Distinct Patterns Of Cytokine Production Research Articles

Development of a dried pre-formulated antibody panel (7 colors/17antibodies) for the identification of human innate lymphoid cells by flow cytometry

Abstract Innate lymphoid cells (ILC) play an important role in innate immunity, lymphoid organogenesis and tissue remodeling. They form a heterogeneous population that can be categorized into 3 groups (i.e. ILC1, ILC2 and ILC3) based on their phenotype and/or distinct patterns of cytokine production. The identification and enumeration of different blood ILC subsets is important to understand immune regulation in disease models such as allergy, cancer and inflammation. Flow Cytometry is the method of choice for analysis of ILCs in human blood. However, as ILC frequencies may range below 0.1% of white blood cells, considerably elevated sample volumes and/or cell-enrichment is required. Furthermore, due to the lack of specific markers for definition of ILCs, at least 15 antibodies are required for mere identification of ILCs, including a lineage-exclusion channel to exclude most of the leukocytes. Based on current knowledge in human ILC biology, we have composed a respective set of markers to design a dry unitized 17 antibodies/7-color panel (CD294/Lineage/CD117/Nkp46/CD127/CD161/CD45) for flow cytometry. The panel in dry DURAClone format allows for the reliable identification of all ILCs subsets in whole blood or peripheral blood mononuclear cell fractions (PBMCs) in concordance with current scientific opinion. Due to the elimination of the pipetting of 17 antibodies, the method can substantially reduce variability and error related to sample preparation. Furthermore, the streamlined workflow can facilitate inter-laboratory transfer of the assays in multi-site studies.

Astaxanthin from Shrimp Cephalothorax Stimulates the Immune Response by Enhancing IFN-γ, IL-10, and IL-2 Secretion in Splenocytes of Helicobacter Pylori-Infected Mice.

Infection with Helicobacter pylori is a critical cause of gastrointestinal diseases. A crucial host response associated with H. pylori infection includes gastric inflammation, which is characterized by a sustained recruitment of T-helper (Th) cells to the site of infection and distinct patterns of cytokine production. Adequate nutritional status, especially frequent consumption of dietary antioxidants, appears to protect against infection with H. pylori. The aim of the present study was to investigate whether astaxanthin (AXT) from shrimp cephalothorax may modulate cytokine release of splenocytes in H. pylori-infected mice (n = 60). Six- to eight-week-old female mice were divided into three groups (n = 20 per group) to receive a daily oral dose of 10 or 40 mg of AXT for six weeks. After six weeks, a trend toward interferon gamma (IFN-γ) upregulation was found (40 mg; p < 0.05) and a significant dose-dependent increase of interleukin 2 (IL-2) and IL-10 (both p < 0.05) was observed. These results suggest that AXT induces higher levels of IL-2 and a shift to a balanced Th1/Th2 response by increasing IFN-γ and augmenting IL-10. We concluded that AXT may influence the pattern of cytokines during H. pylori infection.

Torque teno virus (TTV) in multiple sclerosis patients with different patterns of disease

Torque teno virus (TTV) is highly prevalent in the general population worldwide. The relationship that TTV establishes with the central nervous system (CNS) of infected hosts is not clear but it is suspected that TTV infection of the CNS lead to increased local expression of inflammatory mediators that may play a role in the pathogenesis of multiple sclerosis (MS). The prevalence and load of TTV in serum and cerebrospinal fluid (CSF) of 207 MS patients and 93 age- and sex-matched healthy controls by qPCR designed on the untranslated region were analyzed. TTV DNA was not detected in CSF, TTV prevalence in serum was similar in MS patients (76.8%) compared to healthy controls (76.3%). Sub analyses performed in MS patients stratified on the basis of clinical phenotypes indicated that TTV viremia was significantly lower in individuals with relapsing remitting compared to chronic progressive disease. Notably, viremia was increased in primary progressive, compared to secondary progressive MS patients, and in relapsing remitting MS patients during quiescent compared to relapsing phases of disease. Since TTV interacts with toll-like receptor (TLR) stimulating the production of inflammatory cytokines, TLR9 expression were examined, showing that it was augmented on monocytes of chronic progressive MS patients, in whom higher TTV viremia was present, but this did not correlate with a distinct pattern of cytokine production. Overall these findings suggest that, although TTV infects the same proportion of MS patients and healthy controls, the levels of replication of the virus differ among patients, being correlated with the clinical phenotype of disease.

Role of MAPK Kinase 6 in Arthritis: Distinct Mechanism of Action in Inflammation and Cytokine Expression

Development of p38alpha inhibitors for rheumatoid arthritis has been hindered by toxicity and limited efficacy. Therefore, we evaluated whether MKK6, an upstream kinase that regulates multiple p38 isoforms, might be an alternative therapeutic target in inflammatory arthritis. Wild-type (WT), MKK6(-/-), and MKK3(-/-) mice were administered K/BxN serum to induce arthritis. Articular expression of activated kinases and cytokines was determined by Western blot, qPCR, ELISA, and multiplex analysis. Immunoprecipitation and confocal microscopy experiments were performed to determine the subcellular location of MKK6, P-p38, and MAPKAPK2 (MK2). Arthritis scores were significantly lower in MKK6(-/-) mice compared with WT mice. Joint destruction and osteoclast differentiation were lower in MKK6(-/-), as were articular IL-6 and matrix metalloproteinase-3 expression. Phospho-p38 levels were modestly decreased in the joints of arthritic MKK6(-/-) mice compared with WT but were significantly higher than MKK3(-/-) mice. P-MK2 was low in MKK6(-/-) and MKK3(-/-) mice. Uncoupled p38 and MK2 activation was also observed in cultured, MKK6(-/-) FLS and confirmed using kinase assays. Immunoprecipitation assays and confocal microscopy showed that P-p38 and MK2 colocalized in activated WT but not MKK6(-/-) FLS. Distinct patterns of cytokine production were observed in MKK6(-/-) and MKK3(-/-) cells. MKK6 deficiency suppresses inflammatory arthritis and joint destruction, suggesting it might be a therapeutic target for inflammation. Although MKK3 and MKK6 activate the p38 pathway, they regulate distinct subsets of proinflammatory cytokines. MKK6 appears mainly to facilitate p38 and MK2 colocalization in the nucleus rather than to phosphorylate p38.

TH1/TH2 balance in concomitant immediate and delayed-type hypersensitivity diseases

In spite of the observation of mutual inhibitory properties of TH1 and TH2 CD4+ cells, a group of patients developed simultaneously immediate and delayed-type hypersensitivity reactions that are theoretically antagonistic. Patients presenting concomitant hypersensitivity reactions were evaluated for cytokine production. PBMC from 45 patients and 13 non-atopic individuals were cultured with mite allergen and mitogen and the supernatants obtained were evaluated for cytokine production by ELISA. The analysis of the cytokines levels revealed increased production of pro-inflammatory cytokine TNF-α in the non-atopic individuals after specific and mitogen stimulus. The IL-4 was largely observed on serum samples and IL-5 levels were higher in the double sensitized group (group DerpNi) after PHA stimulus. The IL-13 levels were increased in sensitized groups (Derp and DerpNi groups) after PHA stimuli. Atopic patients (Derp and DerpNi groups) presented lowest levels IFN-γ and the analysis of TGF-β production after rDER P I stimulation have shown increased levels among sensitized patients to Dermatophagoides pteronyssinus mite. IL-10 levels did not differ after antigen stimulation but basal production was higher on Derp and DerpNi groups. Furthermore, negative correlations were observed between IFN-γ levels and IL-4, IL-13 and IL-10. This study has shown patients able to react, concomitantly, to the two types of antigens – rDER P I and NiSO4, present distinct pattern of cytokine production. The increased levels of IL-13 in the sensitive individuals to mite antigen (rDER P I) and IFN-γ in NiSO4 sensitized individuals confirm the role of the type TH2 response in the atopies and TH1 type in DCA.

Inoculation of Bacillus Calmette-Guerin to mice induces an acute episode of sickness behavior followed by chronic depressive-like behavior

Although cytokine-induced sickness behavior is now well-established, the mechanisms by which chronic inflammation and depression are linked still remain elusive. Therefore this study aimed to develop a suitable model to identify the neurobiological basis of depressive-like behavior induced by chronic inflammation, independently of sickness behavior. We chose to measure the behavioral consequences of chronic inoculation of mice with Bacillus Calmette-Guerin (BCG), which has been shown to chronically activate both lung and brain indoleamine 2,3-dioxygenase (IDO), a tryptophan-catabolizing enzyme that mediates the occurrence of depressive-like behavior following acute innate immune system activation. BCG inoculation induced an acute episode of sickness (approximately 5 days) that was followed by development of delayed depressive-like behaviors lasting over several weeks. Transient body weight loss, reduction of motor activity and the febrile response to BCG were dissociated temporarily from a sustained increase in the duration of immobility in both forced swim and tail suspension tests, reduced voluntary wheel running and decreased preference for sucrose (a test of anhedonia). Moreover, we show that a distinct pattern of cytokine production and IDO activation parallels the transition from sickness to depression. Protracted depressive-like behavior, but not sickness behavior, was associated with sustained increase in plasma interferon-γ and TNF-α concentrations and peripheral IDO activation. Together, these promising new data establish BCG inoculation of mice as a reliable rodent model of chronic inflammation-induced depressive-like behaviors that recapitulate many clinical observations and provide important clues about the neurobiological basis through which cytokines may have an impact on affective behaviors.

Differences in innate immune responses upon stimulation with gram‐positive and gram‐negative bacteria

Host recognition pathways for gram-negative and gram-positive bacteria comprise pattern recognition receptors among which Toll-like receptors (TLRs) play a pivotal role. TLRs share common signaling pathways yet exhibit specificity as well. Periodontal disease is initiated and maintained in the first line by gram-negative but also gram-positive bacterial infection of the gingival sulcus. To date only limited information is available on whether gram-positive and gram-negative bacteria induce different host responses (strength or quality). To elucidate these differential effects we focused on proinflammatory cytokine releases by assessing ex vivo stimulation of whole blood with heat-killed gram-negative and gram-positive bacteria and thereof derived microbial products associated with distinct TLRs. Tumor necrosis factor-alpha and interleukin-8 release were measured in the supernatants by enzyme-linked immunosorbent assay. In addition, innate immune responses of peritoneal macrophages from mice lacking TLR2 and TLR4 were tested. We observed that gram-negative and gram-positive species induced distinct patterns of cytokine production. Gram-negative species produced higher amounts of tumor necrosis factor-alpha while gram-positive species released higher amounts of the chemokine interleukin-8. Data from TLR knockout mice and TLR-transfected HEK cells revealed a somehow specific role of TLR4 and TLR2 for the recognition of gram-negative and gram-positive bacteria, respectively, an observation that goes along with the dominant recognition of the respective pathogen associated molecular patterns lipopolysaccharide and lipoteichoic acid. The results show that gram-negative and gram-positive bacterial species induce different patterns of immunoregulatory activity, which might be the result of activation of different TLRs.

A distinct pattern of cytokine production from blood mononuclear cells in multitransfused patients with β-thalassaemia

The unstimulated and induced production of granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), IL-3, IL-6, stem cell factor (SCF), IL-1beta, tumour necrosis factor-alpha (TNF-alpha), TNF-beta, interferon-gamma (IFN-gamma) and transforming growth factor-beta (TGF-beta) was determined after culture of blood mononuclear cells from 22 patients with severe beta-thalassaemia in a regular transfusion programme, five non-regularly transfused patients with beta-thalassaemia intermedia and nine normal persons. A distinct pattern of cytokine production in thalassaemic patients was detected, namely a low unstimulated production of all cytokines and a significant increase in the stimulated production of IFN-gamma, TNF-alpha and IL- 1beta; these abnormalities were more pronounced in the more heavily transfused older patients. The increased production of the above cytokines, which usually characterize the acute response to infectious agents and have a negative effect on erythropoiesis, may explain the deterioration of anaemia found in thalassaemic patients during acute infections.

In Vitro Secretion of Cytokines by Human Bone Marrow: Effects of Age and Estrogen Status1

It has been proposed that cytokines mediate the acceleration of bone loss following menopause. Because of the intimate relationship between bone marrow stromal cells and bone tissue, it is possible that marrow cells and their products contribute to the bone microenvironment and influence the regulation of bone cell differentiation and activity. We examined the production of cytokines by bone marrow stromal cells from a total of 37 women and 15 men undergoing total hip replacement for noninflammatory joint disease. Low-density mononuclear cells were isolated from bone marrow and were cultured in phenol red-free alpha MEM medium supplemented with 10% FBS and antibiotics. Constitutive secretion of interleukin-6 (IL-6) was positively correlated with age in a series of 8 women and 5 men measured by bioassay (r = 0.98; P < 0.01) and in a series of 18 women and 10 men measured by immunoassay (r = 0.56; P < 0.01). The pattern of cytokine production by bone marrow stromal cells was examined in detail in 23 postmenopausal women, aged 49-88 yr. Basal secretion of immunoreactive IL-6 and IL-11, but not granulocyte-macrophage colony-stimulating factor, increased with time in culture. Exogenous IL-1 beta stimulated secretion of IL-6 and IL-11 in a saturable, dose-dependent manner. Secretion of soluble IL-6 receptor was not correlated with secretion of IL-6, either constitutively or in the presence of IL-1 beta. In 4 of 14 samples, IL-1 beta also stimulated secretion of granulocyte-macrophage colony-stimulating factor. IL-1 beta was undetectable in 7 of 9 cultures during the 2-week culture period. IL-6 did not stimulate secretion of IL-1 beta in the 7 cultures tested. Cells were dependent upon serum for viability and growth and were not sustained by a serum substitute (1% insulin-transferrin-selenium-BSA). Cells grown in medium with 10% FBS and supplemented with 1% insulin-transferrin-selenium-BSA secreted 10-fold more IL-6 than cells grown in serum alone. Marrow from 7 women receiving estrogen replacement therapy showed lower constitutive secretion of IL-6 (75%; P < 0.006) and IL-11 (43%; P < 0.05) than marrow from age-matched controls and had blunted stimulation of IL-6 and IL-11 secretion by exogenous IL-1 beta. These data indicate distinct patterns of cytokine production by human marrow stromal cultures dependent upon age and estrogen status.

A distinct pattern of cytokine production from blood mononuclear cells in multitransfused patients with β-thalassaemia: Salsaa B, Zoumbos NC. Clin Exp Immunol 107:589–592, 1997

A distinct pattern of cytokine production from blood mononuclear cells in multitransfused patients with β-thalassaemia: Salsaa B, Zoumbos NC. Clin Exp Immunol 107:589–592, 1997

Human T‐cell responses to Trichophyton tonsurans: inhibition using the serum free medium Aim V

Proteins of the fungus Trichophyton tonsurans have been shown to give strong T cell proliferative responses in vitro using lymphocytes from individuals with immediate or delayed skin tests. Furthermore, Trichophyton-specific T-cell lines produce distinct patterns of cytokine production depending upon the skin-test reactivity of the host. However, skin-test negative individuals generally give limited responses. A recent study has demonstrated dust mite specific proliferation with lymphocytes from atopic and non-atopic subjects using the serum free medium Aim V. Compare the T-cell reactivity to Trichophyton and dust mite antigens in Aim V and media containing 10% pooled AB serum. Peripheral blood mononuclear cells (PBMC) were separated from subjects with different skin-test reactivities and cultured either in media with serum or serum free media. Proliferation to two Trichophyton extracts was decreased in serum free media among subjects with either immediate or delayed hypersensitivity. Trichophyton skin-test negative subjects gave poor proliferative responses in both culture conditions. A similar decrease in proliferation in serum free media was observed in cultures with PHA and tetanus toxoid. In contrast, the majority of individuals showed increased proliferation to dust mite antigens when cultured in serum free media. Cultures in serum free medium produced less IFNgamma, IL-4, or IL-5 compared with cultures in AB medium. In vitro T-cell responses to the dermatophyte fungus T. tonsurans are inhibited by the serum free medium Aim V. This inhibition is seen equally with cells from individuals with delayed and immediate hypersensitivity. Furthermore, the results do not support the view that AB serum is masking T-cell responses in skin-test negative individuals.

In vivo molecular analysis of cytokines in a murine model of ocular onchocerciasis I. Up-regulation of IL-4 and IL-5 mRNAs and not IL-2 and IFNγ mRNAs in the cornea due to experimental interstitial keratitis

Sclerosing keratitis is the major cause of blindness due to onchocerciasis which results from chronic infection with the filarial parasite Onchocerca volvulus. Using a murine model of onchocercal sclerosing keratitis, we have demonstrated previously that predominantly (>85%) CD3 + CD4 + T-cells as well as the IL-2 receptor bearing cells infiltrate into the cornea in vivo during development and progress of the disease. The identification of CD4 + subsets T h 1 and T h 2 based on the cytokine secretion patterns of murine T-lymphocytes has been useful for understanding the immune basis of resistance and pathogenesis in murine models of several parasitic diseases. The present investigation was carried out to demonstrate whether the local immune response at the corneal lesion due to onchocercal interstitial keratitis correlated with such distinct patterns of cytokine production. For that purpose, mRNA was extracted separately from corneas obtained from the diseased eyes and the normal eyes of A J mice with onchocercal interstitial keratitis, reverse transcribed and amplified by the polymerase chain reaction with four different cytokine specific primers. In corneas obtained from the eyes affected with onchocercal interstitial keratitis, mRNAs coding for IL-4 and IL-5 were up-regulated compared to the normal eyes having no lesions from the same animals. However, the levels of mRNAs for IL-2 and IFNγ were found to be the same in the diseased and normal eyes. Taken together, these data suggest that IL-4 and IL-5 producing T h 2-lymphocytes are active at the corneal lesion due to onchocercal interstitial keratitis.

Th1 and Th2 cytokine induction in pulmonary T cells during infection with respiratory syncytial virus.

Helper T (Th) cells can be classified functionally into two main types. Broadly, Th1 cells play a major role in eliminating viral pathogens, while Th2 cells mediate anti-parasite immunity and allergic responses. These functions are thought to depend on characteristic and distinct patterns of cytokine production. Infection with human respiratory syncytial virus, an important common cold virus, causes transient lymphocytic bronchiolitis in mice. Activated T cells are partly responsible for this disease, but also eliminate the virus. To show whether polarized cytokine production occurs in individual cells during viral bronchiolitis, we sampled murine bronchoalveolar lavage and mediastinal lymph node cells before and after infection. RT-PCR of cellular mRNA and flow cytometric analysis of intracellular cytokine production showed a rapid IFN-gamma response at both sites, which persisted for more than 3 weeks in the lung. Most IFN-gamma-producing cells were CD8+. Some early CD4+ IFN-gamma-producing cells also made IL-10. Only low levels of IL-2, IL-4 and IL-5 mRNA or protein expression were detected at any time at either site. No cytokines were detected in B cell populations at either site. These novel techniques show the true complexity of cytokine production patterns on a cell-by-cell basis, allowing T cells to be reclassified according to function.

Enhanced T-helper 2 lymphocyte responses: immune mechanism of Helicobacter pylori infection.

Several lines of evidence implicate Helicobacter pylori (H. pylori) infection in gastroduodenal inflammation. However, the exact pathogenesis of H. pylori infection is not fully understood. T-helper (TH) lymphocytes may be subdivided into TH1 and TH2 cells based on the distinct patterns of cytokine production. TH1 reaction is associated with immunity or resistance to infection, while TH2 reaction is associated with the progression or persistence of infection. The production of interferon-gamma (INF-gamma) and interleukin 2 (IL-2), which are type 1 cytokines, is decreased in H. pylori infection. Enhanced production of type 2 cytokines (IL-4) and IL-6) is observed in individuals with H. pylori infection. Suppressed proliferative responses of peripheral blood and gastric lymphocytes have also been demonstrated in patients with H. pylori colonisation, suggesting that specific T-cell responses may be down-regulated by an enhanced TH2 reaction. Suppressed TH1 and enhanced TH2 responses in H. pylori infection may be involved in the immunopathogenesis of chronic H. pylori infection.

CD4 + T Cell Clones Isolated from Human Renal Cell Carcinoma Possess the Functional Characteristics of Th2 Helper Cells

The underlying cause for the observed poor antitumor activity of lymphocytes resident, in situ, in freshly resected human renal cell carcinoma (RCC) is unknown. To examine the basis for this observation we evaluated 217 T cell clones established from 20 consecutive patients with renal cell carcinoma. Of these, 75% were CD4 +, 22% were CD8 +, and 3% were NK +. Cytotoxic T cell function of CD4 + and CD8 + T cell clones was assessed by antibody-induced triggering of the TcR/CD3 complex. Nearly 93% of the CD4 + clones possessed poor cytolytic activity defined as <50% killing (E:T, 50:1). Alternatively, nearly 80% of CD8 + clones possessed strong cytolytic activity defined as ⩾50% killing. In all T cell clones tested, MHC nonrestricted killing against Daudi or K562 was not observed. Overall, 77% of the T cell clones isolated from patients with RCC in this study were characterized by poor antitumor cytotoxic function; only 22% of the clones displayed strong cytolytic activity. Clones with strong cytotoxic function were tested for cytotoxic function against autologous tumor. However, all clones tested demonstrated little to no tumoricidal activity against autologous tumor. These results indicate that cytolytic T cells, while present in RCC, are not effectively activated by tumor to express CTL function. The immunobiology of CD4 + T cell clones was further evaluated. Thirty-two CD4 + clones from seven patients revealed distinct patterns of cytokine production following TcR/CD3 activation. CD4 + clones could be divided into IL-2 nonproducers and IL-2 producers. A total of 27/32 clones did not produce IL-2 following activation and IFN-γ production by IL-2 nonproducers was move than threefold less than that by IL-2 producers. However, no differences were found in the levels of IL-4, IL-6, GM-CSF, or TNF-α between the two groups. Antitumor cytotoxicity mediated by CD4 + clones did not correlate with cytokine production. These results demonstrate that among T cells resident in human RCC, the predominant type of lymphocyte population consists of noncytolytic helper CD4 + T cells capable of secreting IL-4, but importantly not IL-2, and low levels of IFN-γ following activation, thus resembling murine Th2 cells. Only a minor contribution by Th0-like cells was observed (i.e., CD4 + clones capable of secreting IL-2, IL-4, and IFN-γ following activation). These results suggest that the immunologic failure to contain RCC is related to the infiltration of T cell subsets, the majority of which resemble murine Th2, which cannot provide T cell help for the generation of CTL responses. In addition, although a small fraction of resident lymphocytes are CD8 + T cells that possess cytolytic function, antigen receptor triggering of this cell population by autologous tumor is not an effective stimulus for the induction of cytotoxic effector cell function.

In vivo cytokine profiles in patients with kala-azar. Marked elevation of both interleukin-10 and interferon-gamma.

The immunological mechanisms underlying the susceptibility to disseminated visceral parasitism of mononuclear phagocytes in patients with kala-azar remain undefined. Resistance and susceptibility are correlated with distinct patterns of cytokine production in murine models of disseminated leishmanial disease. To assess lesional cytokine profiles in patients with kala-azar, bone marrow aspirates were analyzed using a quantitative reverse transcriptase PCR technique to amplify specific mRNA sequences of multiple Th1-, Th2-, and/or macrophage-associated cytokines. Transcript levels of IL-10 as well as IFN-gamma were significantly elevated in patients with active visceral leishmaniasis; IL-10 levels decreased markedly with resolution of disease. These findings suggest that IL-10, a potent, pleiotropic suppressor of all known microbicidal effector functions of macrophages, may contribute to the pathogenesis of kala-azar by inhibiting the cytokine-mediated activation of host macrophages that is necessary for the control of leishmanial infection.

Selective induction of Th2 cells in murine Peyer's patches by oral immunization.

We have used three different methods to determine the T helper (Th) cell response, including Th1 and Th2 types, in murine Peyer's patches (PP) following oral immunization with sheep red blood cells (SRBC). These include: (i) use of cytokine-specific (IFN-gamma and IL-5), single cell assays to estimate the frequencies of Th1 and Th2 cells respectively, (ii) cytokine-specific mRNA--cDNA dot blot and Northern gel hybridizations to detect levels of specific mRNA, and (iii) T cell cloning techniques to determine the frequency of Th1 and Th2 clones. Mice were immunized with SRBC by either the oral or i.p. route. The PP and splenic (SP) CD3+ and CD3+ CD4+ T cell subsets were isolated and cultured with antigen, feeder cells, and IL-2, and were assessed at various intervals (days 0, 1, 3, and 6) for numbers of T cells producing either IFN-gamma or IL-5 by use of an enzyme-linked immunospot (ELISPOT) procedure. Cultures of T cells from PP or SP of mice given SRBC by the oral route had a high frequency of IL-5 spot forming cells (SFC), with lower numbers of IFN-gamma SFC. However, cultures of CD3+ T cells and CD3+ CD4+ Th cells from spleens of i.p. immunized mice exhibited predominantly IFN-gamma SFC, with smaller but significant numbers of IL-5 SFC. This distinct pattern of cytokine production was supported by mRNA analysis where high IL-5 specific mRNA levels were noted in PP T cell cultures of orally primed mice, while IFN-gamma mRNA was predominant in the SP CD3+ T cell and CD3+ CD4+ Th cell cultures from i.p. immunized mice. When the frequencies of IFN-gamma or IL-5 SFC were assessed among cloned Th cells from orally- or systemically-immunized mice, 74% of Th cell clones from PP of mice orally immunized with SRBC were IL-5 producers (Th2 type), while 67% of Th cell clones from SP of mice immunized by the i.p. route were IFN-gamma producers (Th1 type). Our studies show that higher frequencies of IFN-gamma producing Th1-type cells occur in SP of mice given antigen by the systemic route, while oral immunization results in predominantly IL-5 producing, Th2-type cells in PP.

Defining protective responses to pathogens: cytokine profiles in leprosy lesions

The immunological mechanisms required to engender resistance have been defined in few infectious diseases of man, and the role of specific cytokines is unclear. Leprosy presents clinically as a spectrum in which resistance correlates with cell-mediated immunity to the pathogen. To assess in situ cytokine patterns, messenger RNA extracted from leprosy skin biopsy specimens was amplified by the polymerase chain reaction with 14 cytokine-specific primers. In lesions of the resistant form of the disease, messenger RNAs coding for interleukin-2 and interferon-gamma were most evident. In contrast, messenger RNAs for interleukin-4, interleukin-5, and interleukin-10 predominated in the multibacillary form. Thus, resistance and susceptibility were correlated with distinct patterns of cytokine production.

Defining Protective Responses to Pathogens: Cytokine Profiles in Leprosy Lesions

The immunological mechanisms required to engender resistance have been defined in few infectious diseases of man, and the role of specific cytokines is unclear. Leprosy presents clinically as a spectrum in which resistance correlates with cell-mediated immunity to the pathogen. To assess in situ cytokine patterns, messenger RNA extracted from leprosy skin biopsy specimens was amplified by the polymerase chain reaction with 14 cytokine-specific primers. In lesions of the resistant form of the disease, messenger RNAs coding for interleukin-2 and interferon-gamma were most evident. In contrast, messenger RNAs for interleukin-4, interleukin-5, and interleukin-10 predominated in the multibacillary form. Thus, resistance and susceptibility were correlated with distinct patterns of cytokine production.

Regulation of interleukin-1 beta and tumor necrosis factor secretion by the human monocytic leukemia cell line, THP-1.

Activated macrophages synthesize and release the potent polypeptides, interleukin-1 (IL-1) and tumor necrosis factor (TNF). In an effort to identify the cellular signals which control cytokine production by activated macrophages, we have developed an in vitro model employing the human THP-1 cell line. In the present study, THP-1 cells "primed" by 1.6 microM phorbol 12-myristate-13-acetate (TPA) for 4 hr demonstrated a dose- and time-dependent release of IL-1 beta and TNF upon activation by 20 micrograms/ml LPS. BSA/anti-BSA-coated latex beads were also a potent stimulus for IL-1 beta secretion. Moreover, the combination of a suboptimal concentration of LPS (200 ng/ml) plus interferon-gamma (0.03-333 U/ml) greatly enhanced IL-1 beta production. Resting THP-1 monocytes not "primed" by TPA did not secrete IL-1 beta or TNF. These distinct patterns of cytokine production may be related to the developmental stages of macrophage activation.