Patterns of immune development in urban preschoolers with recurrent wheeze and/or atopy.

BACKGROUND: Disadvantaged urban children have high rates of allergic diseases and wheezing, which are diseases associated with type 2-biased immunity. OBJECTIVE: We sought to determine whether environmental exposures in early life influence cytokine responses that affect the development of recurrent wheezing illnesses and allergic sensitization. METHODS: A birth cohort of 560 urban families was recruited from neighborhoods with high rates of poverty, and 467 (83%) children were followed until 3 years of age. Cytokine responses were measured in blood cell samples obtained at birth (cord blood) and ages 1 and 3 years. Cytokine responses were examined in relation to personal characteristics and environmental exposures to allergens and endotoxin and to the development of allergic sensitization and recurrent wheeze assessed at age 3 years. RESULTS: Cytokine responses generally increased with age, but responses at birth were poorly predictive for those at ages 1 and 3 years. Exposure to certain allergens (cockroach, mouse, dust mite) was significantly associated with enhanced cytokine responses at age 3 years, including IFN-α and IL-10 responses to certain stimulants and responses to phytohemagglutinin. Regarding the clinical outcomes, reduced LPS-induced IL-10 responses at birth were associated with recurrent wheeze. In contrast, reduced respiratory syncytial virus-induced IL-8 responses and increased 5'-cytosine-phosphate-guanine-3' (CpG)-induced IL-12p40 and allergen-induced IL-4 responses were associated with atopy. CONCLUSIONS: These findings suggest that diverse biologic exposures, including allergens and endotoxin, in urban homes stimulate the development of cytokine responses in early life, and that cytokine responses to specific microbial and viral stimuli are associated with the development of allergic sensitization and recurrent wheeze.

Role of IL-16 in CD4+ T cell-mediated regulation of relapsing multiple sclerosis.

In an important article published in Nature Medicine, Liu and colleagues described a novel CD4(+) FoxA1(+) regulatory T (Treg) cell population as distinct regulators of relapsing-remitting multiple sclerosis (RRMS) and experimental autoimmune encephalomyelitis (EAE). CD4(+) FoxA1(+) Treg cells appear as key regulators of responsiveness to therapy with interferon beta (IFN-ß) in RRMS patients. Data indicate that CD4(+)FoxA1(+) FOXP3(-) Treg cells develop within the central nervous system (CNS), and a potential of cerebellar granule neurons (CGN) in generation of CD4(+)FoxA1(+)PD-L1(hi)FOXP3(-) Treg cells from encephalitogenic CD4(+) T cells. A CD4 co-receptor specific ligand, IL-16, governs trafficking and biological properties of CD4(+) T cells irrespective of their activation state. Functions of IL-16, relevant to Treg cells, include expansion of CD4(+)CD25(+) T cells in long-term cultures with IL-2, de novo induction of FOXP-3 and migration of FOXP-3(+) T cells. IL-16 is highly conserved across species including human and mouse. CGN and neurons in hippocampus contain neuronal-IL-16 (NIL-16), splice variant of immune IL-16, and express CD4 molecule. In a CD4-dependent manner, IL-16 supports cultured CGN survival. Concomitant studies of RRMS lesions and corresponding MOG35-55-induced relapsing EAE in (B6 × JL)F1 (H-2(b/s)) mice discovered similar roles of IL-16 in regulation of relapsing disease. In RRMS and EAE relapse, peak levels of IL-16 and active caspase-3 correlated with CD4(+) T cell infiltration and levels of T-bet, Stat-1(Tyr(701)), and phosphorylated neurofilaments of axonal cytoskeleton [NF (M + H) P], suggesting a role of locally produced IL-16 in regulation of CD4(+) Th1 inflammation and axonal damage, respectively. IL-16 was abundantly present in CD4(+) T cells, followed by CD20(+) B, CD8(+) T, CD83(+) dendritic cells, and Mac-1(+) microglia. Apart from lesions, bioactive IL-16 was located in normal-appearing white matter (NAWM) and normal-appearing grey matter (NAGM) in RRMS brain and spinal cord. A cytokine IL-16 emerges as an important regulator of relapsing MS and EAE. Better understanding of immune cell-neuron interactions mediated by IL-16 will foster development of more specific CD4(+) T cell subset-targeted therapies to prevent or ameliorate progression of neuroinflammation and axonal and neuronal damage. Translational studies necessitate corresponding EAE models.

Emerging role of IL-16 in cytokine-mediated regulation of multiple sclerosis.

Cytokines are pleiotropic soluble mediators of cellular functions. Cytokines are critical in immune pathogenesis of human diseases, including autoimmune CD4(+) T cell mediated chronic inflammatory, demyelinating and neurodegenerative diseases of the central nervous system (CNS), multiple sclerosis (MS). In MS and its experimental model, experimental autoimmune encephalomyelitis (EAE), chronic persistence and/or reoccurrence of inflammation in the CNS causes chronic progressive or relapsing disease, accompanied with demyelination and damage to axons and oligodendrocytes, which ultimately leads to paralysis and disability. As opposed to other cytokines, whose effects are not limited to the CD4(+) T cell subset, IL-16 exerts its biological properties by exclusive binding and signaling through CD4 receptor. IL-16 selectively regulates migration of all CD4 expressing T cells regardless of their activation state, which is of critical importance for immune modulation and potential therapy of MS. Other major biological properties of IL-16 essential for the function of CD4(+) T cells include regulation of: T cell activation, CD25 expression, MHC class II expression, dendritic cell (DC)-T cell cooperation, B cell-T cell and T cell-T cell cooperation, inflammatory cytokine production and modulation of chemokine regulated T cell chemo-attraction. In this article we outline immune pathogenesis of the disease necessary to understand significance of cytokines and IL-16 in MS regulation. We revisit cytokine regulation with emphasis on involvement of IL-16 mechanisms, implicated in MS progression and important for development of new therapies. We emphasize the significance of similar IL-16 mechanisms for other chronic inflammatory CNS diseases.

An NT4/TrkB-dependent increase in innervation links early-life allergen exposure to persistent airway hyperreactivity.

Children who are exposed to environmental respiratory insults often develop asthma that persists into adulthood. In this study, we used a neonatal mouse model of ovalbumin (OVA)-induced allergic airway inflammation to understand the long-term effects of early childhood insults on airway structure and function. We showed that OVA sensitization and challenge in early life led to a 2-fold increase in airway smooth muscle (ASM) innervation (P<0.05) and persistent airway hyperreactivity (AHR). In contrast, OVA exposure in adult life elicited short-term AHR without affecting innervation levels. We found that postnatal ASM innervation required neurotrophin (NT)-4 signaling through the TrkB receptor and that early-life OVA exposure significantly elevated NT4 levels and TrkB signaling by 5- and 2-fold, respectively, to increase innervation. Notably, blockade of NT4/TrkB signaling in OVA-exposed pups prevented both acute and persistent AHR without affecting baseline airway function or inflammation. Furthermore, biophysical assays using lung slices and isolated cells demonstrated that NT4 was necessary for hyperreactivity of ASM induced by early-life OVA exposure. Together, our findings show that the NT4/TrkB-dependent increase in innervation plays a critical role in the alteration of the ASM phenotype during postnatal growth, thereby linking early-life allergen exposure to persistent airway dysfunction.

Relation between stress and cytokine responses in inner-city mothers.

BACKGROUND: Women in poor urban neighborhoods have high rates of stress and allergic diseases, but whether stress or stress correlates such as depression promote inflammatory and type 2 cytokine responses is unknown. OBJECTIVE: To examine associations among external stressors, perceived stress, depression, and peripheral blood mononuclear cell cytokine responses of mothers enrolled in the Urban Environment and Childhood Asthma Study and test the hypothesis that stress would be positively associated with type 2 and selected proinflammatory (tumor necrosis factor-α and interleukin-8) responses. METHODS: Questionnaire data from mothers living in 4 inner cities included information about external stress, stress perception, and depression. The external stress domains (interpersonal problems, housing, and neighborhood stress) were combined into a Composite Stressor score. Peripheral blood mononuclear cells were stimulated ex vivo and cytokine responses to innate, adaptive, and polyclonal immune stimuli were compared with stress and depression scores for 469 of the 606 study participants. RESULTS: There were no significant positive associations between Composite Stressor scores, perceived stress, or depression scores and proinflammatory or type 2 cytokine responses, and these findings were not modified by allergy or asthma status. There were some modest associations with individual stressors and cytokine responses, but no consistent relations were noted. Depression was associated with decreased responses to some stimuli, particularly dust mite. CONCLUSION: Composite measurements of stressors, perceived stress, or depression were not positively related to proinflammatory or type 2 cytokine responses in these young urban women. These data do not support the hypothesis that these factors promote cytokine responses associated with allergy. TRIAL REGISTRATION: ClinicalTrials.gov, identifier NCT00114881.

Mannose-capped lipoarabinomannan from Mycobacterium tuberculosis preferentially inhibits sphingosine-1-phosphate-induced migration of Th1 cells.

Chemokine receptor cross-desensitization provides an important mechanism to regulate immune cell recruitment at sites of inflammation. We previously reported that the mycobacterial cell wall glycophospholipid mannose-capped lipoarabinomannan (ManLAM) could induce human peripheral blood T cell chemotaxis. Therefore, we examined the ability of ManLAM to desensitize T cells to other chemoattractants as a potential mechanism for impaired T cell homing and delayed lung recruitment during mycobacterial infection. We found that ManLAM pretreatment inhibited in vitro migration of naive human or mouse T cells to the lymph node egress signal sphingosine-1-phosphate (S1P). Intratracheal administration of ManLAM in mice resulted in significant increases in T cells, primarily CCR5(+) (Th1) cells, in lung-draining lymph nodes. To investigate the selective CCR5 effect, mouse T cells were differentiated into Th1 or Th2 populations in vitro, and their ability to migrate to S1P with or without ManLAM pretreatment was analyzed. ManLAM pretreatment of Th1 populations inhibited S1P-induced migration but had no effect on Th2 cell S1P-directed migration, suggesting a differential effect by S1P on the two subsets. The PI3K/AKT inhibitor Ly294002 inhibited S1P-directed migration by Th1 cells, whereas the ERK inhibitor U0126 inhibited Th2 cell S1P-directed migration. These observations demonstrate that S1P-induced migratory responses in Th1 and Th2 lymphocytes occurs via different signaling pathways and suggests further that the production of ManLAM during Mycobacterium tuberculosis infection may function to sequester Th1 cells in lung-draining lymph nodes, thereby delaying their recruitment to the lung.

A genome-wide association study of plasma total IgE concentrations in the Framingham Heart Study.

BACKGROUND: Atopy and plasma IgE concentration are genetically complex traits, and the specific genetic risk factors that lead to IgE dysregulation and clinical atopy are an area of active investigation. OBJECTIVE: We sought to ascertain the genetic risk factors that lead to IgE dysregulation. METHODS: A genome-wide association study (GWAS) was performed in 6819 participants from the Framingham Heart Study (FHS). Seventy of the top single nucleotide polymorphisms (SNPs) were selected based on P values and linkage disequilibrium among neighboring SNPs and evaluated in a meta-analysis with 5 independent populations from the Cooperative Health Research in the Region of Augsburg cohort, the British 1958 Birth Cohort, and the Childhood Asthma Management Program cohort. RESULTS: Thirteen SNPs located in the region of 3 genes, FCER1A, signal transducer and activator of transcription 6 (STAT6), and IL13, were found to have genome-wide significance in the FHS cohort GWAS. The most significant SNPs from the 3 regions were rs2251746 (FCER1A, P = 2.11 × 10(-12)), rs1059513 (STAT6, P = 2.87 × 10(-8)), and rs1295686 (IL13, P = 3.55 × 10(-8)). Four additional gene regions, HLA-G, HLA-DQA2, HLA-A, and Duffy blood group, chemokine receptor (DARC), reached genome-wide statistical significance in a meta-analysis combining the FHS and replication cohorts, although the DARC association did not appear independent of SNPs in the nearby FCER1A gene. CONCLUSION: This GWAS of the FHS cohort has identified genetic loci in HLA genes that might have a role in the pathogenesis of IgE dysregulation and atopy. It also confirmed the association of the known susceptibility loci FCER1A, STAT6, and IL13 for the dysregulation of total IgE.

Comparison of the etiology of viral respiratory illnesses in inner-city and suburban infants.

BACKGROUND: The risk of developing childhood asthma has been linked to the severity and etiology of viral respiratory illnesses in early childhood. Since inner-city infants have unique environmental exposures, we hypothesized that patterns of respiratory viral infections would also be distinct. METHODS: We compared the viral etiology of respiratory illnesses in 2 groups: a cohort of 515 infants from 4 inner-city areas and a cohort of 285 infants from mainly suburban Madison, Wisconsin. Nasal secretions were sampled during periods of respiratory illness and at 1 year of age and were analyzed for viral pathogens by multiplex polymerase chain reaction. RESULTS: Overall, inner-city infants had lower rates of viral detection. Considering specific viruses, sick urban infants had lower rates of detectable rhinovirus or respiratory syncytial virus infection and higher rates of adenovirus infection. Every urban site had a higher proportion of adenovirus-positive samples associated with illnesses (10%-21%), compared with Madison (6%). CONCLUSIONS: These findings provide evidence that inner-city babies have different patterns of viral respiratory illnesses than babies who grow up in a more suburban location. These findings raise important questions about the etiology of virus-negative illnesses in urban infants and the possibility of long-term consequences of early life infections with adenovirus in this population.

Mannose-capped Lipoarabinomannan from Mycobacterium tuberculosis induces soluble tumor necrosis factor receptor production through tumor necrosis factor alpha-converting enzyme activation.

Primary Mycobacterium tuberculosis infection results in granuloma formation in lung tissue. A granuloma encapsulates mycobacterium-containing cells, thereby preventing dissemination and further infection. Tumor necrosis factor alpha (TNF-α) is a host-protective cytokine during M. tuberculosis infection due to its role in promoting and sustaining granuloma formation. TNF activity is regulated through the production of soluble TNF receptors (sTNFRI and sTNFRII). Therefore, we examined the potential production of endogenous sTNFRs during M. tuberculosis infection. Using the murine model of aerosol M. tuberculosis infection, we determined that levels of sTNFR production were elevated in bronchoalveolar lavage fluid 1 month following infection. An investigation of M. tuberculosis cell wall components identified that the known virulence factor mannose-capped lipoarabinomannan (ManLAM) was sufficient to induce sTNFR production, with sTNFRII being produced preferentially compared with sTNFRI. ManLAM stimulated the release of sTNFRs without TNF production, which corresponded to an increase in TNF-α-converting enzyme (TACE) activity. To determine the relevance of these findings, serum samples from M. tuberculosis-infected patients were tested and found to have an increase in the sTNFRII/sTNFRI ratio. These data identify a mechanism by which M. tuberculosis infection can promote the neutralization of TNF and furthermore suggest the potential use of the sTNFRII/sTNFRI ratio as an indicator of tuberculosis disease.

Diesel exhaust particulates exacerbate asthma-like inflammation by increasing CXC chemokines.

Particulate matter heavily pollutes the urban atmosphere, and several studies show a link between increased ambient particulate air pollution and exacerbation of pre-existing pulmonary diseases, including asthma. We investigated how diesel exhaust particulates (DEPs) aggravate asthma-like pulmonary inflammation in a mouse model of asthma induced by a house dust extract (HDE) containing cockroach allergens and endotoxin. BALB/c mice were exposed to three pulmonary challenges via hypopharyngeal administration of an HDE collected from the home of an asthmatic child. One hour before each pulmonary challenge, mice were exposed to DEP or PBS. Pulmonary inflammation was assessed by histological features, oxidative stress, respiratory physiological features, inflammatory cell recruitment, and local CXC chemokine production. To prove the role of CXC chemokines in the augmented inflammation, CXC chemokine-specific antibodies were delivered to the lungs before DEP exposure. DEP exacerbated HDE-induced airway inflammation, with increased airway mucus production, oxidative stress, inflammatory cell infiltration, bronchoalveolar lavage concentrations of CXC chemokines, and airway hyperreactivity. Neutralization of airway keratinocyte-derived chemokine and macrophage inflammatory protein-2 significantly improves the respiratory function in addition to decreasing the infiltration of neutrophils and eosinophils. Blocking the chemokines also decreased airway mucus production. These results demonstrate that DEP exacerbates airway inflammation induced by allergen through increased pulmonary expression of the CXC chemokines (keratinocyte-derived chemokine and macrophage inflammatory protein-2).

A homologous form of human interleukin 16 is implicated in microglia recruitment following nervous system injury in leech Hirudo medicinalis.

In contrast to mammals, the medicinal leech Hirudo medicinalis can completely repair its central nervous system (CNS) after injury. This invertebrate model offers unique opportunities to study the molecular and cellular basis of the CNS repair processes. When the leech CNS is injured, microglial cells migrate and accumulate at the site of lesion, a phenomenon known to be essential for the usual sprouting of injured axons. In the present study, we demonstrate that a new molecule, designated HmIL-16, having functional homologies with human interleukin-16 (IL-16), has chemotactic activity on leech microglial cells as observed using a gradient of human IL-16. Preincubation of microglial cells either with an anti-human IL-16 antibody or with anti-HmIL-16 antibody significantly reduced microglia migration induced by leech-conditioned medium. Functional homology was demonstrated further by the ability of HmIL-16 to promote human CD4+ T cell migration which was inhibited by antibody against human IL-16, an IL-16 antagonist peptide or soluble CD4. Immunohistochemistry of leech CNS indicates that HmIL-16 protein present in the neurons is rapidly transported and stored along the axonal processes to promote the recruitment of microglial cells to the injured axons. To our knowledge, this is the first identification of a functional interleukin-16 homologue in invertebrate CNS. The ability of HmIL-16 to recruit microglial cells to sites of CNS injury suggests a role for HmIL-16 in the crosstalk between neurons and microglia in the leech CNS repair.

Human immunodeficiency virus type 1 gp120 reprogramming of CD4+ T-cell migration provides a mechanism for lymphadenopathy.

Infection by human immunodeficiency virus type 1 (HIV-1) is associated with decreases in peripheral CD4(+) T cells and development of lymphadenopathy. The precise mechanisms by which HIV-1 induces these changes have not been elucidated. T-cell trafficking through lymphoid tissues is facilitated by CCL21-mediated entry and sphingosine-1-phosphate (S1P)-mediated egress. Having previously determined that HIV-1 envelop glycoprotein, gp120, directly alters T-cell migration, we investigated whether gp120 without HIV-1 infection could influence the responses of CD4(+) T cells to the signals involved in T-cell trafficking through lymph tissue. Incubation of normal human T cells with gp120 for 1 h resulted in reprogramming of CD4 T-cell migratory responses by increasing sensitivity to CCL20 and CCL21 and complete inhibition of migration to S1P. Incubation of human T cells with gp120 prior to injection into NOD.CB17-Prkdc(scid)/J mice resulted in increases in lymph node accumulation of CD4(+) T cells, with reciprocal decreases in blood and spleen compared to T cells not exposed to gp120. The effects of gp120 required CD4 signaling mediated through p56(lck). These findings suggest that gp120 alone can alter CD4(+) influx and efflux from lymph nodes in a fashion consistent with the development of lymphopenia and lymphadenopathy.

Parental characteristics, somatic fetal growth, and season of birth influence innate and adaptive cord blood cytokine responses.

BACKGROUND: Immunologic responses at birth likely relate to subsequent risks for allergic diseases and wheezing in infancy; however, the influences of parental characteristics and prenatal factors on neonatal immune responses are incompletely understood. OBJECTIVE: This study investigates potential correlations between urban parental, prenatal, and perinatal factors on innate and adaptive stimuli-induced cytokine responses. METHODS: Five hundred sixty and 49 children of parents with and without allergic disease or asthma, respectively, were enrolled into a prospective birth cohort study (Urban Environment and Childhood Asthma). Cord blood mononuclear cells were incubated with innate and adaptive immune stimuli, and cytokine responses (ELISA) were compared with season of birth, parental characteristics, in utero stressors, and fetal growth. RESULTS: Many cytokine responses varied by season of birth, including 2-fold to 3-fold fluctuations with specific IFN-alpha and IFN-gamma responses. Birth weight was inversely associated with IFN-gamma responses to respiratory syncytial virus (R = -0.16), but positively associated with IL-8 responses to a variety of innate stimuli (R = 0.08-0.12). Respiratory syncytial virus-induced cytokine responses were 21% to 54% lower in children of mothers with asthma. Cytokine responses were generally lower in babies born to parents with allergy/asthma. CONCLUSIONS: Innate cytokine responses are associated with parental allergic or airway disease, somatic fetal growth, ethnicity, and season of birth. Collectively, these findings suggest that urban prenatal exposures and familial factors affect the development of the fetal immune system.

Characterization of regulatory T cells in urban newborns.

BACKGROUND: In the United States, asthma prevalence is particularly high among urban children. Although the underlying immune mechanism contributing to asthma has not been identified, having impaired T regulatory (Treg) cells at birth may be a determining factor in urban children. The objective of this study was to compare Treg phenotype and function in cord blood (CB) of newborns to those in peripheral blood (PB) of a subset of participating mothers. METHODS: Treg numbers, expression, and suppressive function were quantified in subjects recruited prenatally from neighborhoods where >/= 20% of families have incomes below the poverty line. Proportion of Treg cells and expression of naïve (CD45RA) or activated (CD45RO, CD69, and HLA-DR) markers in CD4+T cells was measured by flow cytometry. Treg suppressive capacity was determined by quantifying PHA-stimulated lymphocyte proliferation in mononuclear cell samples with and without CD25 depletion. RESULTS: In an urban cohort of 119 newborns and 82 mothers, we found that newborns had similar number of cells expressing FOXP3 as compared to the mothers but had reduced numbers of CD4+CD25+bright cells that predominantly expressed the naïve (CD45RA) rather than the activated/memory (CD45RO) phenotype found in the mothers. Additionally, the newborns had reduced mononuclear cell TGF-beta production, and reduced Treg suppression of PHA-stimulated lymphocyte proliferation compared to the mothers. CONCLUSION: U.S. urban newborns have Treg cells that express FOXP3, albeit with an immature phenotype and function as compared to the mothers. Longitudinal follow-up is needed to delineate Treg cell maturation and subsequent risk for atopic diseases in this urban birth cohort.

Lung lining fluid glutathione attenuates IL-13-induced asthma.

GGT(enu1) mice, deficient in gamma-glutamyl transferase and unable to metabolize extracellular glutathione, develop intracellular glutathione deficiency and oxidant stress. We used intratracheal IL-13 to induce airway inflammation and asthma in wild-type (WT) and GGT(enu1) mice to determine the effect of altered glutathione metabolism on bronchial asthma. WT and GGT(enu1) mice developed similar degrees of lung inflammation. In contrast, IL-13 induced airway epithelial cell mucous cell hyperplasia, mucin and mucin-related gene expression, epidermal growth factor receptor mRNA, and epidermal growth factor receptor activation along with airway hyperreactivity in WT mice but not in GGT(enu1) mice. Lung lining fluid (extracellular) glutathione was 10-fold greater in GGT(enu1) than in WT lungs, providing increased buffering of inflammation-associated reactive oxygen species. Pharmacologic inhibition of GGT in WT mice produced similar effects, suggesting that the lung lining fluid glutathione protects against epithelial cell induction of asthma. Inhibiting GGT activity in lung lining fluid may represent a novel therapeutic approach for preventing and treating asthma.

Immunomodulatory cytokines in asthmatic inflammation.

The development of asthmatic inflammation involves a complex array of cytokines that promote the recruitment and activation of a number of different immune cells. While factors involved in initiating and establishing inflammation are well characterized, the process by which this pro-inflammatory cascade is regulated is less well understood. The identification and characterization of immunomodulatory cytokines in asthma has been a difficult proposition. Many of the putative regulatory factors have pleiotropic bioactivities and have been characterized as pro-inflammatory in association with certain pathologic conditions. This chapter addresses the potential role of several endogenous factors which appear to attenuate asthmatic inflammation. Understanding the integration of these factors into the regulation of the inflammatory process will likely result in novel therapeutic approaches.

Immunoglobulin G from patients with Graves' disease induces interleukin-16 and RANTES expression in cultured human thyrocytes: a putative mechanism for T-cell infiltration of the thyroid in autoimmune disease.

Mechanisms underlying lymphocyte infiltration of the thyroid gland and orbit in Graves' disease (GD) are poorly understood. The IGF-I receptor (IGF-IR) is a newly recognized self-antigen that, when activated in GD fibroblasts by IGF-I or GD-IgGs, provokes the expression of IL-16 and RANTES (regulated upon activation, normal T cell expressed and secreted)-dependent T lymphocyte chemoattraction and hyaluronan synthesis. IL-16 is a CD4(+)-specific ligand, and RANTES is a C-C chemokine. Here we report that IGF-I and GD-IgG could induce IL-16 and RANTES in cultured human thyrocytes in a time-dependent manner. Importantly, human TSH failed to induce either chemoattractant. This induction could be attenuated by dexamethasone. Rapamycin, a specific inhibitor of the FRAP/mammalian target of rapamycin/p70(s6k) pathway, prevented GD-IgG-provoked IL-16 synthesis. IH7, a monoclonal antibody directed at IGF-IR also blocked the induction of chemoattraction as well as RANTES mRNA synthesis. Our findings suggest that thyrocytes can be activated by GD-IgG and IGF-I to express powerful T-cell chemoattractants. These actions of GD-IgG appear to be mediated through pathways independent of the TSH receptor. Thus, in GD, thyrocytes may participate directly in lymphocyte recruitment through their expression of IL-16 and RANTES.

Standardization and performance evaluation of mononuclear cell cytokine secretion assays in a multicenter study.

BACKGROUND: Cryopreservation of peripheral blood mononuclear cells has been used to preserve and standardize immunologic measurements for multicenter studies, however, effects of cryopreservation on cytokine responses are incompletely understood. In designing immunologic studies for a new multicenter birth cohort study of childhood asthma, we performed a series of experiments to determine the effects of two different methods of cryopreservation on the cytokine responses of cord and peripheral blood mononuclear cells. RESULTS: Paired samples of PBMC were processed freshly, or after cryopreservation in a Nalgene container (NC) or a controlled-rate freezer (CRF). Although there were some differences between the methods, cryopreservation inhibited PHA-induced IL-10 secretion and Der f 1-induced IL-2 secretion, and augmented PHA-induced IL-2 secretion and spontaneous secretion of TNF-alpha. In separate experiments, NC cryopreservation inhibited secretion of several cytokines (IL-13, IL-10, IFN-gamma, TNF-alpha) by PHA-stimulated cord blood mononuclear cells. With the exception of PHA-induced IL-13, results from fresh and cryopreserved cord blood samples were not significantly correlated. Finally, in reproducibility studies involving processing of identical cell samples in up to 4 separate laboratories, variances in cytokine responses of fresh cells stimulated at separate sites did not exceed those in cryopreserved cells stimulated at a central site. CONCLUSION: Collectively, these studies indicate that cryopreservation can affect mononuclear cell cytokine response profiles, and that IL-10 secretion and antigen-induced responses may be especially vulnerable. These studies also demonstrate that mononuclear cell responses can be standardized for performance in a small number of laboratories for multicenter studies, and underscore the importance of measuring reproducibility and of testing whether cryopreservation techniques alter specific immunologic outcomes.

Production of IL-16 correlates with CD4+ Th1 inflammation and phosphorylation of axonal cytoskeleton in multiple sclerosis lesions.

BACKGROUND: Multiple sclerosis (MS) is a central nervous system-specific autoimmune, demyelinating and neurodegenerative disease. Infiltration of lesions by autoaggressive, myelin-specific CD4+Th1 cells correlates with clinical manifestations of disease. The cytokine IL-16 is a CD4+ T cell-specific chemoattractant that is biased towards CD4+ Th1 cells. IL-16 precursor is constitutively expressed in lymphocytes and during CD4+ T cell activation; active caspase-3 cleaves and releases C-terminal bioactive IL-16. Previously, we used an animal model of MS to demonstrate an important role for IL-16 in regulation of autoimmune inflammation and subsequent axonal damage. This role of IL-16 in MS is largely unexplored. Here we examine the regulation of IL-16 in relation to CD4+ Th1 infiltration and inflammation-related changes of axonal cytoskeleton in MS lesions. METHODS: We measured relative levels of IL-16, active caspase-3, T-bet, Stat-1 (Tyr 701), and phosphorylated NF(M+H), in brain and spinal cord lesions from MS autopsies, using western blot analysis. We examined samples from 39 MS cases, which included acute, subacute and chronic lesions, as well as adjacent, normal-appearing white and grey matter. All samples were taken from patients with relapsing remitting clinical disease. We employed two-color immunostaining and confocal microscopy to identify phenotypes of IL-16-containing cells in frozen tissue sections from MS lesions. RESULTS: We found markedly increased levels of pro- and secreted IL-16 (80 kD and 22 kD, respectively) in MS lesions compared to controls. Levels of IL-16 peaked in acute, diminished in subacute, and were elevated again in chronic active lesions. Compared to lesions, lower but still appreciable IL-6 levels were measured in normal-appearing white matter adjacent to active lesions. Levels of IL-16 corresponded to increases in active-caspase-3, T-bet and phosphorylated Stat-1. In MS lesions, we readily observed IL-16 immunoreactivity confined to infiltrating CD3+, T-bet+ and active caspase-3+ mononuclear cells. CONCLUSION: We present evidence suggesting that IL-16 production occurs in MS lesions. We show correlations between increased levels of secreted IL-16, CD4+ Th1 cell inflammation, and phosphorylation of axonal cytoskeleton in MS lesions. Overall, the data suggest a possible role for IL-16 in regulation of inflammation and of subsequent changes in the axonal cytoskeleton in MS.

The influence of atopy and asthma on immune responses in inner-city adults.

Asthma in the inner-city population is usually atopic in nature, and is associated with significant morbidity and mortality. However, the underlying immune abnormalities that underlie asthma in urban adults have not been well defined. We investigated the influence of atopy and asthma on cytokine responses of inner-city adult women to define immune abnormalities associated with asthma and atopy. Blood samples were collected from 509 of 606 inner-city women enrolled in the Urban Environment and Childhood Asthma (URECA) study. We tested for associations between atopy and asthma status and cytokine responses in peripheral blood mononuclear cells incubated ex vivo with a panel of innate and adaptive immune stimulants. Atopic subjects had heightened Th2 cytokine responses (IL-4, IL-5, IL-13) to cockroach and dust mite antigens, tetanus toxoid, and phytohemagglutinin (P < 0.05 for all). Differences in cytokine responses were greatest in response to stimulation with cockroach and dust mite. In a multivariate analysis, atopy was broadly related to increased Th2-like responses to all antigens and PHA, while asthma was only weakly related to mitogen-induced IL-4 and IL-5 responses. There were few asthma or allergy-related differences in responses to innate stimuli, including IFN-α and IFN-γ responses. In this inner-city adult female population, atopy is associated with enhanced Th2 responses to allergens and other stimuli, and there was little or no additional signal attributable to asthma. In particular, these data indicate that altered systemic interferon and innate immune responses are not associated with allergies and/or asthma in inner-city women.