response to non-cognate stimulation, while IL-15R expression was dispensable. Interestingly, effector Th1 cells generated by immunization rather than live infection had lower non-cognate activity despite comparable IL-18R and DR3 expression. Mice lacking T cell intrinsic expression of MyD88, an important adapter molecule in non-cognate T cell stimulation, exhibited higher bacterial burdens upon infection with Salmonella, Chlamydia or Brucella, suggesting that non-cognate Th1 stimulation is a critical element of efficient bacterial clearance. Thus, IL-18R and DR3 are critical players in non-cognate stimulation of Th1 cells and this response plays an important role in protection against intracellular bacteria.

Increased adipose tissue expression of IL-18R and its ligand IL-18 associates with inflammation and insulin resistance in obesity.

INTRODUCTION: The proinflammatory cytokine IL-18 is involved in the pathogenesis of metabolic syndrome. While the changes in IL-18 are known, IL-18R expression and relationship with IL-18 and other inflammatory markers in the adipose tissue in obesity/type-2 diabetes (T2D) remain unclear. METHODS: We, therefore, determined the adipose tissue expression of IL-18R and IL-18 mRNA/protein in lean, overweight, and obese individuals with and without T2D, 15 each, using qRT-PCR, immunohistochemistry, and confocal microscopy. Data (mean ± SEM) were analyzed using unpaired t-test and Pearson's correlation (r); all P values ≤0.05 were considered statistically significant. RESULTS: We found the upregulated gene/protein expression of IL-18R and IL-18 in non-diabetic obese/overweight as compared with lean individuals (P < 0.05). BMI correlated positively (P < 0.05) with the adipose tissue expression of IL-18R (mRNA: r = 0.90 protein: r = 0.84) and IL-18 (mRNA: r = 0.84 protein: r = 0.80). Similarly, in T2D individuals, gene and protein expression of IL-18R/IL-18 was significantly higher in obese as compared with overweight/lean individuals. The BMI was associated with the changes in both IL-18R (mRNA: r = 0.55 protein: r = 0.50) and IL-18 (mRNA: r = 0.53 protein: r = 0.57) expression. IL-18R/IL-18 gene expression in the adipose tissue was positively associated (P < 0.05) with local gene expression of other inflammatory markers including CD11c, CD86, CD68, CD163, TNF-α, and CCL5. Homeostatic model assessment of insulin resistance (HOMA-IR) was higher in diabetic/non-diabetic obese and it correlated with BMI (P < 0.05). IL-18R and IL-18 mRNA/protein expression in obesity was associated with HOMA-IR only in non-diabetics. CONCLUSIONS: The adipose tissue IL-18R/IL-18 expression is enhanced in obesity which associates with proinflammatory gene signature and insulin resistance in these individuals.

[Elevated IL-18, IL-18 binding protein and IL-18 receptor in eosinophil-enriched blood cells in patients with asthma].

Objective To investigate the changes of interleukin-18 (IL-18), interleukin 18 binding protein (IL-18BP) and interleukin 18 receptor (IL-18R) expressions in eosinophil-enriched cells from asthmatic patients, and study the correlations among them. Methods Peripheral venous blood from asthmatic patients and normal subjects were collected and stimulated with the extracts of Artemisia pollen, dust mite, and Platanus pollen. The expressions of IL-18, IL-18BP and IL-18R in eosinophil-enriched blood cells were detected by flow cytometry, and the correlations among them were analyzed. Results The proportion of IL-18+ cells in eosinophil-enriched cells from asthmatic patients increased 15-fold compared with normal subjects. The proportion of IL-18+ cells increased about 1.3-fold and 1.5-fold in eosinophil-enriched cells from asthmatic patients whose blood was stimulated with dust mite and Platanus pollen extracts, respectively. The average fluorescence intensity (MFI) of IL-18BP+ cells increased about 1.5-fold and the proportion of IL-18R+ cells increased about 2-fold in eosinophil-enriched cells from asthmatic patients' blood stimulated with Platanus pollen extracts. In addition, the expressions of IL-18BP+ cells and IL-18R+ cells showed a positive correlation (r=0.639) in eosinophil-enriched cells from asthmatic patients stimulated with allergens. However, the expressions of IL-18, IL-18BP and IL-18R in eosinophil-enriched cells did not obviously change in normal subjects. Conclusion IL-18, IL-18R and IL-18BP expressed in eosinophils may be involved in the inflammatory reaction of asthma.

NK cells activated by Interleukin-4 in cooperation with Interleukin-15 exhibit distinctive characteristics.

Natural killer (NK) cells are known to be activated by Th1-type cytokines, such as IL-2, -12, or -18, and they secrete a large amount of IFN-γ that accelerates Th1-type responses. However, the roles of NK cells in Th2-type responses have remained unclear. Because IL-4 acts as an initiator of Th2-type responses, we examined the characteristics of NK cells in mice overexpressing IL-4. In this study, we report that IL-4 overexpression induces distinctive characteristics of NK cells (B220(high)/CD11b(low)/IL-18Rα(low)), which are different from mature conventional NK (cNK) cells (B220(low)/CD11b(high)/IL-18Rα(high)). IL-4 overexpression induces proliferation of tissue-resident macrophages, which contributes to NK cell proliferation via production of IL-15. These IL-4-induced NK cells (IL4-NK cells) produce higher levels of IFN-γ, IL-10, and GM-CSF, and exhibit high cytotoxicity compared with cNK cells. Furthermore, incubation of cNK cells with IL-15 and IL-4 alters their phenotype to that similar to IL4-NK cells. Finally, parasitic infection, which typically causes strong Th2-type responses, induces the development of NK cells with characteristics similar to IL4-NK cells. These IL4-NK-like cells do not develop in IL-4Rα KO mice by parasitic infection. Collectively, these results suggest a novel role of IL-4 in immune responses through the induction of the unique NK cells.

Immunologic Function and Molecular Insight of Recombinant Interleukin-18.

In recent years, cytokine-mediated therapy has emerged as further advance alternative in cancer therapy. Interleukin-18 (IL-18) has exhibited interesting anti-cancer properties especially when combined with IL-12. We engineered IL-18 in order to improve its activity using single point mutagenesis. IL-18 mutants were constructed according to binding residues and polarity which we tried to increase polarity in M33Q and M60Q, enhanced cationicity in E6K, and flexibility in T63A. All IL-18 proteins were expressed in Pichia pastoris, purified, and then measured the activity by treating with the NK-92MI cell line to evaluate interferon-γ (IFN-γ) stimulation. The E6K and T63A mutant forms showed higher activity with respect to native proteins at the concentration of 200 ng mL-1 by inducing the expression of IFN-γ, about factors of 9 and 4, respectively. Meanwhile, M33Q and M60Q had no significant activity to induce IFN-γ. Interestingly, the combination of E6K and T63A mutations could synergize the induction activity of IL-18 to be 16 times at 200 ng mL-1. Furthermore, molecular dynamics studies have elucidated the effect due to mutation on conformation of the binding site of IL-18. The results turn out that E6K provides structural perseverance against mutation, while M33Q and M60Q promote vivid overall change in protein conformation, especially at the binding site. For T63A, mutation yields small difference in structure but clearly increases structural flexibility. However, a small structural change was observed when T63A was combined with E6K. Our research resulted in a novel version of IL-18 which could be a new key candidate for cytokine-mediated therapy.

The role of IL-18 in type 1 diabetic nephropathy: The problem and future treatment.

Diabetic vascular complication is a leading cause of diabetic nephropathy, a progressive increase in urinary albumin excretion coupled with elevated blood pressure leading to declined glomerular filtration and eventually end stage renal failure. There is growing evidence that activated inflammation is contributing factor to the pathogenesis of diabetic nephropathy. Meanwhile, IL-18, a member of the IL-1 family of inflammatory cytokines, is involved in the development and progression of diabetic nephropathy. However, the benefits derived from the current therapeutics for diabetic nephropathy strategies still provide imperfect protection against renal progression. This imperfection points to the need for newer therapeutic agents that have potential to affect primary mechanisms contributing to the pathogenesis of diabetic nephropathy. Therefore, the recognition of IL-18 as significant pathogenic mediators in diabetic nephropathy leaves open the possibility of new potential therapeutic targets.

T cell responses are elicited against Respiratory Syncytial Virus in the absence of signalling through TLRs, RLRs and IL-1R/IL-18R.

Pattern recognition receptors (PRRs) and cytokine receptors are key players in the initiation of immune responses to infection. PRRs detecting viral RNA, such as toll like receptor (TLR)-3, -7/8, and RIG-I like receptors (RLRs; RIG-I and MDA-5), as well as cytokine receptors such as interleukin 1 receptor (IL-1R), have been implicated in responses to RNA viruses that infect the airways. The latter includes respiratory syncytial virus (RSV), a human pathogen that can cause severe lower respiratory tract infections, especially in infants. To evaluate the collective contribution of PRRs and IL-1R signalling to RSV immunity, we generated Myd88/Trif/Mavs(-/-) mice that are deficient in signalling by all TLRs, RLRs and IL-1R, as well as other cytokine receptors such as IL-18 receptor. Early production of pro-inflammatory mediators and lung infiltration by immune cells were completely abrogated in infected Myd88/Trif/Mavs(-/-) mice. However, RSV-specific CD8(+) T cells were elicited and recruited into the lungs and airways. Consistent with these findings, Myd88/Trif/Mavs(-/-) mice survived RSV infection but displayed higher viral load and weight loss. These data highlight an unappreciated level of redundancy in pathways that couple innate virus sensing to adaptive immunity, providing the host with remarkable resilience to infection.

Allergic airway inflammation: unravelling the relationship between IL-37, IL-18Rα and Tir8/SIGIRR.

The hallmarks of allergic bronchial asthma arise from chronic airway inflammation. Thus, elucidating the mechanisms regulating the maintenance of this chronic inflammatory response is key to understanding asthma pathogenesis. To date, it is not clear whether a predominance of proinflammatory factors or a reduced capacity of counterbalancing anti-inflammatory mediators is the pivotal factor predisposing individuals towards asthma development. The IL-1 cytokine family and its receptor systems comprise a variety of proinflammatory cytokines like IL-1ß and IL-18 and anti-inflammatory molecules such as the Toll/interleukin-1 receptor 8/single Ig IL-1 receptor (IL-R)-related molecule (Tir8/SIGIRR) and the recently established cytokine IL-37. This article reviews the functions of these IL-1 cytokine family members in the regulation of allergic airway inflammation and asthma as they have been assessed clinically, in vitro and in mouse models.

Divergent behavior of mucosal memory T cells.

Memory CD4 T cells are strategically positioned at mucosal surfaces to initiate a robust adaptive immune response. The detection of specific antigen via the T-cell receptor causes these memory T cells to unleash a potent antimicrobial response that includes rousing local innate immune populations for tissue-specific defense. Paradoxically, these same memory T cells can also be stimulated by nonantigen-specific signals that are generated by the activity of local innate immune cells. This versatility of mucosal memory T cells in both the initiation and the sensing of local innate immunity could be a vitally important asset during pathogen defense but alternatively could be responsible for initiating and maintaining chronic inflammation in sensitive mucosal tissues.

IL-18 contributes to susceptibility to Leishmania amazonensis infection by macrophage-independent mechanisms.

We evaluated the role of IL-18 during Leishmania amazonensis infection in C57BL/6 mice, using IL-18KO mice. We showed that IL-18 is involved in susceptibility to L. amazonensis, since IL-18KO mice presented reduced lesions and parasite loads. Because macrophages are the host cells of the parasite, we investigated if macrophages were involved in IL-18-mediated susceptibility to L. amazonensis. We showed that macrophages obtained from WT or IL-18KO responded similarly to L. amazonensis infection. Moreover, we showed that C57BL/6 macrophages do not respond to IL-18, since they do not express IL-18R. Therefore, macrophages are not involved in IL-18-mediated susceptibility to L. amazonensis.

A major population of mucosal memory CD4+ T cells, coexpressing IL-18Rα and DR3, display innate lymphocyte functionality.

Mucosal tissues contain large numbers of memory CD4(+) T cells that, through T-cell receptor-dependent interactions with antigen-presenting cells, are believed to have a key role in barrier defense and maintenance of tissue integrity. Here we identify a major subset of memory CD4(+) T cells at barrier surfaces that coexpress interleukin-18 receptor alpha (IL-18Rα) and death receptor-3 (DR3), and display innate lymphocyte functionality. The cytokines IL-15 or the DR3 ligand tumor necrosis factor (TNF)-like cytokine 1A (TL1a) induced memory IL-18Rα(+)DR3(+)CD4(+) T cells to produce interferon-γ, TNF-α, IL-6, IL-5, IL-13, granulocyte-macrophage colony-stimulating factor (GM-CSF), and IL-22 in the presence of IL-12/IL-18. TL1a synergized with IL-15 to enhance this response, while suppressing IL-15-induced IL-10 production. TL1a- and IL-15-mediated cytokine induction required the presence of IL-18, whereas induction of IL-5, IL-13, GM-CSF, and IL-22 was IL-12 independent. IL-18Rα(+)DR3(+)CD4(+) T cells with similar functionality were present in human skin, nasal polyps, and, in particular, the intestine, where in chronic inflammation they localized with IL-18-producing cells in lymphoid aggregates. Collectively, these results suggest that human memory IL-18Rα(+)DR3(+) CD4(+) T cells may contribute to antigen-independent innate responses at barrier surfaces.

Squalene emulsion potentiates the adjuvant activity of the TLR4 agonist, GLA, via inflammatory caspases, IL-18, and IFN-γ.

The synthetic TLR4 agonist glucopyranosyl lipid adjuvant (GLA) is a potent Th1-response-inducing adjuvant when formulated in a squalene oil-in-water emulsion (SE). While the innate signals triggered by TLR4 engagement are well studied, the contribution of SE remains unclear. To better understand the effect of SE on the adjuvant properties of GLA-SE, we compared the innate and adaptive immune responses elicited by immunization with different formulations: GLA without oil, SE alone or the combination, GLA-SE, in mice. Within the innate response to adjuvants, only GLA-SE displayed features of inflammasome activation, evidenced by early IL-18 secretion and IFN-γ production in memory CD8(+) T cells and neutrophils. Such early IFN-γ production was ablated in caspase-1/11(-/-) mice and in IL-18R1(-/-) mice. Furthermore, caspase-1/11 and IL-18 were also required for full Th1 CD4(+) T-cell induction via GLA-SE. Thus, we demonstrate that IL-18 and caspase-1/11 are components of the response to immunization with the TLR4 agonist/squalene oil-in-water based adjuvant, GLA-SE, providing implications for other adjuvants that combine oils with TLR agonists.

Human plasmacytoid dendritic cells regulate IFN-α production through activation-induced splicing of IL-18Rα.

IFN-α production by pDCs regulates host protection against viruses and is implicated in autoimmune pathology. Human pDCs express high levels of IL-18R, but little is known of its role in pDC function. We report that IL-18R signaling negatively regulates IFN-α production through activation-induced splicing of IL-18Rα in human pDCs. Our data reveal two distinct isoforms of IL-18Rα in human pDCs: the known, full-length receptor (IL-18Rα1) and a novel, truncated variant (IL-18Rα2), which functions as a molecular decoy that competitively inhibits the canonical IL-18Rα1/IL-18Rß signaling pathway. Whereas NK cells and pDCs both express IL-18Rα1, pDCs express significantly higher levels of IL-18Rα2, resulting in differential responses of these populations to IL-18. Flu exposure increases IL-18Rα1 expression in pDCs, and the blocking of IL-18R enhances pDC production of IFN-α and IP-10; thus, pDCs use activation-induced splicing to regulate IFN-α production in response to flu. These data demonstrate that IL-18R modulates IFN-α release by human pDCs and suggest that IL-18R signaling may represent a promising therapeutic target.

Analysis of the association of polymorphic loci rs917997 in IL18RAP gene and rs187238 in IL18 gene with the risk for non-Hodgkin's malignant lymphomas in Novosibirsk population.

We analyzed the association of polymorphic variants of rs917997 (G/A) locus in IL18RAP gene and rs187238 (G/C) locus in IL18 gene with the risk of malignant non-Hodgkin's lymphomas in Novosibirsk population. Allele and genotype frequencies of the above loci were determined in patients (243 persons) and control group (371 persons) and compared using χ(2) test. None of the analyzed loci showed statistically significant association with the risk of malignant non-Hodgkin's lymphomas.

IL-18 regulates melanoma VLA-4 integrin activation through a Hierarchized sequence of inflammatory factors.

Very late antigen-4 (VLA-4) is frequently overexpressed on melanoma cells contributing to inflammation-dependent metastasis. Melanoma cell adhesion to endothelium via VLA-4-vascular cell adhesion molecule-1 (VCAM-1) interaction was used to study VLA-4 activation during melanoma cell response to inflammation. Cooperation among major inflammatory mediators was analyzed in melanoma cells exposed to single inflammatory factors in the presence of inhibitors for other assayed mediators. A stepwise cascade of hierarchized molecules heterogeneously made and used during melanoma response to IL-18, induced hydrogen peroxide (H2O2), in turn activating VLA-4 and melanoma cell adhesion to endothelium. The cascade involved prostaglandin E2 (PGE2) production from melanoma induced by IL-18-dependent tumor necrosis factor-α (TNFα); next, PGE2-induced IL-1ß via vascular endothelial growth factor (VEGF) secretion, which in turn induced VLA-4 activation via cyclooxygenase 2-dependent H2O2. This sequence operated in IL-18R/VLA-4/VEGF-expressing murine (B16) and human (A375 and 883) melanomas, but not in those without this phenotype. Separation of active VLA-4-expressing B16 melanoma cells through immobilized VCAM-1 verified their higher IL-18R/TNFR1/VEGFR2 expression and metastatic growth than inactive VLA-4-expressing cells. However, cooperation among melanoma cell sub-populations with heterogeneous cytokine receptor levels may occur through VLA-4-stimulating factors, leading to intratumoral amplification of metastatic potential. Therefore, expression of the VLA-4-stimulating factor sequence may help to predict melanoma prometastatic risk, and offers therapeutic targets for metastatic melanoma deactivation through VLA-4 activation blockade.