Systemic impact of intestinal helminth infections.

In this review, we examine the evidence that intestinal helminths can control harmful inflammatory responses and promote homeostasis by triggering systemic immune responses. Induction of separable components of immunity by helminths, which includes type 2 and immune regulatory responses, can both contribute toward the reduction in harmful type 1 immune responses that drive certain inflammatory diseases. Despite inducing type 2 responses, intestinal helminths may also downregulate harmful type 2 immune responses including allergic responses. We consider the possibility that intestinal helminth infection may indirectly affect inflammation by influencing the composition of the intestinal microbiome. Taken together, the studies reviewed herein suggest that intestinal helminth-induced responses have potent systemic effects on the immune system, raising the possibility that whole parasites or specific molecules produced by these metazoans may be an important resource for the development of future immunotherapies to control inflammatory diseases.

Prevention of type 1 diabetes through infection with an intestinal nematode parasite requires IL-10 in the absence of a Th2-type response.

Helminth infection can prevent type 1 diabetes (T1D); however, the regulatory mechanisms inhibiting disease remain largely undefined. In these studies, nonobese diabetic (NOD) IL-4(-/-) mice were infected with the strictly enteric nematode parasite, Heligmosomoides polygyrus. Short-term infection, 5-7 weeks of age, inhibited T1D onset, as late as 40 weeks of age. CD4(+) T-cell STAT6 phosphorylation was inhibited, while suppressed signal transducer and activator of transcription 1 phosphorylation was sustained, as were increases in FOXP3(-), CD4(+) T-cell interleukin (IL)-10 production. Blockade of IL-10 signaling in NOD-IL-4(-/-), but not in NOD, mice during this short interval abrogated protective effects resulting in pancreatic ß-cell destruction and ultimately T1D. Transfer of CD4(+) T cells from H. polygyrus (Hp)-inoculated NOD IL-4(-/-) mice to NOD mice blocked the onset of T1D. These studies indicate that Hp infection induces non-T-regulatory cells to produce IL-10 independently of STAT6 signaling and that in this Th2-deficient environment IL-10 is essential for T1D inhibition.

Th1- and Th2-cell commitment during infectious disease: asymmetry in divergent pathways.

The development of T helper 1 (Th1) versus Th2 cells is a major branch point in the immune response. It is an important determinant of whether the response to an infectious pathogen will lead to protection of the host or dissemination of the disease. Recent studies have suggested that this process is governed by distinct sets of signals provided by dendritic cells upon interactions with specific infectious agents. A model is proposed that links together the pathogen, the innate response and Th-cell polarization.

Distinct types of T-cell help for the induction of a humoral immune response to Streptococcus pneumoniae.

Studies have indicated that purified soluble polysaccharide antigens can elicit T cell-independent Ig responses in vivo, although these responses can be modulated by T cells in a noncognate manner. Relatively little is known, however, concerning the parameters that regulate polysaccharide-specific, as well as protein-specific, Ig isotype responses to an intact extracellular bacterium. Using the murine in vivo humoral response to intact Streptococcus pneumoniae as a model it can be shown that CD4+ T-cell receptor alphabeta+ T cells deliver help for both polysaccharide- and protein-specific Ig responses. However, these responses differ fundamentally in their mechanism of action.

B7 requirements for primary and secondary protein- and polysaccharide-specific Ig isotype responses to Streptococcus pneumoniae.

The requirements for B7 costimulation during an in vivo humoral response to an intact extracellular bacteria have not been reported. In this study we immunized mice with Streptococcus pneumoniae (R36A) to determine the B7 requirements for induction of Ig, specific for two determinants on R36A, the phosphorylcholine (PC) determinant of C-polysaccharide and pneumococcal surface protein A (PspA). We show that the primary anti-PspA response, the development of PspA-specific memory, and the induction of the secondary anti-PspA response in primed mice were completely dependent upon B7 costimulation. Of note, costimulation was required only briefly after the secondary immunization compared with after the primary immunization for optimal induction of Ig. Blockade of B7 costimulation at the time of secondary immunization also completely abrogated the established state of memory, but did not induce tolerance. In contrast to the anti-PspA response, the primary anti-PC response involved only a very short period of B7 costimulation. Whereas B7-2 alone was required for induction of the primary anti-PspA and anti-PC responses, a redundant role for B7-1 and B7-2 was noted for the PspA-specific secondary response. CTLA4Ig blocked both the anti-PC and anti-PspA responses equally well over a wide range of bacterial doses. These studies demonstrate a critical, but variable, role for B7-dependent costimulation during an Ig response to an extracellular bacteria.

IL-4 suppression of in vivo T cell activation and antibody production.

Injection of mice with a foreign anti-IgD Ab stimulates B and T cell activation that results in large cytokine and Ab responses. Because most anti-IgD-activated B cells die before they can be stimulated by activated T cells, and because IL-4 prolongs the survival of B cells cultured with anti-Ig, we hypothesized that treatment with IL-4 at the time of anti-IgD Ab injection would decrease B cell death and enhance anti-IgD-induced Ab responses. Instead, IL-4 treatment before or along with anti-IgD Ab suppressed IgE and IgG1 responses, whereas IL-4 injected after anti-IgD enhanced IgE responses. The suppressive effect of early IL-4 treatment on the Ab response to anti-IgD was associated with a rapid, short-lived increase in IFN-gamma gene expression but decreased CD4+ T cell activation and decreased or delayed T cell production of other cytokines. We examined the possibilities that IL-4 stimulation of IFN-gamma production, suppression of IL-1 or IL-2 production, or induction of TNF-alpha or Fas-mediated apoptosis could account for IL-4's suppressive effect. The suppressive effect of IL-4 was not reversed by IL-1, IL-2, or anti-TNF-alpha or anti-IFN-gamma mAb treatment, or mimicked by treatment with anti-IL-2Ralpha (CD25) and anti-IL-2Rbeta (CD122) mAbs. Early IL-4 treatment failed to inhibit anti-IgD-induced Ab production in Fas-defective lpr mice; however, the poor responsiveness of lpr mice to anti-IgD made this result difficult to interpret. These observations indicate that exposure to IL-4, while T cells are first being activated by Ag presentation, can inhibit T cells activation or promote deletion of responding CD4+ T cells.

IL-13-mediated worm expulsion is B7 independent and IFN-gamma sensitive.

B7 costimulation is a required component of many type 2 immune responses, including allergy and protective immunity to many nematode parasites. This response includes elevations in Th2 cytokines and associated effector functions including elevations in serum IgG1 and IgE and parasite expulsion. In studies of mice infected with Trichuris muris, blocking B7 ligand interactions inhibited protective immunity, suppressed IL-4 production, and enhanced IFN-gamma production, but unexpectedly did not inhibit production of the Th2 cytokine, IL-13. Blocking both IFN-gamma and B7 restored protective immunity, which was IL-13 dependent, but did not restore IL-4 or associated IgE responses. Although IL-13 was required for worm expulsion in mice in which both IFN-gamma and B7 were blocked, IL-4 could mediate expulsion in the absence of both IL-13 and IFN-gamma. These studies demonstrate that 1) B7 costimulation is required to induce IL-4, but not IL-13 responses; 2) IL-13 is elevated in association with the IFN-gamma response that occurs following inhibition of B7 interactions, but can only mediate IL-4-independent protection when IFN-gamma is also inhibited; and 3) increased IL-13 production, in the absence of increased IL-4 production, is not associated with an IgE response, even in the absence of IFN-gamma.

The development of CD4+ T effector cells during the type 2 immune response.

Multiple pathways may be involved in the development of interleukin 4 (IL-4) producing T helper (Th) cells and the associated type 2 immune response. Increasing evidence suggests that the strength of signals delivered to the T cell may favor the development of the type 2 response. In contrast, antigen-presenting cell- (APC) derived stimuli produced following pattern recognition receptor binding during the innate response promotes the development of interferon-gamma (IFN-gamma) producing cells and the associated type 1 immune response. In many cases, the balance between increased signaling strength and the innate response may determine whether the type 2 response develops. T cell receptor (TCR), CD4, and costimulatory molecule interactions may all contribute to signal strength, but the type 2 immune response may be particularly dependent on the availability of coreceptor and costimulatory molecule interactions. B7 ligand interactions are required for the development of the type 2 immune response and interaction of CD28 with either B7-1 or B7-2 can provide sufficient signals for its initiation. In B7-2-deficient mice, the initial type 2 immune response is intact, but the response is not sustained, suggesting that B7-2 is important at later stages of the type 2 immune response. The roles of CD28 and CTLA-4 during the type 2 response remain unclear. The type 2 response to infectious pathogens is pronounced in CD28-/- mice, suggesting that other costimulatory molecule interactions can substitute for CD28 for the development of IL-4 producing T cells and the associated type 2 immune response.

In vivo polysaccharide-specific IgG isotype responses to intact Streptococcus pneumoniae are T cell dependent and require CD40- and B7-ligand interactions.

In vivo Ig responses to soluble, haptenated polysaccharide (PS) Ags are T cell independent and do not require CD40 ligand (CD40L). However, little is known regarding the regulation of in vivo PS-specific Ig responses to intact bacteria. We immunized mice with a nonencapsulated, type 2 Streptococcus pneumoniae (R36A) and compared the parameters that regulated in vivo Ig isotype responses to the bacterial cell wall C-PS determinant, phosphorylcholine (PC), relative to Ig responses to the cell wall protein, pneumococcal surface protein A. Consistent with previous reports using soluble PS and protein Ags, the anti-PC and anti-pneumococcal surface protein A responses differed in that the anti-PC response was induced more rapidly, had a distinctive Ig isotype profile, and failed to demonstrate boosting upon secondary challenge with R36A. However, in contrast to previous studies, the IgG anti-PC response was TCR-alphabeta+ T cell dependent, required CD40L, and was blocked by administration of CTLA4 Ig. The nature of the T cell help for the anti-PC response had distinct features in that it was only partially blocked by CTLA4 Ig and was dependent upon both CD4+ and CD8+ T cells. Surprisingly, whereas the IgM anti-PC response was largely T cell independent, a strong requirement for CD40L was still observed, suggesting the possibility of an in vivo T cell-independent source for CD40L-dependent help. These data suggest that the regulatory parameters that govern in vivo Ig responses to purified, soluble PS Ags may not adequately account for PS-specific Ig responses to intact bacteria.

Is depression associated with immune activation?

BACKGROUND: Some research immunologists have suggested that major depression amd chronic fatigue syndrome (CFS) are characterized by immune activation. To test this hypothesis, we compared immunological function in patients with major depression and in patients with CFS who developed major depression after the onset of CFS to that of sedentary healthy controls. METHODS: Subjects completed the Centers for Epidemiological Study-Depression (CES-D) questionnaire and allowed venisection. We performed flow cytometric analysis on 13 groups of white blood cells and used a reverse transcriptase PCR method to assay m-RNA of eight cytokines. RESULTS: CES-D scores were high in both patient groups and did not differ significantly. We found no evidence for immune activation in either patient group. Instead the data suggested immunological downregulation in depression. LIMITATIONS: Not all the subjects in the two patient groups were off antidepressants. CONCLUSIONS: The data indicate that immune activation is not necessary in depression--either alone or with CFS.

Immunological response in chronic fatigue syndrome following a graded exercise test to exhaustion.

This study was conducted to evaluate the immunological response to an exhaustive treadmill exercise test in 20 female chronic fatigue syndrome patients compared to 14 matched sedentary controls. Venipuncture was performed at baseline and 4 min, 1 hr, and 24 hr postexercise. White blood cells were labeled for monoclonal antibody combinations and were quantified by FACsan. Cytokines were assayed utilizing quantitative RT/PCR. No group difference was seen in VO2peak (28.6 +/- 1.6 vs 30.9 +/- 1.2 ml.kg-1.min-1; P > 0.05). However, 24 hr after exercise the patients' fatigue levels were significantly increased (P < 0.05). The counts of WBC, CD3+ CD8+ cells, CD3+ CD4+ cells, T cells, B cells, natural killer cells, and IFN-gamma changed across time (P's < 0.01). No group differences were seen for any of the immune variables at baseline or after exercise (P's > 0.05). The immune response of chronic fatigue syndrome patients to exhaustive exercise is not significantly different from that of healthy nonphysically active controls.

B7-2 is required for the progression but not the initiation of the type 2 immune response to a gastrointestinal nematode parasite.

T cells require CD28/CTLA-4 costimulatory molecule interactions in addition to Ag-specific signals through the TCR for in vivo effector Th cell function. Some studies have suggested that the ligands for these costimulatory molecules may differentially influence effector T cell function with B7-2 favoring a type 2 response and B7-1 favoring a type 1 response, while other studies have suggested that these molecules may be redundant. The recent development of B7-2-deficient mice permits the direct analysis of the requirement of B7-2 during a type 2 immune response to an infectious pathogen. We have examined, in B7-2-deficient mice, effector Th cell function and the associated type 2 immune response following infection with Heligmosomoides polygyrus, a natural murine parasitic nematode. Elevations in cytokine gene expression and protein secretion were pronounced and comparable in inoculated B7-2-/- and B7-2+/+ mice at day 8 after H. polygyrus inoculation. However, by day 14 after infection, increases in T cell cytokine expression were markedly inhibited in H. polygyrus-inoculated B7-2-/- mice. Furthermore, elevations in serum IgE and germinal center formation were inhibited at later stages of the immune response, while elevations in serum IgG1 persisted. These findings suggest that certain T-dependent components vary in their B7-2-dependency during the type 2 immune response. They further demonstrate that B7-2 interactions are not necessary for the initiation of the type 2 immune response, but are instead required for its progression after the development of effector T cells.

Vasculitis in the Palmerston North mouse model of lupus: phenotype and cytokine production profile of infiltrating cells.

OBJECTIVE: To define the phenotype of cells in the perivascular and vascular infiltrates of Palmerston North (PN) mice and the cytokines that those cells produce. METHODS: Immunohistologic analysis, flow cytometric analysis, and reverse transcriptase-polymerase chain reaction (RT-PCR) studies were performed on tissues and cells from female PN mice and age-matched and sex-matched DBA/2 mice. RESULTS: With aging, PN mice developed a female-predominant, lupus-like disease, with a severe systemic mononuclear cell perivasculitis and vasculitis. The perivasculitis involved arteries and veins in kidney, liver, brain, and lung; the vasculitis predominantly involved veins and venules. The perivascular and vascular infiltrates in female PN mice were composed mainly of an unusual cell type that expressed phenotypic markers characteristic of both T cells (Thy1+, CD3+, CD4+, T cell receptor + [TCR+]) and B cells (B220+). In addition, the infiltrates contained a smaller number of conventional CD4+,B220- T cells and macrophages. Very few CD8+ T cells or surface Ig+ B cells were seen. Unlike the Thy1+,B220+ T cells present in MRL/lpr mice, most of which were CD4-,CD8- and TCRalpha/beta+, the majority of the Thy1+,B220+ T cells in the perivascular/vascular infiltrates of PN mice were CD4+ and expressed either TCRalpha/beta or TCRgamma/delta. By immunohistologic staining, the cells in the perivascular and vascular infiltrates in the kidneys of older PN mice were shown to produce interleukin-4 (IL-4), IL-6, and IL-10, but not IL-2, interferon-gamma, transforming growth factor beta, tumor necrosis factor alpha, or IL-1beta. By RT-PCR, the kidneys of older PN mice were found to express high levels of IL-4, IL-6, and IL-10 messenger RNA. CONCLUSION: The vascular and perivascular infiltrates in PN mice are composed predominantly of an unusual subpopulation of T cells that are Thy1+,B220+,CD4+,CD8-, express either TCRalpha/beta or TCRgamma/delta, and produce mainly type 2 cytokines. The exact role of these cells in the immunopathogenesis of lupus-like disease in PN mice remains to be elucidated.

Changes in immune parameters seen in Gulf War veterans but not in civilians with chronic fatigue syndrome.

The purpose of this study was to evaluate immune function through the assessment of lymphocyte subpopulations (total T cells, major histocompatibility complex [MHC] I- and II-restricted T cells, B cells, NK cells, MHC II-restricted T-cell-derived naive and memory cells, and several MHC I-restricted T-cell activation markers) and the measurement of cytokine gene expression (interleukin 2 [IL-2], IL-4, IL-6, IL-10, IL-12, gamma interferon [IFN-gamma], and tumor necrosis factor alpha [TNF-alpha]) from peripheral blood lymphocytes. Subjects included two groups of patients meeting published case definitions for chronic fatigue syndrome (CFS)-a group of veterans who developed their illness following their return home from participating in the Gulf War and a group of nonveterans who developed the illness sporadically. Case control comparison groups were comprised of healthy Gulf War veterans and nonveterans, respectively. We found no significant difference for any of the immune variables in the nonveteran population. In contrast, veterans with CFS had significantly more total T cells and MHC II+ T cells and a significantly higher percentage of these lymphocyte subpopulations, as well as a significantly lower percentage of NK cells, than the respective controls. In addition, veterans with CFS had significantly higher levels of IL-2, IL-10, IFN-gamma, and TNF-alpha than the controls. These data do not support the hypothesis of immune dysfunction in the genesis of CFS for sporadic cases of CFS but do suggest that service in the Persian Gulf is associated with an altered immune status in veterans who returned with severe fatiguing illness.

Mouse running activity is lowered by Brucella abortus treatment: a potential model to study chronic fatigue.

Chronic fatigue syndrome, which can occur after acute infection and last for years, is characterized by severe and persistent fatigue. Others have reported decreases in mouse running activity following infection and have suggested this may provide an animal model for studying chronic fatigue. Voluntary running is a highly motivated activity in mice, which will often run 5-7 mi/day in our laboratory. Following 2 weeks of acclimation to running wheels with food and water available ad lib, female BALB/c mice received 0.2-mL tail vein injections of killed Brucella abortus (BA) or saline vehicle. Subsequently the effects on voluntary running and grooming behavior were determined. Injection of BA caused an immediate large decrease in running and a lack of grooming. Vehicle injections produced no changes in behavior. After the first several days of reduced running behavior, levels of running and grooming slowly returned back to normal over the next 2-4 weeks, with substantial individual differences in the rate of recovery. The pattern of running during recovery was intriguing in that BA mice first ran at normal levels just after the lights went out, but they stopped after only 1-2 h. As recovery proceeded, they gradually increased the duration of the running bout during the night. Because this model uses voluntary exertion and the ability to run for longer periods of time characterizes recovery, the model may be a good one for studying the biologic underpinnings of chronic fatigue.

Development of murine allergic asthma is dependent upon B7-2 costimulation.

Allergic asthma is thought to be mediated by CD4+ T lymphocytes producing the Th2-associated cytokines, IL-4, and IL-5. Recently, the costimulatory molecules B7-1 and B7-2, which are expressed on the surface of APC, have been suggested to influence the development of Th1 vs Th2 immune responses. We examined the in vivo role of these costimulatory molecules in the pathogenesis of Th2-mediated allergen-induced airway hyperresponsiveness in a murine model of asthma. In this model, OVA-sensitized A/J mice develop significant increases in airway responsiveness, pulmonary eosinophilia, and pulmonary Th2 cytokine expression following aspiration challenge with OVA as compared with PBS-control animals. Strikingly, administration of anti-B7-2 mAb to OVA-treated mice abolished allergen-induced airway hyperresponsiveness, pulmonary eosinophilia, and elevations in serum IgG1 and IgE levels. Anti-B7-2 treatment of OVA-treated mice reduced both total lung IL-4 and IL-5 mRNA and bronchoalveolar lavage fluid IL-4 and IL-5 protein levels, with no significant changes in IFN-gamma message or protein levels. In contrast, treatment with anti-B7-1 mAbs had no effect on allergen-induced airway hyperresponsiveness, IgE production, or cytokine production, however, it significantly suppressed pulmonary eosinophilia. We conclude that B7-2 provides the necessary costimulatory signal required for the development of in vivo allergic responses to inhaled allergen exposure.

Do effector and memory T helper cells also need B7 ligand costimulatory signals?

Blocking B7 ligand-costimulatory molecules can inhibit a primary T-dependent immune response, but whether these interactions also mediate ongoing or memory immune responses is less clear. Development of immunotherapies based on blocking B7 ligand interactions would be limited if they were effective only at the initiation of an immune response. We discuss the conditions under which T helper effector and memory cells may or may not require B7 ligand interactions for their function.

Kinetics of cytokine expression and regulation of host protection following infection with molecularly cloned Venezuelan equine encephalitis virus.

In the mouse model, the arbovirus Venezuelan equine encephalitis virus (VEE) replicates in lymphoid tissues prior to either inducing protective immunity (attenuated VEE mutant) or progressing to lethal encephalitis (virulent parent VEE). To investigate the mechanism of the protective response, cytokine gene expression was examined during the course of the primary in vivo immune response to molecularly cloned, virulent VEE and a single-site attenuated VEE mutant, using a quantitative reverse transcriptase-polymerase chain reaction assay. VEE-induced cytokine gene expression was 100-fold elevated over that of untreated controls for IFN-gamma and IL-6 and 10-fold increased for IL-12, IL-10, and TNF-alpha. There was no qualitative difference in cytokine gene induction comparing mice infected with the attenuated and the virulent VEE; however, there were significant differences in the cytokine gene expression kinetics. In mice infected with the attenuated VEE, elevated cytokine gene expression was delayed 24 hr when compared to mice infected with the virulent parent VEE clone at the same dose. Further, IFN-gamma protein secretion by cells from the draining lymph node mimicked the pattern of IFN-gamma gene induction by cells harvested from the same site. IFN-gamma gene expression was elevated at an earlier time point in mice given virulent V3000 24 hr after attenuated V3032 injection compared to mice infected with virulent V3000 alone. The combined V3000/V3032 infection resulted in host protection. Treatment of mice with IL-12 prior to infection with virulent VEE failed to reduce the severity of infection, while anti-IL-12 antibody did not prevent the early protective effect of attenuated virus. In contrast, administration of anti-IFN-alpha/beta antibody prior to VEE infection worsened virulent VEE disease. These results indicate that the attenuated VEE strain elicits a similar but delayed cytokine response compared to the virulent strain, suggesting that the kinetics of cytokine expression and the particular cytokine produced may influence the development of a host protective response. Furthermore, IFN-alpha/beta, but not IL-12, seems to be a major factor in the induction of early protection against VEE infection and disease.

CD28 dependence of T cell differentiation to IL-4 production varies with the particular type 2 immune response.

T cell differentiation to effector cell function is required for the development of a type 2 immune response. The T cell surface molecule, CD28, is widely considered to be the principal costimulatory molecule involved in T cell differentiation to effector function, including IL-4 production, although this has been difficult to directly examine in vivo. We have studied in vivo differentiation to T cell effector function during two type 2 immune responses in CD28 knockout mice: the systemic immune response to goat anti-mouse IgD Ab and the mucosal immune response following oral inoculation with the nematode parasite, Heligmosomoides polygyrus. Our results show that in C57BL/6 CD28 knockout mice elevations in IL-4 gene expression and protein secretion are blocked during the immune response to goat anti-mouse IgD, and associated increases in serum IgG1 and IgE are also inhibited to untreated control levels. In marked contrast, T cell differentiation to IL-4 production is comparable in C57BL/6 CD28 -/- and CD28 +/+ H. polygyrus-inoculated mice, and elevations in both serum IgG1 and IgE levels occur. These results indicate that the specific kind of type 2 immune response determines whether T cell differentiation to IL-4 production is CD28 dependent.