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Studying the cytokine profiles in animal models or patients with sepsis provides an experimental basis for the identification of diagnostic biomarkers and therapeutic targets. In this study, we used a liquid protein chip (LiquiChip), also known as flexible multi-analyte profiling technology, to perform quantitative analyses of several cytokines and chemokines (e.g., IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, TNF-α, IFN-γ, granulocyte-macrophage colony-stimulating factor, keratinocyte chemoattractant, monocyte chemoattractant protein, monokine induced by gamma interferon, IFN-γ-inducible protein 10, and macrophage inflammatory protein 1 alpha). The levels of these cytokines and chemokines were determined both in the blood and in tissues, including the lung, liver, heart, kidney, spleen, brain, stomach, intestine and muscle, of mice challenged with LPS. Our data showed variable production levels of LPS-induced cytokines in different mouse organs, and the cytokine in the blood did not correlate with those in the organs. We also showed that the levels of most cytokines peaked within 1 to 6 h and decreased rapidly afterward. A variety of inflammatory cytokines might be related to the damage in different organs during septic shock. Our data also suggest that the spleen might be an important target organ in the development of systemic inflammatory response syndrome and sepsis.

Despite the use of combined antiretroviral therapy, HIV-infected individuals have a higher risk of developing B-cell lymphoma compared to the general population. We aim to explore whether lymphocyte activation, increase in Th1 response as well as markers of EBV reactivation, may precede lymphoma diagnosis.Thirteen cases and 26 controls matched on CD4(+) T-cell count and HIV plasma viral load were identified. Samples were collected 0 to 5 years prior to B-cell lymphoma diagnosis. Seven out of 13 (54 %) and 16/26 (61.5 %) of cases and controls were receiving antiretroviral therapy at the time of sampling, respectively. CD8(+) T-cell activation and Th1 cytokine concentrations were measured before lymphoma onset, together with IgG antibodies directed against viral capsid antigen (VCA) and serum levels of EBV DNA.A higher level of CD8(+) T-cell activation was observed in patients developing lymphoma. Four out of seven Th1 cytokine serum concentrations were significantly higher in patients with lymphoma than in the control group: IL-2R, IL-12p40/70, IFN-γ-inducible protein 10 (IP-10) and monokine induced by IFN-γ (MIG). Anti-VCA IgG level were significantly higher in cases than in controls. Four cases (30 %) but no controls had detectable EBV DNA in serum.A higher level of T-cell activation, Th1 cytokine serum concentration and markers of EBV replication, preceded B-cell lymphoma diagnosis. This may suggest that viral antigen stimulation is associated with the genesis of lymphoma in HIV-infected patients.

The tyrosine kinase inhibitor dasatinib exerts immunosuppressive effects on T-cells and NK-cells in vitro. However, in some dasatinib-treated leukemia patients, clonal lymphocytosis with large granular lymphocyte (LGL) morphology develops, and this is associated with enhanced therapeutic responses. To elucidate the mechanistic basis for this paradoxical observation, we conducted detailed phenotypic and functional analyses of T-cell and NK-cell populations from 25 dasatinib-treated leukemia patients. All tested patients with LGL expansions (15/16) were cytomegalovirus (CMV) immunoglobulin (IgG) seropositive with high frequencies of CMV-specific CD8(+) T-cells; 5/16 LGL patients also experienced symptomatic CMV reactivation during dasatinib therapy. Expanded T-cell and NK-cell populations exhibited late differentiated (CD27(-)CD57(+)) phenotypes; this was associated with a predisposition to apoptosis within the T-cell compartment and impaired NK-cell cytotoxicity. Only 3/9 non-LGL patients were CMV IgG seropositive. Dasatinib inhibited in vitro lymphocyte functions, similarly in LGL patients and controls. Notably, distinct CD8(high) and CD8(low) T-cell subsets were observed in LGL patients; this phenotypic dichotomy was also apparent in CMV-specific CD8(+) T-cell populations, and exhibited features consistent with antigen-driven activation. In addition, plasma levels of IP-10, IL-6, monokine induced by interferon-γ and interleukin-2R were significantly increased in LGL patients. These data provide evidence that dasatinib-associated LGL expansion is linked to CMV reactivation and suggest a potential mechanism for this phenomenon.

To determine the clinical utility of serum levels of chemokines and cytokines for the evaluation of disease activity in patients with systemic sclerosis (SSc), concentrations of four chemokines (interferon γ-inducible protein-10 [IP-10, CXCL10], monokine induced by interferon γ [MIG/CXCL9], monocyte chemoattractant protein-1 [MCP-1/CCL2], interleukin 8 [IL-8/CXCL8]) and six cytokines (IL-2, IL-4, IL-6, IL-10, tumor necrosis factor [TNF]-α, interferon [IFN]- γ) were measured using cytometric beads array kits in serum samples from 31 Japanese patients with SSc and 20 normal controls. Clinical and laboratory data and serum chemokine and cytokine levels were assessed for each patient at their first visit and each subsequent year for 3 years. Among these chemokines and cytokines, serum levels of IP-10, MIG and MCP-1 were significantly elevated in SSc patients compared with normal controls at their first visit. Serum MCP-1 levels declined year and year, along with improvement for skin sclerosis. The variations of MCP-1, but not IP-10 and MIG, were significantly associated with the variations of skin thickness score and vital capacity during 3 years. These results suggest that MCP-1 is a serological indicator of the activity of skin and lung involvement in patients with SSc. However, a longer-term prospective study in a larger population will be needed to confirm its clinical utility as predictors of outcomes.

Here, we investigated the antitumor effect of adenovirus-mediated gene transfer of LIGHT, the tumor-necrosis factor (TNF) superfamily member also known as TNFSF14, in the murine A20 B-cell lymphoma. LIGHT gene modification resulted in upregulated expression of Fas and the accessory molecule--intercellular adhesion molecule-1 (ICAM-1) on A20 cells and led to enhanced A20 cell apoptosis. LIGHT-modified A20 cells effectively stimulated the proliferation of T lymphocytes and interferon (IFN)-gamma production in vitro. Immunization of BALB/c mice with a LIGHT-modified A20 cell vaccine efficiently elicited protective immunity against challenge with the parental tumor cell line. Adenovirus-mediated gene transfer of LIGHT by intratumoral injection exerted a very potent antitumor effect against pre-existing A20 cell lymphoma in BALB/c mice. This adenovirus-mediated LIGHT therapy induced substantial splenic natural killer (NK) and cytotoxic T lymphocyte (CTL) activity, enhanced tumor infiltration by inflammatory cells and increased chemokine expression of CC chemokine ligand 21 (CCL21), IFN-inducible protein-10 (IP-10) and monokine induced by IFN-gamma (Mig) from tumor tissues. Thus, adenovirus-mediated LIGHT therapy might have potential utility for the prevention and treatment of B-cell lymphoma.

The intracerebral formation of inflammatory infiltrates is a complex process, which may be regulated by chemokines. This study defines the kinetics and cellular sources of T cell- and macrophage-attracting chemokines in murine Toxoplasma encephalitis (TE) by ribonuclease protection assay, reverse transcription-PCR, in situ hybridization, and immunohistochemistry. Whereas astrocytes were the major source of interferon (IFN)-gamma-inducible protein-10 (CRG-2/IP-10) and monocyte chemoattractant protein (MCP)-1, microglia expressed RANTES, monokine induced by IFN-gamma (MuMIG) and occasionally CRG-2/IP-10 RNA. Despite being ubiquitously activated, only astrocytes and microglia confined to inflammatory infiltrates expressed chemokine genes. Intracerebral leukocytes transcribed RANTES, MuMIG, and occasionally CRG-2/IP-10 and MCP-1. IFN-gamma-deficient mice failed to produce CRG-2/IP-10, MuMIG, RANTES and expressed macrophage inflammatory protein (MIP-1)alpha, MIP-1 beta, and MCP-1 mRNA at reduced levels, functionally resulting in a strongly reduced recruitment of leukocytes across the blood-brain barrier and prevented their further invasion of the brain parenchyma. Since T cells are the single source of IFN-gamma in TE, these findings indicate that T cells pave the way of leukocytes to parenchymatous parasites via IFN-gamma.

Probiotic lactobacilli have been proposed as a potential oral bacteriotherapeutic means of modulating immune phenotype expression in vivo, via their ability to promote cytokine production. This study investigated the ability of a known interferon (IFN)gamma-promoting probiotic (Lactobacillus rhamnosus HNOOI) to modulate cytokine production in mice expressing an on-going Th2-type immune response. BALB/c mice were primed to ovalbumin in alum adjuvant to invoke antigen-specific Th2 cytokine-secreting cell populations. Mice that were fed Lb. rhamnosus HN001 during antigen sensitization produced higher levels of lymphocyte-derived IFNgamma, but also interleukin (IL)-4 and IL-5, in comparison to control animals. Although HN001 was additionally shown to induce pro-IFNgamma monokine (IL-12, IL-18) secretion in macrophages in vitro, its ability to invoke mixed lymphocyte cytokine production during an on-going Th2-type immune response in vivo suggests that this probiotic is a general immunostimulatory agent, in contrast to the pro-Th1/anti-Th2 immunoregulation reported for some strains of IFNgamma-promoting lactobacilli.

Seventy newly diagnosed Caucasian male patients with head and neck squamous cell carcinomas (HNSCC) were included in the study. All patients were less than 80 years of age, with no cachexia or auto-immune disease, and they were not taking immuno-active medications. Monocytes from these patients were cultured in vitro and supplemented with autologous serum under ex vivo conditions or cultured with serum-free medium. Comparison was made to monocytes from 59 patients with benign HN diseases. Similar physical activity levels prior to testing as well as a minimum stress load were ensured in both groups. Increased monocyte supernatant levels were determined under ex vivo conditions of interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6) and tumor necrosis factor (TNF)-alpha, but not of interleukin-12 (IL-12) with endotoxin stimulated monocytes of HNSCC patients compared to control conditions. Increased monokine levels were not present with mononuclear cell cultures stimulated with a T-cell general stimulatory agent or with purified monocytes not specifically stimulated. With endotoxin-stimulated monocytes under in vitro conditions, an increased supernatant was shown for TNF-alpha, but not IL-6. With serum from the different patients cultured with monocytes employed from a healthy control, no difference between the groups was shown in the IL-6 and TNF-alpha response to endotoxin stimulation. The differences in IL-1 beta and TNF-alpha, but not IL-6 levels were differentiated statistically from the smoking and alcohol histories of the patients. These findings suggest that the function of monocytes in general, and thus possibly all mononuclear phagocyte system cells in HNSCC patients, are altered.

Whole-cell pertussis found in diphtheria–tetanus–pertussis (DTP) vaccine can produce symptoms reminiscent of biological responses to circulating proinflammatory monokines such as IL-6, IL-1β, and TNFα. Therefore the ability of pertussis-containing vaccines and several heat-killed Bordetella pertussis preparations to stimulate cytokine production in a human monocytic cell line, THP-1, were examined. The whole-cell pertussis vaccine induced significantly more IL-6, IL-1β, and TNFα production than did the acellular pertussis or diphtheria–tetanus-only vaccine. Polymyxin B was able to inhibit most of the IL-6 induced by pertussis endotoxin and a heat-killed preparation of B. pertussis containing a null mutation in bvgAS, a regulatory locus required for expression of all known protein virulence factors synthesized by this organism. However, it only partially inhibited IL-6 production induced by other pertussis-containing preparations, including DTP vaccine. These results indicate that in vitro whole-cell vaccine is a potent stimulator of IL-6, IL-1β, and TNFα. They also suggest that although endotoxin is a major inducer of IL-6, other components of B. pertussis also contribute to IL-6 production by monocytes.

Recombinant human interleukin-4 (rhIL-4) and rhIL-1 alpha each produced a rapid down-modulation of tumour necrosis factor receptor (TNFR) on rheumatoid synovial fibroblasts (RSF) in vitro. This was associated with a staurosporine-resistant increase in p55 soluble TNFR levels, in culture media, suggesting that down-modulation was due to enhanced receptor shedding via a protein kinase C-independent mechanism. Pretreatment with rhIL-4 reduced the subsequent tumour necrosis factor alpha (TNF alpha) stimulation of prostaglandin E (PGE) and matrix metalloproteinase-3 (MMP-3) production by RSF. Thus, the potential anti-synovial monokine properties of rhIL-4 are not confined to inhibiting monokine production but also include the ability to interfere with their action on cells that constitute a substantial proportion of the rheumatoid synovium.

In this study, we demonstrate that mononuclear cells of human milk have a potential for production of many different cytokines. We applied a technique for cytokine detection at the single-cell level using cytokine specific MAb and immunofluorescence. The characteristic staining pattern obtained represents intracellular cytokine production, which allows for the assessment of the cellular origin of production. Milk mononuclear cells were mitogen-stimulated in vitro and cultured for 4 h and then stained for 13 cytokines. Lipopolysaccharide stimulation induced extensive production of the following monokines: IL-1 alpha, IL-1 beta, IL-1ra, IL-6, IL-8, and tumor necrosis factor-alpha. IL-10 and granulocyte-macrophage colony-stimulating factor were smaller products, although detectable in most samples. The abundant monokine production correlated with the high number of macrophages in milk. Spontaneous monokine production in unstimulated cells could be detected in six out of 11 samples. The highest incidence was evident for IL-8. No spontaneous lymphokine production was detected. Considering the low proportion of lymphocytes, stimulation with phorbol myristate acetate in combination with ionomycin resulted in considerable production of the following lymphokines: IL-2, IL-3, IL-4, IL-10, interferon-gamma, tumor necrosis factor-alpha. Macrophages contributed to the high production of tumor necrosis factor-alpha and GM-CSF. IL-5 synthesis was detectable in only one sample. This work reveals that human milk mononuclear cells are potent producers of cytokines when mitogen stimulated in vitro. The in vivo implications of these findings remain to be investigated further.

To determine the age-related changes in monokine secretion, the serum concentrations of tumor necrosis factor alpha (TNF), interleukin-1 alpha (IL-1α) and interleukin-1 beta (IL-1β) were measured in 31 young healthy subjects (21–35 years old) and 19 healthy elderly subjects (65–83 years old). There were no differences between the two groups with respect to serum concentrations of these cytokines. We also measured in a subgroup of young (n=8–11) and elderly (n=8–10) subjects the in vitro secretion of TNF and IL-1β in response to adding to the medium lipopolysaccharide (LPS, 2 ng/ml), interferon gamma (50 U/ml) or advanced glycosylation endproduct of bovine serum albumin (250 ug/ml). Only LPS-stimulated IL-1β production was significantly reduced in elderly subjects (456.7 ± 229.9 vs 1118.8 ± 494.8 pg/ml). It is concluded that with the possible exception of LPS-stimulated IL-1β production, monokine secretion measured in this study is well preserved in elderly subjects.

Background Chronic obstructive pulmonary disease (COPD) is a heterogeneous syndrome characterized by varying degrees of airflow limitation and diffusion impairment. There is increasing evidence to suggest that COPD is also characterized by systemic inflammation. The primary goal of this study was to identify soluble proteins in plasma that associate with the severity of airflow limitation in a COPD cohort with stable disease. A secondary goal was to assess whether unique markers associate with diffusion impairment, based on diffusion capacity of carbon monoxide (DLCO), independent of the forced expiratory volume in 1 second (FEV1). Methods A cross sectional study of 73 COPD subjects was performed in order to examine the association of 25 different plasma proteins with the severity of lung function impairment, as defined by the baseline measurements of the % predicted FEV1 and the % predicted DLCO. Plasma protein concentrations were assayed using multiplexed immunobead-based cytokine profiling. Associations between lung function and protein concentrations were adjusted for age, gender, pack years smoking history, current smoking, inhaled corticosteroid use, systemic corticosteroid use and statin use. Results Plasma concentrations of CCL2/monocyte chemoattractant protein-1 (CCL2/MCP-1), CCL4/macrophage inflammatory protein-1β (CCL4/MIP -1β), CCL11/eotaxin, and interleukin-13 (IL-13) were inversely associated with the % FEV1. Plasma concentrations of soluble Fas were associated with the % DLCO, whereas CXCL9/monokine induced by interferon-γ (CXCL9/Mig), granulocyte- colony stimulating factor (G-CSF) and IL-13 showed inverse relationships with the % DLCO. Conclusion Systemic inflammation in a COPD cohort is characterized by cytokines implicated in inflammatory cell recruitment and airway remodeling. Plasma concentrations of IL-13 and chemoattractants for monocytes, T lymphocytes, and eosinophils show associations with increasing severity of disease. Soluble Fas, G-CSF and CXCL9/Mig may be unique markers that associate with disease characterized by disproportionate abnormalities in DLCO independent of the FEV1.

We have previously reported that rat peritoneal macrophages stimulated with LPS release a factor (MNCF) which induces neutrophil migration which is not blocked by glucocorticoids. The initial characterization of MNCF showed that it did not correspond to any of the current known chemotactic cytokines or to known chemotactic agents. Recent studies have shown that neutrophil migration to the inflammatory focus may involve protein molecules with carbohydrate-binding sites, the LEC-CAMs. This fact prompted us to investigate the possible lectin-like nature of MNCF. Thus, the supernatants of macrophage monolayers stimulated with LPS were submitted to affinity chromatography on immobilized sugar columns. We observed that the D-galactose (D-gal) binding fraction retained the MNCF activity. This fraction, consisting of four protein components, was submitted to chromatography on Sephadex G-75, yielding a homogeneous preparation of the active component. MNCF has a MW of 54 KDa (gel filtration and SDS-PAGE) and a pI of 2-3 (isoelectrofocusing and chromatofocusing). A direct mechanism of action of MNCF on neutrophil migration initially suggested by the absence of blockade of the phenomenon in vivo by glucocorticoids, was confirmed by the observation that a protein-carbohydrate interaction is involved in this activity. Homogeneous preparation of MNCF induced neutrophil migration in vivo and in vitro. The in vitro effect was blocked by D-gal which did not interfere with the chemotactic effects of known interleukins (IL-1β, TNF-α, IL-6 and IL-8). The present results strengthen our previous suggestion that MNCF is yetundescribed monokine which induces neutrophil migration through a direct mechanism involving the D-gal binding site of the molecule.

Introduction Excessive mechanical loading of intervertebral discs (IVDs) is thought to alter matrix properties and influence disc cell metabolism, contributing to degenerative disc disease and development of discogenic pain. However, little is known about how mechanical strain induces these changes. This study investigated the cellular and molecular changes as well as which inflammatory receptors and cytokines were upregulated in human intervertebral disc cells exposed to high mechanical strain (HMS) at low frequency. The impact of these metabolic changes on neuronal differentiation was also explored to determine a role in the development of disc degeneration and discogenic pain. Methods Isolated human annulus fibrosus (AF) and nucleus pulposus (NP) cells were exposed to HMS (20% cyclical stretch at 0.001 Hz) on high-extension silicone rubber dishes coupled to a mechanical stretching apparatus and compared to static control cultures. Gene expression of Toll-like receptors (TLRs), neuronal growth factor (NGF) and tumour necrosis factor α (TNFα) was assessed. Collected conditioned media were analysed for cytokine content and applied to rat pheocromocytoma PC12 cells for neuronal differentiation assessment. Results HMS caused upregulation of TLR2, TLR4, NGF and TNFα gene expression in IVD cells. Medium from HMS cultures contained elevated levels of growth-related oncogene, interleukin 6 (IL-6), IL-8, IL-15, monocyte chemoattractant protein 1 (MCP-1), MCP-3, monokine induced by γ interferon, transforming growth factor β1, TNFα and NGF. Exposure of PC12 cells to HMS-conditioned media resulted in both increased neurite sprouting and cell death. Conclusions HMS culture of IVD cells in vitro drives cytokine and inflammatory responses associated with degenerative disc disease and low-back pain. This study provides evidence for a direct link between cellular strain, secretory factors, neoinnervation and potential degeneration and discogenic pain in vivo.

Monokine induced by IFN-γ (Mig) is also called CXCL9. It is a CXC-chemokine and reported to have myelosuppressive activity just as many other CXC-chemokines. Murine mig was identified in our large scale gene expression array screening for candidate regulators of bone marrow (BM) regeneration. We hypothesized that Mig could suppress the proliferation of hematopoietic stem/progenitor cells in vivo, through which it may protect BM from cancer chemotherapy drugs such as 5-fluorouracil (5-Fu) that causes BM damage as its major dose-limiting and life-threatening side effect. The recombinant murine Mig (rMuMig) administrated to normal mice was demonstrated to reduce the number of BM cells significantly by inhibition of G1-to-S phase transition of the cells. When the protein was injected into the mice before 5-Fu, it protected up to 60% of the mice from dying of high dose 5-Fu, compared to 10% of the PBS control. Furthermore, the BM hematopoiesis of the rMuMig treated mice recovered significant quicker than the control group according to the peripheral blood cell counts, BM cell counts, and the BM histology. We further investigated the mechanisms of myeloprotective role of rMuMig. FACS analysis revealed that rMuMig inhibited BM cells cycling into S-phase. The endogenous Mig and its receptor CXCR3 expression were up-regulated after 5-Fu treatment of the mice, indicating the Mig/CXCR3 signaling pathway is a natural hematopoietic protective mechanism. Neutralization the endogenous Mig with anti-Mig antibody accelerated the recovery of BM and increased the mice survival rate after 5-Fu treatment. In conclusion, we have identified that Mig/CXCR3 is an endogenous BM protection system. By mechanismbased proper use of the natural BM protective system, we demonstrate that rMuMig as well as rHuMig (recombinant human Mig) deliver the BM protection against the BM suppression side effect of cancer chemotherapy. Our studies suggest that Mig may be developed as a new chemo-protecting drug and may contribute greatly to the improvement of cancer therapy in combination with chemotherapy.

Background: SLC/CCL21, normally expressed in high endothelial venules and in T cell zones of spleen and lymph nodes, strongly attracts T cells and dendritic cells (DC). We have previously shown that SLC/CCL21-mediated anti-tumor responses are accompanied by significant induction of IFNγ and the CXC chemokines, monokine induced by IFNγ (MIG/CXCL9) and IFNγ-inducible protein-10 (IP-10/CXCL10). Results: We assessed the importance of IFNγ, IP-10/CXCL10 and MIG/CXCL9 in SLC/CCL21 therapy. In vivo depletion of IP-10/CXCL10, MIG/CXCL9 or IFNγ significantly reduced the antitumor efficacy of SLC/CCL21. Assessment of cytokine production at the tumor site showed an interdependence of IFNγ, MIG/CXCL9 and IP-10/CXCL10; neutralization of any one of these cytokines caused a concomitant decrease in all three cytokines. Similarly, neutralization of any one of these cytokines led to a decrease in the frequency of CXCR3 +ve T cells and CD11c +ve DC at the tumor site. Conclusion: These findings indicate that the full potency of SLC/CCL21-mediated anti-tumor responses require in part the induction of IFNγ, MIG/CXCL9 and IP-10/CXCL10.

Methotrexate (MTX) and mycophenolic acid (MPA) are used clinically for their immunosuppressive properties. MTX is widely used for the treatment of RA. MPA is used to prevent graft rejection and is now experimentally used in SLE and RA. The precise mechanism of action is still debated. Both drugs, though in different ways, inhibit the de novo synthesis of DNA and RNA. We have analysed cytokine production in short cell cultures in whole blood and isolated cells by ELISA. We have shown before that both drugs inhibit the production of several cytokines after T-cell stimulation, and we concluded that MTX leads to irreversible elimination of activated T cells by apoptosis, whereas MPA reversibly prevents activation of resting T cells. We now show that when monocytes are stimulated by SAC or lipopolysaccharide, both MTX and MPA decrease TNF-α, IL-6 and IL-8 production. However the inhibition is not as profound as after T-cell stimulation. An exception is the effect on the production of the proinflammatory cytokine IL-1β. The production of IL-1β is not influenced by MTX after SAC or LPS stimulation, whereas the production is increased by MPA when cells are stimulated with LPS, but not with SAC. We have investigated the cause for this increase. The expression of IL-1β mRNA is not influenced by MPA. Immunoprecipitation and ELISA show that MPA leads to a decrease in the intracellular proform of IL-1β. We conclude that MPA leads to enhanced cleavage of pro-IL-1β.

There is a sufficient amount of data that TNF-alpha, the monokine produced by macrophages after bacterial stimulation might be an important factor responsible for irreversible tissue damage during the septic shock. In an attempt to elucidate the possible function of TNF-alpha in the pathomechanism of sepsis the assesment of TNF-alpha activity in the blood of newborns with bacterial septicaemia was performed. A sample of 21 newborns with sepsis (13 with Staph.epi., 1 with Staph.aur., 7 with G(-) bacteria) was studied. A group of 22 infection-free infants matched by gestational age, birthweight and sex, were used as a controls. TNF-alpha in the blood was determined by ELISA method. For statistic analysis a non-parametric (Wilcoxon) test was used. Comparisons: A:B(p

Augmented IgE production and increased infections are often seen in allergic patients. As monocytes (MN) and polymorphonuclear leukocytes (PMN) are involved in both immune regulation and inflammatory reaction, MN function in terms of monokine production stimulated with lipopolysaccharide (MN supernatant; MN-sup) and its biological activity and the response of PMN to MN-sup and recombinant interleukin-1 (rIL-1) regarding chemotactic activity and expression of IgG Fc receptor (FcR) were studied in 26 normal children and 28 new and 22 hyposensitized (HS) asthmatic children. The results showed the following. There was no difference in IL-1 production, as assayed by thymocyte proliferation, among the three groups. All MN-sup from the three groups could enhance IL-2 production, but that of new patients was less efficient. In the absence of PWM, MN-sup of new patients greatly augmented the production of IgG, IgA, IgM, and IgE, but that of HS patients could enhance only IgE synthesis. MN-sup of patients enhanced less efficiently the chemotactic activity and FcR expression of PMN from healthy volunteers, and PMN from asthmatics responded much less vigorously to rIL-1 regarding the above-mentioned functions. The number of PMN with membrane IL-1 was much lower in allergic patients. Thus the abnormal MN and PMN functions may be used to explain partly the augmented IgE production and increased infections in allergic patients.

Recombinant human interleukin-1 beta (IL-1) and tumor necrosis factor-alpha (TNF) synergistically stimulated BALB/c 3T3 cells to produce a chemotactic factor for rat polymorphonuclear leukocytes (PMNs), whereas an addition of 10(-11)-10(-8) M IL-1 or TNF alone to the cell culture resulted in a slight increase in the production of chemotactic factor. The partially purified factor was not a chemokinetic but chemotactic factor for PMNs when analyzed by checkerboard analysis. The partially purified factor was trypsin sensitive and heat stable; its isoelectric point was 8.5-10, and its molecular weight was about 10 kDa as estimated by gel filtration. These results suggest that fibroblasts may participate in PMN migration to the inflammatory site where both IL-1 and TNF are released by activated inflammatory cells, including macrophages.

The effects of a highly purified tumor necrosis factor (TNF) on transplanted methylcholanthrene (Meth A)-induced murine tumors were compared with those of lipopolysaccharide (LPS). TNF caused immediate subepidermal edema and hyperemia followed 2 h later by fibrin thrombi in tumor blood vessels. Finally hemorrhagic necrosis with dispersal of tumor cells occurred. LPS produced similar hemorrhagic necrotizing changes. However, the necrotic action of LPS was delayed and complete tumor regression was not achieved with LPS. These findings suggest that tumor necrosis induced by TNF is due to circulatory disturbance associated with a microvascular injury in the tumor manifested by hyperemia and multiple fibrin thrombi.

When peripheral blood lymphocytes from patients with chronic active hepatitis were stimulated with liver specific lipoprotein (LSP), considerably higher frequencies of lymphocyte transformation and MIF production were induced. Peritoneal macrophages from guinea pigs were activated by lymphokine-containing lymphocyte culture supernatant and produced a cytotoxic (or cytostatic) factor acting on isolated hepatocytes in culture. The cytotoxic (or cytostatic) factor, which was fractionated by Sephadex G-75 column gel filtration followed by DEAE-cellulose column chromatography, had cytotoxic effect on isolated liver cells and produced a significant amount of lipid peroxide. These results suggested the possibility that the cytotoxic effects may be caused at least partially by the lipid peroxide formation.

The properties of synovial cells are altered in vitro by monocyte-macrophage polypeptides (monokines), and these changes could explain some of the properties of the inflamed synovium in rheumatoid disease. Purified monokines have become available only recently for testing on the target synovial cells. We report here that purified human interleukin (IL)-1 beta and recombinant human IL-1 alpha stimulate the extracellular activity of the lysosomal hydrolase, N-acetyl-beta-glucosaminidase (NAG), of human synovial fibroblast-like cells. In contrast, another monokine, synovial activator, does not increase the NAG activity. Thus NAG is another cellular activity which can be modulated by interleukin-1.

In vitro stimulation of mononuclear cells from human peripheral blood with mitogens causes the release of factors (monokines and lymphokines) which possess distinct biological activities. One such factor, termed 22K, can induce production of human beta-interferon (HuIFN-beta) in cultured human fibroblasts, thereby rendering these cells resistant to virus infection. Here we report the complete purification and partial sequencing (39 N-terminal amino acids) of this factor, whose relative molecular mass was estimated by SDS-polyacrylamide gel electrophoresis to be 17,000 (17K). In addition to an antiviral effect, the pure protein exhibits several other biological activities. Most significantly, intravenous (i.v.) injection of the factor in rabbits caused fever and granulopenia at doses of 0.1-1 microgram per kg, effects which we attribute to a monokine called endogenous pyrogen (EP). In vitro, the protein was scored as positive in a LAF (lymphocyte-activating factor) assay at 0.1-1 ng ml-1. LAF and EP are considered to be members of one family of monokines, called interleukin-1 (IL-1). For this reason, and also because the amino-acid sequence of the 22K factor is at least partially homologous to a complementary DNA-derived IL-1 sequence, we postulate that the 22K factor also belongs to the IL-1 family.

Interleukin-1 (IL-1) describes two inflammatory proteins, IL-1 alpha and IL-1 beta, produced by activated macrophages and other cell types and encoded by two genes. Their amino acid sequences have only 26% similarity, but their biological activities are comparable, with a few exceptions; indeed, both molecules appear to act at the same receptor. As IL-1 release prostaglandins which sensitize nociceptors in man and in experimental animals, we tested IL-1 alpha and IL-1 beta in rats for hyperalgesic (nociceptive) activity. Our results show that IL-1 beta given systemically is an extremely potent hyperalgesic agent with a probable peripheral site of action; IL-1 alpha is approximately 3,000 times less active than IL-1 beta. We have delineated the region of IL-1 beta mediating the hyperalgesic effect and developed an analgesic tripeptide analogue of IL-1 beta which antagonizes hyperalgesia evoked by IL-1 beta and by the inflammatory agent carrageenan.

Tumor necrosis factor (TNF)-alpha is a cytokine with multiple biological properties, particularly proinflammatory, apart from the induction of tumor necrosis. In order to elucidate the role of TNF-alpha in the pathogenesis of psoriasis, we have carried out bioassay and enzyme-linked immunosorbent assay for TNF-alpha in suction blister fluids and horny tissue extracts from psoriatic skin. Although bioassay showed some activities in the suction blister fluids and horny tissue extracts, there were no significant differences between the levels of activities from normal and psoriatic skin. They were at the background level and pretreatment of the samples with anti-TNF-alpha antiserum failed to abrogate the activities. ELISA confirmed the absence of TNF-alpha. Therefore, the present study could not verify that TNF-alpha plays a major role in the pathogenesis of psoriasis.

We had previously established a murine bone marrow-derived cell line, designated JBM phi 1.1, which displayed properties of normal macrophages, including the ability to perform macrophage-mediated cytolysis. It was also found that these cells could be induced by lipopolysaccharide (LPS) to produce reproducibly high levels of a cytolytic factor (CF) resembling tumor necrosis factor (TNF). This cell line was therefore selected for further studies on macrophage-mediated tumor cell lysis and CF production. Moreover, the CF production during incubation with LPS was higher in the absence of serum than in its presence, with a maximum at days 2-3 following the addition of LPS. A factor inhibitory to CF production (CIF) was detected in our laboratory in the supernatant of embryonic fibroblast cultures. We established the experimental conditions required for the optimal production and suppressive effect of CIF. High levels of CIF activity were obtained under conditions that promote fibroblast proliferation. Addition of embryonic fibroblast culture supernatant to the macrophages shortly before LPS suppressed both LPS-induced CF production and tumoricidal activity. CIF did not affect macrophage protein synthesis in the presence or absence of LPS. However, LPS-induced interleukin 1 release was partially (55%) suppressed by embryonic fibroblast culture supernatant. Our results show that CIF does not exert a general inactivating effect on the macrophages, although it may possibly affect other functions in addition to CF production and tumor cell lysis. The strong inhibition of both the latter properties further indicates that TNF-like CF is an important mediator in macrophage-mediated tumor cell lysis.

Human blood monocytes were isolated by counter-flow centrifugal elutriation from healthy donors and these noncytotoxic monocytes were rendered tumoricidal to allogeneic melanoma (A375) cells by activation with a synthetic acyltripeptide (FK-565), as assessed by measuring release of [125I]iododeoxyuridine in 72 h. When monocytes were treated with FK-565 for 16 h, and then fixed with paraformaldehyde, they showed cytotoxicity to A375 melanoma cells. The fixed-monocyte-mediated cytotoxicity to A375 cells was induced by the synergistic actions of FK-565 and recombinant interferon-gamma (rIFN-gamma), but not other cytokines [rIFN-alpha A, rIFN-beta, tumor necrosis factor (TNF), interleukin (IL)-2, -3 and -6]. For synergistic activation of monocytes with induction of a membrane-associated antitumor monokine, the monocytes had to be incubated first with rIFN-gamma and then with FK-565. FK-565 also acted synergistically with rIFN-gamma to stimulate monocytes to produce membrane-associated IL-1 activity, which induced C3H/HeJ thymocyte blastogenesis in response to phytohemagglutinin P. The tumoricidal and thymocyte-stimulating activities of the fixed monocytes were almost completely inhibited by a specific anti-(IL-1 alpha) antiserum, but not by a specific anti-(IL-1 beta) antiserum or monoclonal anti-TNF antibody. These results suggest that membrane-associated IL-1 alpha of human blood monocytes can be induced by two activation signals (rIFN-gamma then FK-565) at their suboptimal concentrations.

In this report, we examine the antigen nonspecific immunoregulating activity of macrophages isolated from the murine methylcholanthrene-induced fibrosarcoma FSA. These cells were shown to enhance the primary anti-CRBC PFC response of whole normal spleen cells in a dose-dependent fashion. This function was associated with a subpopulation of large Ia-negative macrophages and was mediated by a soluble macrophage-derived factor that appeared to act by stimulating the proliferation and/or differentiation of antigen-reactive T cells. The relationship of this factor to previously described monokines is discussed.