Genome-Wide Association Study of Genetic Variants in LPS-Stimulated IL-6, IL-8, IL-10, IL-1ra and TNF-α Cytokine Response in a Danish Cohort

Background

Cytokine response plays a vital role in various human lipopolysaccharide (LPS) infectious and inflammatory diseases. This study aimed to find genetic variants that might affect the levels of LPS-induced interleukin (IL)-6, IL-8, IL-10, IL-1ra and tumor necrosis factor (TNF)-α cytokine production.

Methods

We performed an initial genome-wide association study using Affymetrix Human Mapping 500 K GeneChip® to screen 130 healthy individuals of Danish descent. The levels of IL-6, IL-8, IL-10, IL-1ra and TNF-α in 24-hour LPS-stimulated whole blood samples were compared within different genotypes. The 152 most significant SNPs were replicated using Illumina Golden Gate® GeneChip in an independent cohort of 186 Danish individuals. Next, 9 of the most statistical significant SNPs were replicated using PCR-based genotyping in an independent cohort of 400 Danish individuals. All results were analyzed in a combined study among the 716 Danish individuals.

Results

Only one marker of the 500 K Gene Chip in the discovery study showed a significant association with LPS-induced IL-1ra cytokine levels after Bonferroni correction (P<10⁻⁷). However, this SNP was not associated with the IL-1ra cytokine levels in the replication dataset. No SNPs reached genome-wide significance for the five cytokine levels in the combined analysis of all three stages.

Conclusions

The associations between the genetic variants and the LPS-induced IL-6, IL-8, IL-10, IL-1ra and TNF-α cytokine levels were not significant in the meta-analysis. This present study does not support a strong genetic effect of LPS-stimulated cytokine production; however, the potential for type II errors should be considered.     

Expression of PE_PGRS 62 protein in Mycobacterium smegmatis decrease mRNA expression of proinflammatory cytokines IL-1β, IL-6 in macrophages

Involvement of mitochondrial and nuclear gene mutations in the development of type 2 diabetes (T2D) has been established well in various populations around the world. Previously, we have found the mitochondrial A>G transition at nucleotide position 3243 and 8296 in the T2D patients of Coimbatore population. This study is aimed to screen for the presence of various mitochondrial and nuclear DNA mutations in the T2D patients of Coimbatore to identify most prevalent mutation. This helps in identifying the susceptible individuals based on their clinical phenotype in future. Blood samples were collected from 150 unrelated late-onset T2D patients and 100 age-matched unrelated control samples according to World Health Organization criteria. Genotyping for the selected genes was done by polymerase chain reaction–single strand confirmation polymorphism, direct sequencing, and polymerase chain reaction–restriction fragment length polymorphism. The mitochondrial T>C transition at 8356 and nuclear-encoded GLUT1 gene mutation were found in the selected T2D patients. The T8356C mutation was found in two patients (1.3%), and the clinical characteristics were found to be similar in both the patients whereas GLUT1 gene mutation was found in seven patients. Four out of seven patients showed homozygous (?) genotype and three patients showed heterozygous (±) genotype for the mutant allele XbaI. Among these three patients, one patient was found to have elevated level of urea and creatinine with the history of kidney dysfunction and chronic T2D. Our results suggest that the T8356C and GLUT1 gene mutations may have an important role in developing late-onset T2D in Coimbatore population. Particularly, individuals with GLUT1 gene may develop kidney dysfunction at their later age.     

Effects of Dietary Manganese on Cu, Fe, Zn, Ca, Se, IL-1β, and IL-2 Changes of Immune Organs in Cocks

Manganese (Mn) is an essential trace element required for normal development and bodily function. However, little is known about the effect of excessive amounts of Mn in immune organs of poultry. The aim of this study was to investigate the effect of dietary Mn on the content of trace elements, such as copper (Cu), iron (Fe), zinc (Zn), calcium (Ca), and selenium (Se), and the mRNA level of IL-1β and IL-2 in immune organs (spleen, thymus, and bursa of Fabricius) and the content of IL-1β and IL-2 in serum of poultry. Fifty-day-old male Hyline cocks were fed either a commercial diet or a Mn-supplemented diet containing 600, 900, and 1,800 mg/kg. The immune organs were collected at 30, 60, and 90 days, respectively, and the content of trace elements and the mRNA level of IL-1β and IL-2 were examined; the serum were collected and the IL-1β and IL-2 contents detected. The results showed that Mn content in immune organs increased and Fe, Zn, and Ca contents decreased; however, Cu and Se contents showed no difference. IL-1β and IL-2 mRNA levels and IL-1β and IL-2 contents decreased. The present study demonstrates that excess exposure to Mn results in metal accumulations in immune organs. Manganism can disturb the balance of trace elements in immune organs and induce immune suppression in the molecular level; therefore, the immune function of cocks are also suppressed after manganism.     

Immunohistochemistry of IL-1β, IL-6 and TNF-α in spleens of mice treated with gold nanoparticles

Gold nanoparticles (AuNPs) possess considerable biocompatibility and therefore gaining more attention for their biomedical applications. Previous studies have shown the transient increase in pro-inflammatory cytokines expression in different organs of rats and mice exposed to AuNPs. Structural changes in the spleen of mice treated with AuNPs have also been reported. This investigation was aimed to study the immunostaining of IL-1β, IL-6 and TNF-α in mice treated with different sizes of AuNPs. The animals were divided into 7 groups of 4 animals in each group. One group received saline and served as control. Two sets of three groups were treated with 5 nm, 20 nm and 50 nm diameter AuNPs. One set was sacrificed on day 1 and the other on day 7 following the AuNPs injections. Spleens were dissected out and promptly fixed in formalin for 3 days and then processed for IL-1β, IL-6 and TNF-α immunostaining using target-specific antibodies. The immunoreactivities of IL-1β and IL-6 were increased with the increase of AuNP size. The immunostaining of IL-1β in spleen of 20 nm AuNP treated mice was subsequently decreased on day 7 whereas it persisted in 50 nm AuNP group. The increase in the immunoreactivity of IL-6 on day 1 was decreased on day 7 in the spleens of mice treated with 20 nm or 50 nm AuNPs. The immunostaining of TNF-α was found to be negative in all the treatment groups. In conclusion, the size of AuNPs plays an important role in the expression of proinflammatory cytokines in mouse spleen; small size (5 nm) AuNPs caused minimal effect, whereas larger (50 nm) AuNPs produced intense immunostaining.     

Effect of recombinant Mce4A protein of Mycobacterium bovis on expression of TNF-α, iNOS, IL-6, and IL-12 in bovine alveolar macrophages

CD38 is a type II transmembrane protein with 25% of its molecular mass consisting of glycosyl moieties. It has long been predicted that the carbohydrate moieties of glycoproteins play important roles in the physical function and structural stability of the proteins on cell surfaces. To determine the structural/functional significance of glycosylation of the human CD38, the four potential N-linked glycosylation sites asparagine residues, N100, N164, N209 and N219 were mutated. The mutant (CD38mu) and wild-type (CD38wt) were expressed separately in Escherichia coli, HeLa, and MCF-7 cells. SDS-polyacrylamide gel electrophoresis under reducing conditions and western blotting indicated that the molecular mass of the CD38wt is 45 kDa, and that of the CD38mu is 34 kDa in HeLa cells. Importantly, the CD38mu protein expressed in HeLa cells, showed the high molecular weight oligomers in addition to the 34 kDa monomeric form. Similarly, in E. coli, the CD38wt formed dimers and other oligomers besides the monomeric form. Moreover, MCF-7 cells stably transfected with CD38wt cDNA, also revealed the presence of cross-linked oligomers when treated with a N-linked glycosylation inhibitor tunicamycin (TM). These results suggested that the N-linked glycosylation of CD38 plays a crucial role in the structure stability by preventing the formation inter-molecular cross-links. In addition, immunostaining, enzyme activity (cyclase), and western blotting data revealed that the glycosylation of human CD38 protein is not required for its localization to the cell membrane.     

Effects of adiponectin in TNF-α, IL-6, and IL-10 cytokine production from coronary artery disease macrophages

Adiponectin, an adipose tissue secreted protein, exhibits anti-inflammatory and antiatherogenic properties. We examined the effects of the globular and full-length adiponectin on cytokine production in macrophages derived from Coronary Artery Disease (CAD) patients and control individuals. Adiponectin's effects in human macrophages upon lipopolysaccharide (LPS) treatment were also examined. Full length adiponectin acted differently on TNF-α and IL-6 production by upregulating TNF-α and IL-6 protein production, but not their mRNA expression. Additionally, full length adiponectin was unable to abrogate LPS proinflammatory effect in TNF-α and IL-6 mRNA expression in CAD and NON-CAD macrophages. In contrast, globular adiponectin appeared to have proinflammatory properties by potently upregulating TNF-α and IL-6 mRNA and protein secretion in human macrophages while subsequently rendered cells resistant to further proinflammatory stimuli. Moreover, both forms of adiponectin powerfully suppressed scavenger MSR-AI mRNA expression and augmented IL-10 protein release, both occurring independently of the presence of LPS or CAD. These data indicate that adiponectin could potentially protect human macrophages via the elevated IL-10 secretion and the suppression of MSR-AI expression. It can also be protective in CAD patients since the reduced adiponectin-induced IL-6 release in CAD macrophages compared to controls, could be beneficial in the development of inflammation related atherosclerosis.     

Genetic Variations of IL-12B,IL-12Rβ1, IL-12Rβ2 in Behcet's Disease and VKH Syndrome

Purpose

To investigate the associations of single nucleotide polymorphisms (SNPs) of three genes (IL-12B, IL-12Rβ1 and IL-12Rβ2) in Behcet''s disease (BD) and Vogt-Koyanagi-Harada (VKH) syndrome in a Chinese Han population.

Methods

A total of 806 BD cases, 820 VKH patients, and 1600 healthy controls were involved in this study. The first investigation included 400 BD patients, 400 VKH cases, and 600 healthy individuals. A second confirmatory study included a separate set of 406 BD patients, 420 VKH cases and another 1000 normal controls. Genotyping was carried out by PCR-restriction fragment length polymorphism assay and results were validated by using direct sequencing. The χ2 test was performed to compare the allele and genotype frequencies between cases and healthy controls.

Results

This study comprised two phases. In the first phase study, a significantly increased frequency of the rs3212227/IL-12B genotype CC and C allele was found in BD patients as compared to controls (Bonferroni corrected p value (p_c) = 0.009, OR 1.8; p_c = 0.024, OR 1.3, respectively). Moreover, the frequency of the C allele of rs3212227/IL-12B was also significantly increased in VKH patients (p_c = 0.012, OR 1.3, 95% CI 1.1 to 1.6). No associations were found for the other seven tested SNPs either in BD or VKH disease. The second study as well as the combined data confirmed the significant association of rs3212227/IL-12B with BD (CC genotype: combined p_c = 6.3×10⁻⁷, OR = 1.8; C allele: combined p_c = 2.0×10⁻⁵, OR = 1.3, respectively) and the C allele frequency of rs3212227/IL-12B as the risk factor to VKH patients (combined p_c = 2.5×10⁻⁵, OR 1.3, 95% CI 1.2 to 1.5).

Conclusions

Our study revealed that the IL-12B gene is involved both in the susceptibility to BD as well as VKH syndrome.     

Serum TNF-α, sTNFR1, IL-6, IL-8 and IL-10 levels in Weil's syndrome

Studies on cytokine levels in Weil's syndrome are lacking. In this study, TNF-α, sTNFR1, IL-6, IL-8 and IL-10 levels were measured in 44 serum samples of patients diagnosed with Leptospira interrogans serovar icterohaemorrhagiae infection. TNF-α levels linked with pulmonary hemorrhagic implications, while elevated sTNFR1 and IL-10 levels linked with fatal cases. IL-6 and IL-8 did not seem to affect the outcome of the disease. Immune response pattern in Weil's syndrome bears resemblance to other patterns described for hemorrhagic fevers. IL-10/TNF-α ratio is proposed as a marker for prognosis.     

Combined Analysis of IFN-γ, IL-2, IL-5, IL-10, IL-1RA and MCP-1 in QFT Supernatant Is Useful for Distinguishing Active Tuberculosis from Latent Infection

The QuantiFERON®-TB Gold In-Tube test (QFT), an interferon-γ release assay, is used to diagnose Mycobacterium tuberculosis, but its inaccuracy in distinguishing active tuberculosis from latent infection is a major concern. There is thus a need for an easy and accurate tool for achieving that goal in daily clinical settings. This study aimed to identify candidate cytokines for specifically differentiating active tuberculosis from latent infection. Our study population consisted of 31 active TB (tuberculosis) patients, 29 LTBI (latent tuberculosis infection) patients and 10 healthy control subjects. We assayed for 27 cytokines in QFT supernatants of both specific antigen-stimulated blood samples (TBAg) and negative-control samples (Nil). We analyzed their specificities and sensitivities by creating receiver operating characteristic (ROC) curves and measuring the area under those curves (AUCs). In TBAg–Nil supernatants, IL-10, IFN-γ, MCP-1 and IL-1RA showed high AUCs of 0.8120, 0.7842, 0.7419 and 0.7375, respectively. Compared with each cytokine alone, combined assay for these top four cytokines showed positive rates in diagnosing active TB, and GDA analysis revealed that MCP-1 and IL-5 are potent in distinguishing active TB from LTBI, with Wilk’s lambda = 0.718 (p < 0.001). Furthermore, utilizing the unique characteristic of IL-2 that its TBAg–Nil supernatant levels are higher in LTBI compared to active TB, the difference between IFN-γ and IL-2 showed a large AUC of 0.8910. In summary, besides IFN-γ, IL-2, IL-5, IL-10, IL-1RA and MCP-1 in QFT supernatants may be useful for distinguishing active TB from LTBI. Those cytokines may also help us understand the difference in pathogenesis between active TB and LTBI.     

Zinc Suppressed the Airway Inflammation in Asthmatic Rats: Effects of Zinc on Generation of Eotaxin, MCP-1, IL-8, IL-4, and IFN-γ

Airway epithelium is rich in labile zinc (Zn), which may have an important protective role in the airway epithelium. The aim of this study is to investigate the effects of Zn on the airway inflammation and the generation of eotaxin, monocyte chemoattractant protein-1 (MCP-1), interleukin-8 (IL-8), interleukin-4 (IL-4), and interferon-?? (IFN-??) in rat models of ovalbumin (OVA)-induced allergic airway inflammation. For this purpose, animal model of asthma was established by OVA challenge and zinc-deficient and zinc-supplemented diets were given. Thirty-two Sprague?CDawley rats were divided into four groups: zinc-deficient diet with OVA treatment group, zinc-supplemented diet with OVA treatment group, zinc-normal diet with OVA treatment group, and zinc-normal diet with saline treatment group. Twenty-four hours after asthma was induced, lung histomorphological changes, cells in bronchoalveolar lavage fluid (BALF), contents of eotaxin, MCP-1, and IL-8 in BALF, and the expression of IFN-?? and IL-4 mRNAs were observed. Compared with the group of zinc-normal diet with OVA challenge rats, the group of zinc-deficient rats had higher numbers of eosinophils, neutrophils, and monocytes in BALF, as well as higher contents of eotaxin and MCP-1 in BALF and lower expression of lung IFN-?? mRNA. Conversely, Zn supplementation would decrease the numbers of eosinophils, neutrophils, and monocytes in BALF; suppress eotaxin and MCP-1 protein secretion; and increase lung IFN-?? mRNA expression. No significant difference was observed in IL-8 and IL-4 among OVA-challenged rats with different zinc diets. These studies suggested that Zn may be an important anti-inflammatory mediator of airway inflammation.     

IL-1β, IL-6, and RANTES as Biomarkers of Chikungunya Severity

Background

Little is known about the immunopathogenesis of Chikungunya virus. Circulating levels of immune mediators and growth factors were analyzed from patients infected during the first Singaporean Chikungunya fever outbreak in early 2008 to establish biomarkers associated with infection and/or disease severity.

Methods and Findings

Adult patients with laboratory-confirmed Chikungunya fever infection, who were referred to the Communicable Disease Centre/Tan Tock Seng Hospital during the period from January to February 2008, were included in this retrospective study. Plasma fractions were analyzed using a multiplex-microbead immunoassay. Among the patients, the most common clinical features were fever (100%), arthralgia (90%), rash (50%) and conjunctivitis (40%). Profiles of 30 cytokines, chemokines, and growth factors were able to discriminate the clinical forms of Chikungunya from healthy controls, with patients classified as non-severe and severe disease. Levels of 8 plasma cytokines and 4 growth factors were significantly elevated. Statistical analysis showed that an increase in IL-1β, IL-6 and a decrease in RANTES were associated with disease severity.

Conclusions

This is the first comprehensive report on the production of cytokines, chemokines, and growth factors during acute Chikungunya virus infection. Using these biomarkers, we were able to distinguish between mild disease and more severe forms of Chikungunya fever, thus enabling the identification of patients with poor prognosis and monitoring of the disease.     

Detection of IL-1β, VEGF and IL-4 with their novel genetic variations in breast cancer patients

Interleukin-1β (IL-1β), vascular endothelial growth factor (VEGF), and IL-4 serum levels and new genetic mutations in breast cancer (BC) patients were assessed in the current study. The serum levels of the examined cytokines in 40 BC patients and 40 control subjects were assessed using the ELISA technique. In order to identify genotype variants of the IL-1β, IL-4, and VEGF genes in 40 Formalin Fixed Paraffin Embedded (FFPE) samples with BC and 10 FFPE samples from healthy women's breast tissue, Sanger sequencing was used. According to this study, BC patients had significantly lower serum concentrations of IL-4 and significantly higher quantities of the tumor markers, CA15-3, IL-1β, and VEGF. In terms of genotype alterations, a total of 21 mutations in three trialed genes (eight in IL-1β, 10 in IL-4, and three in VEGF) were found in BC patients. The results of the current investigation suggested that angiogenesis and the development of BC may be significantly influenced by the genetic differences and higher levels of the examined cytokines.     

Membrane-bound/soluble IL-2 receptor (IL-2R) and levels of IL-1α, IL-2, and IL-6 in the serum and in the PBMC culture supernatants from 17 patients with hematological malignancies

The present study investigated the peripheral blood mononuclear cells (PBMC) blastic responses to PHA, PHA plus recombinant IL-2 (rIL-2) and rIL-2 alone; the expression of membrane-bound IL-2R on PHA-stimulated PBMC; and the levels of IL-1α, IL-2, IL-6, and sIL-2R in serum and in culture supernatants from PHA-stimulated PBMC in 17 patients with hematological malignancies (mean age 58.5 yr, range 22–82): 6 with non-Hodgkin’s lymphoma (NHL), 4 with Hodgkin’s lymphoma (HL), 5 with Hairy cell leukemia, 1 with chronic myelogenous leukemia, and 1 with chronic lymphocytic leukemia. The patients with HL and NHL with active disease (AD) were separated from those in clinical remission. The patients with AD were studied at diagnosis (obviously before therapy) and the patients in clinical remission were out of therapy since at least 6 mo. The lymphocyte blastogenic response to PHA was significantly lower in patients with HL and NHL with AD than in the control group. The response to rIL-2 alone was in the same range in the control group and in HL and NHL AD patients. By adding rIL-2 to PHA there was an increase of the blastogenic response of the same patients. The percentage of CD25 expressed on PHA-stimulated lymphocytes from patients with HL and NHL AD and from normal subjects is in the same range. Serum levels of IL-2, IL-6, and sIL-2R were significantly higher in HL and NHL AD patients than in controls as well as in all other hematological malignancies. Supernatants derived from PHA-stimulated PBMC were assessed for the presence of cytokines and sIL-2R by ELISA. The levels of IL-2, IL-6, and sIL-2R were significantly lower in HL and NHL AD patients than in controls as well as in all other hematological malignancies.     

Effect of cytokines (IL-2, IL-7, and IL-15) having common γ-chain of receptors on differentiation and maturation of CD4+/CD8+ T-cells in a CD45RA T-lymphocyte population in vitro

The effect of cytokines (IL-2, IL-7, and IL-15) having a common γ-chain of receptors on the maturation and differentiation of CD3⁺CD45RA⁺CD4⁺/CD8⁺ lymphocytes in homeostatic cultivation model in vitro was analyzed. It was found that the maximum IL-2 concentration in the helper CD45RA⁺-T-cell population mediates an increase in the number of CD45RA⁺CD4⁺ T lymphocytes with the phenotype of mature and immature terminally differentiated TEMRA T cells. IL-15 leads to the production of lymphocytes with CD27^–CD62L⁺ phenotype (presumably, TEMRA, in which the CD62L expression persists). In the CD45RA⁺CD8⁺ T lymphocyte populations, the studied cytokines (IL-2, IL-7, and IL-15) initiate the production of mature TEMRA (E) T lymphocytes and memory T cells with the CD45RA^?CD27⁺CD62L⁺ central phenotype (TCM).     

Effects of Sodium Selenite on Aflatoxin B1-Induced Decrease of Ileac T cell and the mRNA Contents of IL-2, IL-6, and TNF-α in Broilers

The aim of this work was to assess the protective effect of sodium selenite on the ileum mucosal immunologic toxicity induced by aflatoxin B₁ (AFB₁). One hundred and eighty one-day-old healthy male avian broilers were divided into four groups of three replicates and 15 birds per replicate and fed with basal diet (control group), 0.3 mg/kg AFB₁ (AFB₁ group), 0.4 mg/kg Se (+Se group), and 0.3 mg/kg AFB₁?+?0.4 mg/kg Se (AFB₁+Se group), respectively. The ileac T-cell subsets were determined by the methods of flow cytometry (FCM), and the mRNA contents of interleukin-2 (IL-2), interleukin-6(IL-6), and tumor necrosis factor-alpha (TNF-α) by quantitative real-time PCR. Compared with those in control group, the percentages of CD₃ ⁺, CD₃ ⁺CD₄ ⁺, CD₃ ⁺CD₈ ⁺ intraepithelial lymphocytes (IELs) and LPLs, the CD4⁺/CD8⁺ ratio of IELs, and the mRNA contents of IL-2, IL-6, and TNF-α were decreased in AFB₁ group. However, compared with those in AFB₁ group, these parameters of AFB₁+Se group were increased to be close to those in control group. It was concluded that 0.3 mg/kg AFB₁ could reduce the cellular immune function of the ileum mucosa, but 0.4 mg/kg supplemented dietary selenium showed protective effects on AFB₁-induced immunologic injury.     

Short communication: Concentration of TGF-β1, IL-10 and IL-6 in boar seminal plasma and TGF-β1 level in different fractions of ejaculates

Levels of the cytokines transforming growth factor (TGF)-β1, interleukin (IL)-10 and IL-6 in the boar seminal plasma (SP) as well as TGF-β1 level in different fractions of ejaculate were studied. These cytokines was chosen because of their expected effect on tissue immune response, i.e. suppressive (TGF-β1 and IL-10) and pro-inflammatory (IL-6). Three whole ejaculates from five boars A-E, (n=15) were sampled weekly to evaluate the levels of seminal plasma TGF-β1, IL-10 and IL-6 as well as their fluctuations over time. The effect of different storage temperatures, -20°C or -80°C, on the level of seminal plasma TGF β1 was also tested (three boars, two fractions in one ejaculate). In addition, in 4 different fractions of ejaculates: the pre-sperm-rich (Pre-SRF), first 10 ml of sperm-rich (10SRF), the rest of the sperm-rich fraction (Rest-SRF) and the rest of the ejaculate (RE) fraction, were collected from three boars (A-C) on four different occasions for TGF-β1 evaluation. In the whole ejaculates (n=15), a wide range in the concentration of the cytokines TGF-β1 (20.4 - 766.5 pg/mL) and IL-10, (73.7 - 837.3 pg/mL), was found. For IL-6, the concentration was low (range 11.5 - 30.9 pg/ml) and only detected in four out of 15 collections (from two boars). The mean levels of TGF-β1 and IL-10 between individual boars varied but were not statistical different. The level of TGF-β1 in Pre-SRF, Rest-SRF and RE fractions was significantly lower in boar A than the other boars. A significantly higher concentration of TGF-β1 was found in the 10SRF than in the other fractions. Different storage temperatures (-20°C or -80°C) did not affect the seminal plasma TGF-β1 level after one year of storage. To conclude: Boar seminal plasma contained TGF- β1 and IL-10 but with high individual variation. IL-6 was low or undetectable. The TGF- β1 level was highest in the first 10 mL of the sperm-rich fraction of the ejaculate. Further studies are needed on the role of different levels of cytokine in boar semen on porcine female reproductive tissue, especially for TGF- β1.     

Potential contribution of naïve immune effectors to oral tumor resistance: role in synergistic induction of VEGF,IL-6, and IL-8 secretion

The aim of this study is to identify the phenotype of resistant oral tumors, and to delineate the contribution of immune effectors to resistance of oral tumors. UCLA-1 oral tumors which were resistant to NK cell mediated cytotoxicity secreted increased amounts of IL-6, IL-1β, GM-CSF, and IL-8 when cultured with or without immune effectors. In addition, the levels of vascular endothelial growth factor (VEGF) secretion in the co-cultures of naïve immune effectors with UCLA-1 rose significantly when compared to tumor cells alone. IL-2 activated NK cells decreased VEGF secretion in all tumor cells. However, NK cells which were induced to undergo cell death with anti-CD16 antibody were not only unable to decrease VEGF secretion, but they also contributed further to the increase in VEGF secretion by oral tumors. Overall, we show in this paper that naïve as well as non-viable immune effectors may contribute to the growth and resistance of oral tumors by triggering the secretion of key tumor cell growth factors.