Ildy M. Katona

The importance of Th2 cytokines in protective immunity to nematodes

The immune response is characterized by its high degree of specificity. B lymphocytes capable of producing antibodies that react with an antigen are selected to proliferate and to difierentiate as a result of their binding antigens that are directly stimulatory (Moller 1975) and as a result of their presentation of antigens to antigen-specific helper T lymphocytes (Noelle & Snow 1991). T lymphocytes similarly are selected to proliferate and to differentiate as a result of signals received when they are presented with an antigen-derived peptide/self MHC complex by antigen-presenting cells, such as B lymphocytes, dendritic cells or macrophages (Lanzavecchia 1990). Specificity of the immune response is maintained because only those lymphocytes that are capable of reacting with a determinant on the immunogen are induced to clonally expand and differentiate into mature effector cells. There is, however, a second, less completely understood aspect to immune specificity. Different effector mechanisms are recruited in response to different antigens or pathogens. In the mouse, for example, viral infections typically induce antibody responses of the IgG2a isotype (Coutelier et al. 1987), and are accompanied by activation of macrophages and the generation of cytotoxic T lymphocytes (Leist et al. 1989). In contrast, helminth parasites typically

Role of STAT6 and Mast Cells in IL-4- and IL-13-Induced Alterations in Murine Intestinal Epithelial Cell Function

Gastrointestinal nematode infections generally invoke a type 2 cytokine response, characterized by the production of IL-4, IL-5, IL-9, and IL-13. Among these cytokines, IL-4 and IL-13 exhibit a functional overlap that can be explained by the sharing of a common receptor or receptor component (IL-4Rα). Binding of IL-4 by either the type 1 or 2 IL-4R, or of IL-13 by the type 2 IL-4R, initiates Jak-dependent tyrosine phosphorylation of the IL-4Rα-chain and the transcription factor, STAT6. In the present study, we investigated: 1) whether IL-13 has effects on intestinal epithelial cells similar to those observed with IL-4, and 2) whether the effects of IL-4 and IL-13 depend on STAT6 signaling and/or mast cells. BALB/c, STAT6−/−, and mast cell-deficient W/Wv mice or their +/+ littermates were treated with a long-lasting formulation of recombinant mouse IL-4 (IL-4C) or with IL-13 for seven days. Segments of jejunum were mounted in Ussing chambers to measure mucosal permeability; chloride secretion in response to PGE2, histamine, 5-hydroxytryptamine, or acetylcholine; and Na+-linked glucose absorption. IL-4C and IL-13 increased mucosal permeability, decreased glucose absorption, and decreased chloride secretion in response to 5-hydroxytryptamine. These effects were dependent on STAT6 signaling. Responses to PGE2 and histamine, which were dependent on mast cells and STAT6, were enhanced by IL-4C, but not by IL-13. The effects of IL-4 and IL-13 on intestinal epithelial cell function may play a critical role in host protection against gastrointestinal nematodes.

Heligmosomoides polygyrus: CD4+ but not CD8+ T cells regulate the IgE response and protective immunity in mice

Oral inoculation of BALB/c mice with infective larvae of Heligmosomoides polygyrus resulted in chronic infection characterized by the release of parasite eggs in the feces for several months. The actual number of eggs per gram of feces was dependent on the dose of the inoculum. Serum IgE in infected mice peaked at a level of greater than 70 micrograms/ml during Weeks 3 through 6 following inoculation, and high levels of IgE (greater than 40 micrograms/ml) persisted for over 14 weeks. Protective immune responses resulted in reduced egg production and the development of markedly fewer adult worms in the small intestines following a challenge inoculation. The role of CD4+ and CD8+ T cells in these responses was examined by depletion in vivo of either T cell subpopulation with rat mAb specific for the appropriate determinants. Mice treated with anti-CD4 during a primary infection had increased EPG which was due primarily to an increase in worm fecundity (eggs produced per adult female). A challenge inoculation of mice that had been cleared of the primary infection with an anthelmintic drug induced a protective response that reduced development of new adult worms by 70-80% and their fecundity by greater than 90%. This protective response was abrogated by injection of mice with anti-CD4. Serum IgE diminished when adult worms were removed after anthelmintic treatment. A more precipitous drop in serum IgE followed successive treatments of mice with an anthelmintic and anti-CD4. In addition, the anamnestic serum IgE response to a challenge inoculation was reduced by over 80% in anti-CD4-treated mice. Anti-CD8 treatment had no appreciable effect on the immunological or parasitological parameters measured following a challenge inoculation with H. polygyrus. Thus, CD4+ T cells regulate host protective immunity, worm fecundity, and IgE levels in an H. polygyrus infection. This experimental system may be particularly suitable for analysis of chronic nematode infections of humans and livestock because of the responsiveness of the parasite in vivo to changes in host immune function.

Enteric Nematodes Induce Stereotypic STAT6-Dependent Alterations in Intestinal Epithelial Cell Function

Infection with gastrointestinal nematodes exerts profound effects on both the immune and physiological responses of the host. We showed previously that the Th2 cytokines, IL-4 and IL-13, induce STAT6-dependent changes in intestinal epithelial cell permeability, absorption, and secretion that are similar to those observed in a secondary infection with Heligmosomoides polygyrus. In the current study we investigated whether nematode-induced effects on epithelial cell function were 1) generic, 2) dependent upon STAT6, and 3) attributable to direct effects on the epithelial cells themselves or mediated by effects on enteric nerves. Our results demonstrate that infection of BALB/c mice with three different gastrointestinal nematodes (H. polygyrus, Nippostrongylus brasiliensis, and Trichinella spiralis) alters intestinal epithelial cell function by decreasing resistance, glucose absorption, and secretory responses to 5-hydroxytryptamine and acetylcholine, two critical mediators in the submucosal reflex pathway. These modified responses are dependent on STAT6 and are the result of both direct effects and indirect effects mediated through enteric nerves.

Development of validated disease activity and damage indices for the juvenile idiopathic inflammatory myopathies: I. Physician, parent, and patient global assessments. Juvenile Dermatomyositis Disease Activity Collaborative Study Group.

OBJECTIVE To determine the reliability, content validity, and responsiveness of physician global assessments of disease activity and damage in the juvenile idiopathic inflammatory myopathies (IIM), and to investigate concordance among physician, parent, and patient global ratings. METHODS Sixteen pediatric rheumatologists rated 10 juvenile IIM paper patient cases for global disease activity and damage, and assessed the importance of 51 clinical and laboratory parameters in formulating their global assessments. Then, 117 juvenile IIM patients were enrolled in a protocol to examine the relationship between Likert and visual analog scale global assessments, their sensitivity to change, and the comparability of physician, parent, and patient global ratings. RESULTS Pediatric rheumatologists demonstrated excellent interrater reliability in their global assessments of juvenile IIM disease activity and damage (97.7% and 94.7% agreement among raters, respectively), and agreed on a core set of clinical parameters in formulating their judgments. Likert scale ratings correlated with those on a visual analog scale, and both were comparable in responsiveness (standardized response means -0.56 for disease activity, 0.02 [Likert] and 0.14 [visual analog] for damage, measured over 8 months). Parent global ratings of disease activity correlated with physician assessments, but were not colinear (Spearmans correlation [r] = 0.41-0.45). Patient global disease activity assessments correlated with those done by parents (r = 0.57-0.84) and physicians (r = 0.37-0.63), but demonstrated less responsiveness (standardized response means -0.21 and -0.12, respectively, over 8 months). CONCLUSION Physician global assessments of juvenile IIM disease activity and damage demonstrated high interrater reliability and were shown to be comprehensive measures. Both physician and parent disease activity assessments should be considered valuable as quantitative measures for evaluating therapeutic responses in juvenile IIM patients.

LOCAL TH1 AND TH2 RESPONSES TO PARASITIC INFECTION IN THE INTESTINE : REGULATION BY IFN-GAMMA AND IL-4

Control of parasitic infections is dependent on the production of cytokines that activate mechanisms which limit invasion, reproduction or survival of the parasite. In contrast, conditions that induce inappropriate cytokine responses facilitate the spread of infection and ultimately exacerbate the level of disease. Measurement of local cytokine responses to different gastrointestinal parasites, such as the intracellular protozoan, Cryptosporidium parvum, and luminal dwelling nematodes like Nippostrongylus brasiliensis and Heligmosomoides polygyrus, reveal stereotype response patterns. In general, intracellular parasites stimulate type 1 responses where IFN-gamma is the predominant immune activator, while extracellular parasites stimulate type 2 responses where IL-4 plays a prominent role in elevating humoral immune mechanisms. Cytokines alter cellular function and the milieu of the intestinal lumen to affect the outcome of an infection. The importance of a particular response during the course of an infection can be studied by selective enhancement with an excess of exogenous recombinant cytokine or cytokine antagonists. For example, exogenous IL-12 enhances resistance to C.parvum, but suppresses the normally rapid cure of an infection with N. brasiliensis. Both mechanisms are dependent on expression of IFN-gamma. At the molecular level, exogenous IL-12 stimulates IFN-gamma production which elevates a protective type 1 response to C. parvum but converts the normally anti-worm type 2 response to a type 1 response that inappropriately regulates the infection. Alternatively, excess IL-4 plays a prominent role in modulating effector elements that change intestinal physiology to create a hostile environment for worm parasites. Exogenous IL-4 can cure chronic worm infection, while IL-4 antagonists interfere with protective responses to infection. These observations provide a paradigm for analysis of stereotype responses to different gastrointestinal parasites, and demonstrate how cytokine-induced immune system-dependent and independent effector mechanisms can limit parasitic infection, while inappropriate cytokine responses can exacerbate the state of disease.

Mononuclear Phagocytic Cells in Human Milk: HLA-DR and FcγR Ligand Expression

The study of the cellular immune components of human milk is essential in the understanding of the role human milk may play in protecting the nursing infant against infection. We have investigated some phenotypic characteristics of breast milk macrophages (BMM) and have compared them to the characteristics of adult peripheral blood monocytes (PBM) by using dual parameter flow microfluorometry. Most BMM expressed the monocyte/macrophage markers Leu-M3 and Leu-M5. The latter marker was present in high density (bright) on BMM, but the density of expression of Leu-M3 was higher on PBM than on BMM [median fluorescence intensity (MFI) 409 +/- 105 versus 203 +/- 106, p = 0.02]. The percentage of BMM (98 +/- 2) that expressed the HLA-DR antigen did not differ significantly from PBM, but the density of expression was higher on BMM (MFI 318 +/- 56 versus 264 +/- 41, p = 0.03). The HLA-DR expression of BMM was further enhanced after incubation with interferon-gamma for 36 h; however, receptor for interleukin-2 could not be induced on BMM by this treatment. The expression of the three classes of Fc gamma R was lower on BMM than on PBM, in percentage (Fc gamma RI 56 +/- 23 versus 79 +/- 17%, p = 0.02), density of expression (Fc gamma RIII MFI 71 +/- 20 versus 153 +/- 73, p = 0.002), or both (Fc gamma RII 74 +/- 22% versus 94 +/- 12%, p = 0.02, and MFI 115 +/- 53 versus 202 +/- 59, p = 0.003).(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of Early Activation Events in Cord Blood B Cells after Stimulation with T Cell-Independent Activators

Human neonates are immunologically immature, particularly in their humoral antibody responses to T cell-independent antigens, as exemplified by their increased susceptibility to infections with polysaccharide-encapsulated bacteria. To clarify the mechanism(s) underlying the unresponsiveness of neonates to polysaccharide antigens, we used an in vitro model with neonatal cord blood cells that has been shown to mimic surface Ig-dependent signaling in the adult by T cell-independent antigens. We studied the ability of cord blood human B cells to become activated after ligation of their surface Ig by unconjugated anti-Ig, dextran-conjugated anti-Ig, and Staphylococcus aureus Cowan A1, and compared their response with that of adult B cells. After the addition of nanogram concentrations of anti-Ig-dextran, neonatal cord blood B cells proliferated at levels comparable to that observed with adult B cells. The majority of cord blood B cells showed a marked rise in intracellular calcium, increased surface expression of human leukocyte antigen DR, and an increase in cell size. Direct activation of protein kinase C by phorbol esters in neonatal B cells led to cellular proliferation, and when combined with anti-Ig, a synergistic effect on proliferation was observed. These data suggest that the unresponsiveness of human neonates to polysaccharide antigens does not represent an inability of these antigens to induce early activation events in circulating B cells.

Membrane Antigen and Ligand Receptor Expression on Neonatal Monocytes

The monocyte/macrophage cell lineage is an essential component of host defense. Functional deficiencies have been described in neonatal monocytes, but knowledge of membrane antigen and receptor ligand expression in neonatal monocytes is incomplete. In this study, antigen and receptor ligand expression of cord blood monocytes (CBM) was examined and compared to adult peripheral blood monocytes (PBM). Leu-M3 and Leu-M5 antigens were shown to be present on all CBM. Using dual fluorescence microfluorometry, the percentage and intensity of expression of HLA-DR, CD4 antigens, Fc gamma and IL-2 receptors (IL-2R) on Leu-M3+ and Leu-M5+ CBM were compared to PBM. A lower percentage of expression of HLA-DR+ (87 +/- 3% vs. 95 +/- 1%, p = 0.02) and FC gamma RII+ (96 +/- 1% vs. 99 +/- 0.2%, p = 0.04) was noted on CBM. CD4, FC gamma RI, and FC gamma RIII expression on CBM were comparable to PBM. LPS stimulation of CBM induced IL-2R expression and enhanced HLA-DR antigen expression as seen previously on PBM. These findings indicate that CBM are phenotypically comparable to adult PBM with deficiencies localized only to a few specific areas.

Klinefelter's syndrome and juvenile chronic arthritis

Klinefelter’s syndrome (KS) is the most common disorder of sexual differentiation in males and the most common disorder of sex chromosomes in humans. In KS, primary male hypogonadism is caused by developmental testicular defects due to the presence of two or more X-chromosomes. The classic karyotype for KS is 47XXY, but the mosaic form, 46XY/47XXY occurs in about 10% of cases. Other extreme variants such as 48XXYY, 48XXXY, and 49XXXXY also exist. It is estimated that the frequency of KS is about 1/500 at conception and 1/1000 at birth due to in utero fetal loss. First described in full by Dr. Harry Klinefelter in 1942 [1], the full syndrome begins to manifest in adolescence and its characteristics include gynecomastia, testicular atrophy, azoospermia and sparse facial and body hair. Increased leg length and armspan are also typical of the syndrome. The phenotypic expression is variable, however, and some individuals are not diagnosed until adulthood. Decreased secretion of androgens, an increased estradiol/testosterone ratio (a relative increase in estrogen effect) and elevated gonadotropin levels are noted in KS. The patients evaluated and described by Dr. Klinefelter demonstrated normal to moderately reduced function of the Leydig cells, increased excretion of follicle-stimulating hormone, and reduced excretion of 17-ketosteroids. As a result of the hormonal abnormalities, clinical findings of hypogonadism, azoospermia, and gynecomastia are noted. Small testes, measuring less than 2–3 cm are the most consistent physical finding. Additional physical features are small phallus and decreased upper to lower body ratio [2]. Laboratory evaluation generally demonstrates elevated FSH and LH and a decreased testosterone levels. KS patients generally need to be started on testosterone replacement therapy at about 12 years of age, with increasing dosages over time that simulate age-appropriate concentrations. Klinefelter’s syndrome has been noted to have an association with several autoimmune diseases including, but not limited to, systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), autoimmune diseases of the thyroid, and ankylosing spondylitis (AS). It has been speculated that some of these diseases, which normally show a female predominance, are seen concomitant with KS due to a relative excess of estrogens and lack of androgens in these patients. An association between KS and SLE is well described [3]. It has been suggested that the peripheral venous and arterial vascular abnormalities seen in KS (such as commonly seen leg ulcers) may be related to immune mechanisms involving anticardiolipin antibodies in KS patients [3]. Many reports of KS in association with autoimmune disease reflect a continuing interest in the complex relationship between sex hormones and autoimmune processes. Complicating this question, however, is the ‘‘Take home message’’: Pediatric patients with Klinefelter’s syndrome, a genetic condition characterized by primary male hypogonadism, should be considered at potential risk for the development of autoimmune diseases, including juvenile chronic arthritis.