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1.  Deficiency of regulatory B cells increases allergic airway inflammation 
To investigate the effect of the X-linked immunodeficiency (Xid) B cell defect on the response to the cockroach allergen in mice.
Two cockroach allergen immunization and challenge protocols were employed to sensitize CBA/J wild-type and CBA/CaHN-btk(−/−)xid/J (Xid) mice. Blood and tissue samples were collected 24 and 48 hrs after the last intratracheal antigen challenge and were analyzed for several parameters of allergic inflammation.
Nearly equivalent amounts of serum IgE were detected in Xid and CBA/J mice after short-term antigen challenge despite the B cell deficiency in Xid mice. A decreased concentration of IgE was detected in CBA/J mice after repeated allergen challenges but not in the Xid mice. Correlating with the discrepancy in serum IgE levels, higher levels of IL-13, IL-5, IL-10 and CCL5 were measured in whole lung homogenates from allergen-challenged Xid mice compared to CBA/J mice. In addition, draining lymph node cells from Xid mice expressed elevated levels of IL-4, IL-5, IL-10 and IFNγ mRNA compared to cells from CBA/J mice after in vitro culture with cockroach antigen. An increase in lung inflammation, interstitial eosinophilia and mucus production was also observed in allergen-challenged Xid mice. CD95L expression increased on B-1a cells following allergen challenge, which was accompanied by an increase in lung CD4+ Th cell apoptosis in wild-type CBA/J mice. In contrast, Xid mice did not have an increase in CD4+ T cell apoptosis following allergen challenge.
These data suggest a regulatory role for B-1a cells in reducing cytokine production, pulmonary inflammation, and CD4+ T cell survival during cockroach allergen-induced airway inflammation.
PMCID: PMC3533497  PMID: 16389573
Allergy; B cells; T cells; Inflammation; Apoptosis
2.  Chemokine C10 Promotes Disease Resolution and Survival in an Experimental Model of Bacterial Sepsis 
Infection and Immunity  2000;68(11):6108-6114.
Previous studies have suggested that the C-C chemokine C10 is involved in the chronic stages of host defense reactions. The present study addressed the role of C10 in a murine model of septic peritonitis, induced by cecal ligation and puncture (CLP). Unlike other C-C chemokines, C10 levels in the peritoneal wash were increased approximately 30-fold above baseline levels at 48 h after CLP surgery. Immunoneutralization of peritoneal C10 levels with polyclonal anti-C10 antiserum during CLP-induced peritonitis negatively impacted mouse survival over 4 days. In contrast, when 500 ng of recombinant murine C10 was administered immediately after CLP surgery, the 4-day survival rate increased from 20% to over 60%. The C10 therapy appeared to facilitate a rapid and significant enhancement of the levels of tumor necrosis factor alpha (TNF-α) and monocyte chemoattractant protein-1 (MCP-1) and a later increase in interleukin-13 (IL-13) levels in the peritoneal cavity. In vitro studies showed that the combination of IL-1β and C10 markedly augmented TNF-α synthesis by peritoneal macrophages and that C10 synthesis was induced in these cells following their exposure to IL-13. At 24 h after CLP surgery, only 25% of C10-treated mice were bacteremic versus 85% of the control group that exhibited dissemination of bacteria into the circulation. The lack of bacteremia in C10-treated mice appeared to be related, in part, to in vitro evidence that C10 significantly enhanced the bacterial phagocytic activity of peritoneal macrophages. In addition, in vivo evidence suggested that C10 therapy significantly reduced the amount of material that leaked from the damaged gut. Taken together, the results of this study demonstrate that the C10 chemokine rapidly promotes disease resolution in the CLP model through its direct effects on the cellular events critically involved in host defense during septic peritonitis.
PMCID: PMC97687  PMID: 11035713
3.  Differential regulation of C-C chemokines during fibroblast-monocyte interactions: adhesion vs. inflammatory cytokine pathways. 
Mediators of Inflammation  1998;7(4):269-274.
The cell-to-cell interactions during chronic inflammatory diseases likely contribute to leukocyte accumulation leading to increased pathology and organ dysfunction. In particular, there is a paucity of information relating to the maintenance of chronic fibrotic diseases. Using a lung fibroblast line and enriched monocyte populations, we have investigated the activational events which contribute to the production of two C-C chemokines, macrophage inflammatory protein-1 alpha (MIP-1alpha) and monocyte chemoattractant protein-1 (MCP-1), during fibroblast-monocyte interactions. Neither the fibroblast cell line (16lu) nor isolated monocytes alone produced significant levels of MIP-1alpha or MCP-1. However, when isolated monocytes were layered onto 16 lu fibroblast monolayers a significant increase in MIP-1alpha and MCP-1 production was observed. The use of fixed cell populations indicated that the MIP-1alpha was derived from monocytes and MCP-1 from both cell populations. To examine the molecules which were required for chemokine production during the interaction, specific antibodies were used in the co-cultures. Blocking beta3-integrin interactions significantly inhibited MIP-1alpha production. In contrast, beta-integrin interactions had no effect on the MCP-1 production, while, neutralization of TNF significantly decreased MCP-1 production during the co-culture. These data indicate that fibroblast-monocyte interactions induce chemokine production through different mechanisms and a combination of these responses may contribute to the maintenance of the mononuclear cell accumulation during disease progression.
PMCID: PMC1781852  PMID: 9792337
4.  Chemokine-induced eosinophil recruitment. Evidence of a role for endogenous eotaxin in an in vivo allergy model in mouse skin. 
Journal of Clinical Investigation  1997;100(7):1657-1666.
Selective eosinophil recruitment into tissues is a characteristic feature of allergic diseases. Chemokines are effective leukocyte chemoattractants and may play an important role in mediating eosinophil recruitment in various allergic conditions in man. Here, we describe a novel mouse model of eosinophil recruitment in which we have compared the in vivo chemoattractant activity of different C-C chemokines. Furthermore, we describe the use of antibodies to chemokines and receptor blockade to address the endogenous mechanisms involved in eosinophil recruitment in a late-phase allergic reaction in mouse skin. Intradermal injection of mEotaxin and mMIP-1alpha, but not mMCP-1, mRANTES, mMCP-5, or mMIP-1beta, induced significant 111In-eosinophil recruitment in mouse skin. Significant 111In-eosinophil recruitment was also observed in an active cutaneous anaphylactic reaction. Pretreatment of skin sites with antieotaxin antiserum, but not an antiMIP-1alpha antibody, suppressed 111In-eosinophil recruitment in this delayed-onset allergic reaction. Similarly, desensitization of the eosinophil eotaxin receptor CCR3 with mEotaxin, or blockade of the receptor with metRANTES, significantly inhibited 111In-eosinophil recruitment in the allergic reaction. These results demonstrate an important role for endogenous eotaxin in mediating the 111In-eosinophil recruitment in allergic inflammation, and suggest that blockade of the CCR3 receptor is a valid strategy to inhibit eosinophil migration in vivo.
PMCID: PMC508348  PMID: 9312163
5.  Elevated levels of macrophage inflammatory protein 2 in severe murine peritonitis increase neutrophil recruitment and mortality. 
Infection and Immunity  1997;65(9):3847-3851.
We hypothesized that chemokines may play important roles in a cecal ligation and puncture (CLP) model of septic peritonitis in CD-1 mice. Concentrations of C-X-C (macrophage inflammatory protein 2 [MIP-2] and ENA-78) and C-C (MIP-1alpha and JE) chemokines were measured (by enzyme-linked immunosorbent assay) in serum, peritoneal lavage fluid, lung, and liver at 4, 8, 24, 48, and 96 h after CLP. Significant elevations in all measured chemokines occurred in peritoneal fluid after CLP (P < 0.05). MIP-2, in particular, increased dramatically (>400-fold, P < 0.001) in peritoneal fluid, serum, and to a lesser extent lung and liver (P < 0.05). Increased MIP-2 was correlated with severity of sepsis (P < 0.001). To determine the significance of this finding, mice were passively immunized prior to CLP with polyclonal antibody to MIP-2, which decreased mortality from 85 to 38% at 96 h (P < 0.01). To further understand the mechanism of the effect of MIP-2, additional measurements demonstrated that anti-MIP-2 prior to CLP decreased the percent neutrophils in peritoneal fluid (55% +/- 12%, compared with 82% +/- 10% in controls), but no significant changes in tumor necrosis factor alpha, interleukin-6, or interleukin-10 occurred. MIP-2 contributes to the inflammatory response and overall mortality in this model of severe septic peritonitis, possibly by increasing recruitment of neutrophils, which clear bacteria but may also injure the host.
PMCID: PMC175549  PMID: 9284162
6.  Balance of inflammatory cytokines related to severity and mortality of murine sepsis. 
Infection and Immunity  1996;64(11):4733-4738.
We tested the hypothesis that, during sepsis, the balance of pro- and anti-inflammatory cytokines is related to severity and survival. Cecal ligation and puncture (CLP) with a large (18-gauge)-, intermediate (21-gauge)-, or small (26-gauge)-diameter needle, or sham laparotomy, was performed on outbred CD-1 mice. Concentrations of tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6), and the anti-inflammatory cytokine IL-10 were measured (by enzyme-linked immunosorbent assay) in serum, peritoneal lavage fluid, and liver and lung samples at 4, 8, 24, 48, and 96 h. As the diameter of the CLP needle decreased, the mortality rate decreased (at 48 h: large, 80%; intermediate, 40%; small, 20%; P < 0.05), the TNF-alpha and IL-6 concentrations decreased, and the time-to-peak TNF-alpha expression increased. In contrast, IL-10 concentration increased compared with baseline (serum at 24 h: large, 2.3-fold +/- 1.6-fold; intermediate, 2.0-fold +/- 0.5-fold; small, 49.9-fold +/- 8.3-fold; P < 0.05). Administration of IL-10 (5 microg, intraperitoneal) prior to CLP decreased mortality (P < 0.001). Administration of polyclonal anti-IL-10 serum prior to CLP (0.5 ml intraperitoneal) had the opposite effect and increased mortality (P < 0.001) and TNF-alpha, IL-6, and TNF-alpha mRNA expression compared with controls. Thus, severe sepsis is associated with a largely unopposed inflammatory response, and a largely unopposed inflammatory response (with anti-IL-10) results in severe sepsis and death. Less severe sepsis is associated with greater anti-inflammatory mediator expression, and greater anti-inflammatory mediator expression (with IL-10) results in less severe sepsis. Thus, the balance of inflammatory mediators is related to the severity and mortality of murine sepsis.
PMCID: PMC174439  PMID: 8890233
8.  TNF-induced IL-8 and MCP-1 production in the eosinophilic cell line, EOL-1 
Mediators of Inflammation  1996;5(3):218-223.
The role of eosinophils in inflammation and their mode of activation is not well understood. Eosinophil accumulation and subsequent expression of cytokines at the site of inflammation may play a role in exacerbation of inflammatory responses. In the present study, we have examined the role of TNF-α in eosinophil activation and chemokine production using a human leukaemic eosinophil cell line, EOL-1. Initial studies demonstrated that TNF-α induced the upregulation of IL-8 and MCP-1 mRNA and protein. Kinetic studies indicated production of chemokines, IL-8 and MCP-1, as early as 4 h post-activation, with peak levels of chemokine produced at 8 h, and decreasing by 24 h post-TNF-α activation. When IL-10, a suppressive cytokine, was incubated with TNF-α and EOL-1 cells, no effect was observed on IL-8 and MCP-1 production. However, dexamethasone, a glucocorticoid, demonstrated potent inhibitory effects on the EOL-1-derived chemokines. These studies indicate that eosinophils may be a significant source of chemokines capable of participating in, and maintaining, leukocyte recruitment during inflammatory responses, such as asthma.
PMCID: PMC2365785  PMID: 18475720
9.  Interleukin-10 expression and chemokine regulation during the evolution of murine type II collagen-induced arthritis. 
Journal of Clinical Investigation  1995;95(6):2868-2876.
In the enclosed study we have examined the expression and contribution of specific chemokines, macrophage inflammatory protein 1 alpha (MIP-1 alpha) and macrophage inflammatory protein 2 (MIP-2), and interleukin 10 (IL-10) during the evolution of type II collagen-induced arthritis (CIA). Detectable levels of chemotactic cytokine protein for MIP-1 alpha and MIP-2 were first observed between days 32 and 36, after initial type II collagen challenge, while increases in IL-10 were found between days 36 and 44. CIA mice passively immunized with antibodies directed against either MIP-1 alpha or MIP-2 demonstrated a delay in the onset of arthritis and a reduction of the severity of arthritis. On the contrary, CIA mice receiving neutralizing anti-IL-10 antibodies demonstrated an acceleration of the onset and an increase in the severity of arthritis. Interestingly, anti-IL-10 treatment increased the expression of MIP-1 alpha and MIP-2, as well as increased myeloperoxidase (MPO) activity and leukocyte infiltration in the inflamed joints. These data suggest that MIP-1 alpha and MIP-2 play a crucial role in the initiation and maintenance, while IL-10 appears to play a regulatory role during the development of experimental arthritis.
PMCID: PMC295974  PMID: 7769128
10.  Cloning of TH0- and TH2-type helper lymphocytes from liver granulomas of Schistosoma mansoni-infected mice. 
Infection and Immunity  1994;62(3):994-999.
The pathological manifestations of schistosomiasis mansoni are primarily induced by circumovum hepatic granuloma formation and fibrosis. The growth and modulation of the granulomas are regulated by T lymphocytes and their products. In the present study, we isolated T-lymphocyte clones from lesions at the acute and chronic stages of murine infection. All of the T-cell clones were characterized by immunofluorescence as CD4+CD8- helper cells. Three T-cell clones derived from vigorous granulomas produced interleukin 2 (IL-2), IL-4, and gamma interferon (IFN-gamma) and were therefore classified as TH0-type T lymphocytes. Of the three clones derived from immunomodulated lesions, two produced IL-2 or IL-4 and/or IFN-gamma (TH0 type) and the other one did not produce IFN-gamma but did produce IL-4 and was therefore characterized as a TH2-type helper clone. The clones were further characterized by their responsiveness to soluble egg antigen fractions. The acute infection-derived clones responded to the lower-molecular-mass (60- to 66-kDa and 25- to 30-kDa) fractions, whereas the immunomodulated-granuloma-derived clones responded to the 60- to 66-kDa and higher-molecular-mass (70- to 90-, 93- to 125-, and > 200-kDa) fractions. Upon adoptive transfer into naive mice, both the TH0- and TH2-type clones were capable of inducing granuloma formation of similar magnitude around antigen-coated beads and/or freshly isolated parasite eggs. The present study revealed the presence of TH0-type precursor helper cells within the liver granulomas. The findings underscore the complexity of the granulomatous response at the T-cell level and demonstrate that both TH0- and TH2-type granuloma lymphocytes play a role in parasite egg-induced granuloma formation.
PMCID: PMC186215  PMID: 8112875
11.  Utilization of fractionated soluble egg antigens reveals selectively modulated granulomatous and lymphokine responses during murine schistosomiasis mansoni. 
Infection and Immunity  1992;60(8):3209-3216.
Worm eggs deposited in the livers and intestines of Schistosoma mansoni-infected mice secrete soluble egg antigens (SEA) and induce T cell-mediated circumoval granulomas. In the present study, we fractionated crude SEA by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and tested the fractions for granuloma elicitation and lymphokine production at different stages of the infection. SEA fraction-coupled beads were used to elicit artificial pulmonary granulomas. Acutely infected mice responded with granulomas to seven fractions (less than 21-, 25- to 30-, 32- to 38-, 60- to 66-, 70- to 90-, 93- to 125-, and greater than 200-kDa fractions) of SEA, whereas chronically infected mice responded to four fractions (60- to 66-, 70- to 90-, 93- to 125-, and greater than 200-kDa fractions). In response to both crude and fractionated SEA, granuloma T cells produced high levels of gamma interferon at the preacute (6-week) stage of infection, but production subsequently diminished. Interleukin-2 (IL-2) and IL-4 production peaked at the acute (8-week) stage of infection and concurrently decreased at the chronic (20-week) stage. At the acute stage of the infection, the granulomagenic SEA fractions also elicited IL-2 and IL-4 production; at the chronic stage, IL-2 production and, to a lesser degree, IL-4 production corresponded to SEA fractions that elicited granulomas. Isolated SEA proteins from the 32- to 38-kDa fraction demonstrated differential lymphokine responses: predominant gamma interferon and IL-2 production was elicited by the 32-kDa fraction, whereas the 35- and 38-kDa proteins elicited predominant gamma interferon and IL-4 production. However, all three proteins elicited granuloma formation. The present study reveals changes in granulomatous responses to SEA fractions during the acute and chronic stages of the infection as well as distinct phases of gamma interferon, IL-2, and IL-4 lymphokine production throughout the infection. Based on these results, it is concluded that granuloma formation and IL-2 and IL-4 production are interrelated.
PMCID: PMC257303  PMID: 1639491
12.  Identification of larval cross-reactive and egg-specific antigens involved in granuloma formation in murine schistosomiasis mansoni. 
Infection and Immunity  1991;59(9):3237-3242.
Cross-reactive humoral immune responses between antigens of different developmental stages of the worm Schistosoma mansoni have previously been demonstrated. In contrast, information on antigenic cross-reactivity at the T-cell level is still very sparse. The present study examined the cross-reactive T-cell responses to eggs and crude and fractionated soluble egg antigens (SEA) in infected mice prior to (from 0 to 4 weeks of infection) and after (5 weeks and onwards) egg deposition. Splenic lymphocyte proliferation to unfractionated SEA was detected as early as 2 weeks postinfection and increased rapidly by 4 weeks postinfection. Injections of live eggs into the lungs of infected mice at 4 weeks postinfection demonstrated enhanced granuloma formation, indicating the presence of primed T cells that respond to egg antigens. Further experiments with the artificial granuloma model and polyacrylamide gel electrophoresis-separated SEA fractions demonstrated that in mice infected for 4 weeks the 60- to 66-, 93- to 125-, and greater than 200-kDa SEA fraction-coated beads elicited significant pulmonary granulomas. By 6 weeks postinfection, when eggs are deposited in the livers, in addition to the cross-reactive fractions (60 to 66, 93 to 125, and greater than 200 kDa), beads coated with fractions of 25 to 30, 32 to 38, and 70 to 90 kDa also elicited significant granulomatous reactions. These antigenic fractions are considered to have elicited egg stage-specific T-cell responsiveness. In addition hepatic granuloma T cells from the 6th week of infection demonstrated the strongest blastogenic response to the 60- to 66-kDa cross-reactive fraction. Thus, in vitro and in vivo experiments demonstrated T-cell cross-reactivity between the larval and egg stages of the worm. On the basis of these observations, the appearance of the primary circumovum granulomatous response in infected mice is considered to represent the sum of larval cross-reactive and egg-specific T-cell responsiveness.
PMCID: PMC258158  PMID: 1908830
13.  Splenic and granuloma T-lymphocyte responses to fractionated soluble egg antigens of Schistosoma mansoni-infected mice. 
Infection and Immunity  1991;59(3):941-948.
Soluble egg antigens (SEA) secreted by the eggs of Schistosoma mansoni worms induce a T-cell-mediated granulomatous response that is principally responsible for the pathology of the disease. In the present study sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated SEA proteins were divided into nine fractions (less than 21, 25 to 30, 32 to 38, 40 to 46, 50 to 56, 60 to 66, 70 to 90, 93 to 125, and greater than 200 kDa), electroeluted, and utilized in in vitro lymphoproliferation assays. T-cell-enriched spleen cells from acutely infected mice responded to all nine fractions, while those from chronically infected mice responded to only the 50- to 56- and the 60- to 66-kDa fractions. Depletion of the CD4+ T-cell subset among acute and chronic-infection spleen cells abrogated the response. Depletion of the CD8+ T-cell population resulted in increased proliferation in response to fractions by acute-infection T cells and facilitated responsiveness to hitherto-inactive SEA fractions in chronic-infection T cells. Acute-infection CD4+ granuloma T cells responded to the 40- to 46-, 50- to 56-, 70- to 90-, 93- to 125-, and greater than 200-kDa fractions, while the chronic-infection granuloma T cells responded only to the greater than 200-kDa fraction of SEA. Selective depletion of the CD4+ T-cell subset when acute-infection granuloma lymphocytes were tested abrogated proliferation, whereas subset depletions when chronic-infection granuloma cells were tested indicated that both CD4+ and CD8+ T cells respond to the greater than 200-kDa fraction. The present study reveals differences between acute- and chronic-infection splenic and granuloma T cells in the pattern of T-cell blastogenic responses to fractionated SEA.
PMCID: PMC258350  PMID: 1900066

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