Tear cytokines in acute and chronic ocular allergic inflammation

PURPOSE OF REVIEW: Elevated levels of inflammatory cytokines have been reported in tears from ocular allergic disease states. The purpose of this review is to assimilate recent research contrasting tear cytokine concentrations in non-allergic subjects versus subjects with acute (seasonal allergic conjunctivitis) and chronic (giant papillary conjunctivitis, vernal keratoconjunctivitis, atopic keratoconjunctivitis) ocular allergic inflammation to discover whether the cytokine profiles could provide useful insight into disease mechanisms and therapeutic targets. RECENT FINDINGS: Recent studies have revealed distinct differences in the cytokine/chemokine concentrations in tears between the various manifestations of ocular allergy. The acute (seasonal allergic conjunctivitis) and iatrogenic (giant papillary conjunctivitis) forms of ocular allergic inflammation are characterized by an overall lack of significant cytokine changes in tears compared with chronic disease (vernal keratoconjunctivitis, atopic keratoconjunctivitis). Chronic ocular allergic inflammation produces increased concentrations of T helper 1 and 2, and proinflammatory cytokines as well as chemokines. However, vernal and atopic keratoconjunctivitis portray distinct differences in the patterns of tear cytokines/chemokines expressed. SUMMARY: The plethora of increased cytokines and chemokines in vernal and atopic keratoconjunctivitis compared with non-allergic, seasonal allergic conjunctivitis and giant papillary conjunctivitis provides a new perspective into the complex inflammatory processes occurring on the ocular surface in chronic disease. The ability to measure multiple cytokines in tears, combined with knowledge obtained from in-vitro analysis of the individual and combined effects of these cytokines on various conjunctival cells (i.e. mast cells, epithelial cells, fibroblasts) has facilitated further understanding of specific processes contributing to maintenance of inflammation and progression of vision-threatening disease and paved the way toward new therapeutic targets.     

Ocular allergic disease

PURPOSE OF REVIEW: This review will focus on recent advances in our understanding of the pathogenesis of allergic eye diseases. Common findings in acute allergic conjunctivitis (seasonal and perennial) and chronic allergic conjunctivitis (vernal keratoconjunctivitis, atopic keratoconjunctivitis, and giant papillary conjunctivitis) include evidence of mast cell activation and eosinophil attraction and activation. Cytokine levels found in tears, conjunctival impression cytology and biopsy specimens, and serum have been evaluated as markers of disease, and as targets of therapeutic intervention. RECENT FINDINGS: Human conjunctival epithelial cells respond to tumor necrosis factor alpha, interleukin-1 beta, and interferon-gamma individually and in combination. Intracellular adhesion molecule-1 expression is upregulated by interleukin-1 beta and tumor necrosis factor alpha. Conjunctival epithelial cells release interleukin-8 in response to interleukin-1 beta and tumor necrosis factor alpha but not interferon-gamma. Supernatants from activated mast cells cause increased adhesion of eosinophils to conjunctival epithelium. Tear levels of tumor necrosis factor alpha were elevated in vernal keratoconjunctivitis patients compared with normal controls. T cell lines from chronic allergic eye disease patients showed inconsistent production of cytokines in atopic and vernal keratoconjunctivitis and low levels in giant papillary conjunctivitis. Vernal keratoconjunctivitis patients have differing levels of eosinophil cationic protein in their serum if they were serum specific immunoglobulin E positive compared to serum specific immunoglobulin E negative patients. SUMMARY: Recent findings continue to expand our basic knowledge of mechanisms and differences between seasonal and perennial allergic conjunctivitis and atopic and vernal keratoconjunctivitis. Understanding the complex interactions and cross talk between cells, cytokines and other mediators is relevant for new therapeutic approaches directed at specific disease entities.     

Measurement of IL-4 in tears of patients with seasonal allergic conjunctivitis and vernal keratoconjunctivitis.

To elucidate the mechanism of ocular surface allergic disease, we focused on IL-4, which is one of the key factors in regulating IgE production, and thus determined the concentration of IL-4 in tears. IL-4 concentration was determined in the tears of 15 patients with seasonal allergic conjunctivitis, 15 vernal keratoconjunctivitis (VKC), 10 giant papillary conjunctivitis (GPC), 10 patients with non-allergic conjunctivitis and post-cataract surgical conjunctivitis as intermediate conjunctivitis, and 10 normal subjects using a highly sensitive sandwich ELISA. The mean level of IL-4 in normal controls was low, and seasonal allergic conjunctivitis, VKC and GPC showed a significant elevation (P < 0.05), respectively. IL-4 of VKC and GPC were also significantly higher than allergic conjunctivitis, and non-allergic conjunctivitis and post-cataract surgical conjunctivitis were not higher than normal. These results raise the possibility that the increased level of IL-4 in tears could play a role in allergic disease and its severity in patients.     

Tear levels of interferon-γ, interleukin (IL) -2, IL-4 and IL-5 in patients with vernal keratoconjunctivitis, atopic keratoconjunctivitis and allergic conjunctivitis

BACKGROUND: A predominance of TH2 activity in chronic allergic diseases, such as vernal keratoconjunctivitis (VKC) and atopic keratoconjunctivitis (AKC), has been suggested recently. However, there is no published study on tear levels of cytokines of the two different subgroups, TH1 and TH2, in patients with ocular allergy. OBJECTIVES: We measured interferon (IFN)-gamma, interleukin (IL)-2, IL-4 and IL-5 levels in tears by ELISA, to determine whether the levels of these cytokines are elevated in allergic ocular diseases when compared among patient groups and normal controls. METHODS: Tear levels of IL-2, IFNgamma, IL-4 and IL-5 were measured by ELISA using samples from patients with VKC, AKC (AKC-NP, without proliferative lesions; and AKC-P, with proliferative lesions), allergic conjunctivitis (AC) and normal subjects. The levels of these cytokines in tears and the clinical severity of AD were also compared. RESULTS: Tear IL-4 level in patients with AKC was significantly higher than those in VKC, AC and controls, and tear IL-4 levels in patients with AKC-P vs VKC differed significantly. Tear IL-5 levels in patients with diseases associated with proliferative lesions, VKC and AKC-P, were higher than those in AC and normal controls. However, tear level of IL-5 in patients with AKC-P was significantly higher than that in AKC-NP. Although the dermatological severity of AD correlated significantly with tear IL-4 level, IFNgamma, IL-2 and IL-5 levels did not correlate with dermatological severity of AD. CONCLUSION: These results indicate that the TH2-like cytokines play an important pathophysiological role in severe ocular allergic conditions such as AKC and VKC and that tear level of IL-5 may be a candidate marker to evaluate the clinical status of ocular allergy. The different patterns of tear levels of IL-4 and IL-5 among ocular allergic diseases may reflect the origin and immunological basis of these cytokines.     

Allergic eye disease – a clinical challenge

Currently, six basic allergic eye diseases are recognized. In seasonal (SAC) and perennial allergic conjunctivitis (PAC), the allergic response is mediated predominantly by mast cells, whereas the more severe conditions, vernal (VKC) and atopic keratoconjunctivitis (AKC) and giant papillary conjunctivitis (GPC), are associated with a preponderance of T cells. Acute allergic conjunctivitis (AAC) occurs when a large quantity of allergen inoculates the eye and is usually self-limiting. SAC, the most common ocular allergy, is the ocular component of hayfever. PAC in the UK is most commonly caused by the house-dust mite (HDM); diagnosis is confirmed by skin-prick tests, eosinophils in the conjunctival smear, and raised tear or serum total IgE. SAC and PAC can usually be managed with chromone eyedrops and antihistamines. VKC usually presents in children under 10 years of age and mainly affects boys. Sufferers frequently have a personal or family history of atopy. Corneal involvement can occur in VKC, making it potentially sight-threatening. AKC occurs in atopic adults, and like VKC it affects the cornea. VKC and AKC require steroid treatment under specialist supervision; minimization of the steroid dose can often be achieved with use of a chromone. GPC occurs due to repeated contact of the conjunctival surface with a foreign surface, such as contact lenses. Attention to lens hygiene or switching to different lenses and treatment with a chromone are frequently effective. In all allergic eye diseases contact with the precipitating allergen should be avoided as far as possible.     

Increased numbers of Th2-like CD4+ T cells in target organs and in the allergen-specific repertoire of allergic patients. Possible role of IL-4 produced by non-T cells.

Phytohemagglutinin (PHA)-induced human T cell clones (TCC) derived from conjunctival flogistic tissues of 3 patients with vernal conjunctivitis produced unusually high amounts of interleukin-4 (IL-4) and no, or limited amounts of, gamma-interferon (IFN-gamma). Allergen (Dermatophagoides pteronyssinus or Lolium perenne group I)-specific TCC derived from peripheral blood of two atopic donors produced significantly higher amounts of IL-4 and significantly lower amounts of IFN-gamma than TCC specific for bacterial antigens (tetanus toxoid and PPD) contemporarily established from the same donors. These data provide evidence for a compartimentalization of Th2-like helper T cells in target organs and in the allergen-specific T cell repertoire of allergic patients. Non-B, non-T bone marrow cells could produce IL-4, but not IL-2 or IFN-gamma, in response to cross-linkage of Fc epsilon type I receptors. These cells may further contribute to the maintenance and amplification of allergic inflammation.     

Toll-like receptor 2 expression on human conjunctival epithelial cells: a pathway for Staphylococcus aureus involvement in chronic ocular proinflammatory responses.

BACKGROUND: Staphylococcus aureus colonization is common in atopic keratoconjunctivitis, potentially activating epithelial cells via toll-like receptor 2 (TLR-2) and the receptor for platelet-activating factor (PAFR). OBJECTIVES: To examine human conjunctival epithelial cells for the expression of TLR-2 in vitro and in vivo and to evaluate the role of TLR-2 in S aureus-mediated activation of these cells. METHODS: Conjunctival epithelial cells isolated from cadaveric tissues were stimulated with interferon gamma (IFN-gamma) or a commercial S aureus cell wall extract (Staphylococcus aureus-CWE) (with or without anti-TLR-2 blocking antibody or PAFR antagonist) and were analyzed for tumor necrosis factor alpha (TNF-alpha) and interleukin 8 (IL-8) release; surface expression of TLR-2, intercellular adhesion molecule-1, HLA, and CD14; and TLR-2 messenger RNA expression. Ocular surface cells collected via impression cytology were examined for TLR-2 expression via flow cytometry. RESULTS: Expression of TLR-2 was up-regulated on conjunctival epithelial cells by IFN-gamma and Staphylococcus aureus-CWE. Expression of TLR-2 messenger RNA was increased by IFN-gamma. Staphylococcus aureus-CWE up-regulated intercellular adhesion molecule 1, HLA, and CD14 expression and increased TNF-alpha and IL-8 release in a dose-dependent manner. Anti-TLR-2 significantly inhibited TNF-alpha release, whereas PAFR antagonist significantly inhibited IL-8 release. Toll-like receptor 2 was expressed on conjunctival epithelial cells from 4 of 5 patients with atopic keratoconjunctivitis, 3 of 5 with seasonal allergies, and 0 of 3 without allergies. CONCLUSIONS: Conjunctival epithelial cells express TLR-2 and may play an active role in the chronic ocular inflammatory response to S aureus through pathways that involve TLR-2 and PAFR.     

Elevated levels of substance P in tears of patients with allergic conjunctivitis and vernal keratoconjunctivitis

Background Recetit studies have suggested that the nervous system may participate in inflammatory processes. Substance P (SP) acts as a chemical mediator as well as a neurotransmitter. Objective In order to clarify the pathogenesis of ocular allergic diseases, we assessed the concentration of SP in tears. Methods Using a highly sensitive and specific double-antibody enzyme immunoassay (EIA), we determined the SP concentration in tears of 10 patients with seasonal allergic conjunctivitis, 10 with atopic dermatitis without keratoconjunctivitis (AD), 13 with vernal keratoconjunctivitis (VKC) and 65 normal controls. Giemsa's staining for brush cytology samples and histocytological study by immunocytochemical staining of giant papillary conjunctival cells from VKC and normal controls was conducted. Results The mean SP level was low in the normal controls and AD, whereas patients with seasonal allergic conjunctivitis and VKC showed significant elevation of SP (P<0.01). Brush cytology samples showed conjunctival epithelial cells with lymphocytes, neutrophils and eosinophils that were not seen in normal subjects. Histocytological examination demonstrated SP positive cells in the conjunctiva of patients with VKC, but not in normal controls. Conclusion This study suggests that the increased level of SP in tears may contribute to the pathogenesis and severity of ocular allergic diseases.     

T-cell cytokines in chronic allergic eye disease

Background: The pathophysiology of chronic allergic eye disease cannot be explained by type I hypersensitivity alone, and T cell–mediated inflammation has been strongly implicated as a possible additional mechanism. Previous studies suggested that T_H2–like T cells play an important role in one form of chronic allergic eye disease. Objectives: This study examined the cytokine profile of T cells in different clinical groups of subjects with chronic allergic eye disease (i.e., vernal keratoconjunctivitis [VKC], atopic keratoconjunctivitis [AKC], and giant papillary conjunctivitis [GPC]) and normal control subjects. Methods: In situ hybridization was used to identify cytokine messenger RNA (mRNA), and two-color immunohistochemical analysis was used to demonstrate cytokine immunoreactivity localizing to T cells in the conjunctiva. Results: Allergic tissue expressed increased levels of mRNA for IL-3, IL-4, and IL-5 when compared with normal tissue. There was significantly greater IL-2 mRNA expression in subjects with AKC than in those with VKC (p = 0.004) and those with GPC (p = 0.02). Immunoreactivity for T-cell IL-5 was present more frequently in subjects with VKC (p = 0.004), GPC (p = 0.02), and AKC (p = 0.04) than in normal control subjects. However, T-cell IFN-γ protein expression was greater in subjects with AKC than in subjects with VKC (p = 0.01), GPC (p = 0.01), and control subjects (p = 0.005). Conclusions: These results show a TH₂–like T-cell cytokine array in subjects with VKC and GPC but a shift in cytokine profile toward a T_H1-like pattern, potentially because of differences in chronicity of the disorders, in subjects with AKC. These important functional T-cell variations in chronic allergic eye conditions are likely to be important in understanding differences in clinical characteristics and therapeutic responses. (J Allergy Clin Immunol 1997;100:817-24.)     

Therapeutic Considerations: Symptoms, Cells and Mediators

Immuno-histopathological studies of conjunctival tissue biopsied from patients with non-sight-threatening allergic conjunctivitis or with sight-threatening allergic keratoconjunctivitis should lead to more effective management of these eye conditions, based on the specific cellular involvement. The major difference between these two categories of eye disease was the occurrence of T-lymphocytes, which were absent in the former but prominent in the sight-threatening disorders. Seasonal and perennial allergic conjunctivitis both showed a heavy mast cell increase, due to infiltration of mucosal type mast cells, and allergen challenge studies linked mast cell histamine release to the early phase reaction occurring within 20 minutes. A second histamine peak at six hours after challenge might implicate basophils (or refractory mast cells) and was accompanied by a rise in eosinophil cationic protein. In sight-threatening, chronic allergic keratoconjunctivitis the responses were clearly directed by T-cells, themselves the primary effector cell in atopic keratoconjunctivitis, whereas vernal keratoconjunctivitis displayed a T-cell driven eosinophilia, with increased expression of the adhesion molecules involved in tissue invasion by these cells. Appropriate therapies for each different category of conjunctivitis should be based on the specific immunopathology, and directed at the activated cell types that are primarily responsible for the disease process.     

The Pathophysiology of Ocular Allergy: Current Thinking

Seasonal allergic conjunctivitis is the only ocular disease to involve solely Type-1 hypersensitivity, the other main forms of ocular allergy - perennial allergic conjunctivitis, vernal and atopic keratoconjunctivitis and giant papillary conjunctivitis - each having a more complex immunological basis and a chronic inflammatory component. Involvement of secondary inflammatory cells, particularly eosinophils, in addition to the mast cells resident in the conjunctival substantia propria, can lead to more serious corneal damage with vision-threatening potential. Thoughtful management of allergic conjunctivitis is needed in order to control the ocular inflammation without incurring steroid-induced side-effects, and patient education is also an important factor in maintaining optimal allergen avoidance, especially in the more severe and chronic cases. Laboratory models can be helpful in assessing the potential of new drugs, and SWR mice (topically sensitised and challenged with short ragweed) show clinical signs of allergic conjunctivitis, together with mast cell and eosinophil involvement, remarkably similar to the human pathophysiology. The antiinflammatory activity of both steroids and nedocromil sodium observed in this animal model supports therapeutic evidence of the usefulness of second-generation mast cell stabilising drugs in the treatment of ocular allergy.     

Mechanisms of deficient interferon-γ production in atopic diseases

BACKGROUND: The mechanisms responsible for an imbalanced cytokine response in atopic diseases are still not understood. While impaired interferon-gamma (IFN-gamma) production may be the result of a pathological T-cell/antigen-presenting cell (APC) interaction, evidence was provided that the T cell itself may have an intrinsic defect to produce IFN-gamma. OBJECTIVE: To clarify whether impaired IFN-gamma production by T cells from patients with atopic dermatitis (AD) represents an intrinsic defect in producing IFN-gamma. METHODS: Effector T cells were generated from CD4+ CD45RA+-naive precursors from patients with AD and healthy control individuals by activation with anti-CD3+ anti-CD28 MoAbs. Following restimulation, IFN-gamma production was measured by ELISA and flow cytometry. RESULTS: IFN-gamma production by atopic T cells was decreased compared with healthy T cells. IL-12 present at priming or high doses of IL-2 during the culture period, even in the absence of IL-12, completely restored IFN-gamma production. Conversion of naive CD45RA+ to CD45R0+ effector cells did not differ between atopic and healthy donors' T cells. CONCLUSION: Impaired IFN-gamma production by T cells from atopic individuals is not the result of an intrinsic, genetically fixed, defect to produce sufficient amounts of IFN-gamma. The data provides evidence that correction of an impaired TH1 response in AD may be successful at the precursor T cell level.     

CCR 4 and CCR 5 expression in conjunctival specimens as differential markers of T(H)1/ T(H)2 in ocular surface disorders

BACKGROUND: Accurate inflammatory mechanisms in chronic ocular surface diseases (OSDs) cannot routinely be assessed. New techniques for investigating ocular surface inflammatory pathways are of major importance. OBJECTIVE: To investigate the expressions of CCR 4 and CCR 5, known to be related to the T(H)2 and T(H)1 systems, respectively, and HLA-DR in conjunctival impression cytology specimens from patients with chronic OSDs. METHODS: In this case-controlled study, impression cytology specimens were taken in a series of patients with vernal keratoconjunctivitis (n=21), giant papillary conjunctivitis (n=6), or keratoconjunctivitis sicca (KCS; n=17), or receiving topical antiglaucoma treatments (n=31), and from 20 normal subjects. Conjunctival cells were incubated with mAbs to CCR 4, CCR 5, CD45, and HLA-DR to quantify conjunctival inflammation in a masked manner using flow cytometry. RESULTS: HLA-DR was higher in the glaucoma and KCS groups than in allergic and normal eyes. CCR 4 was overexpressed in allergy and glaucoma, whereas CCR 5 was higher in the KCS and glaucomatous groups. CD45 was expressed by only few cells in all groups, with almost no significant differences. CCR 4 expression was negatively correlated with CCR 5 and HLA-DR, whereas CCR 5 was positively correlated with HLA-DR. CONCLUSION: This study confirms the overexpression of chemokine receptors by the conjunctival epithelium in OSDs. CCR 4 and CCR 5 expression may vary according to the immune pathway involved. Accurate mechanisms in ocular surface inflammatory reactions-that is, those related to the T(H)1 or T(H)2 systems-could be differentiated by CCR 4/CCR 5 profiles. Our results also suggest that long-term use of topical treatments may stimulate both systems.     

IL-16 inhibits IL-5 production by antigen-stimulated T cells in atopic subjects

BACKGROUND: We have previously shown increased expression of the CD4+ cell chemoattractant IL-16 at sites of airway allergic inflammation. Little is known about the significance of IL-16 in allergic inflammation and its role in allergen-driven T-cell cytokine responses. Because IL-16 interacts specifically with CD4+ T cells, we hypothesized that IL-16 released at sites of inflammation may modulate the pattern of cytokines produced by CD4+ T cells. OBJECTIVE: We investigated the effects of exogenous rhIL-16 on cytokine production of PBMCs from atopic and nonatopic subjects in response to antigen and PHA. METHODS: Primary cultures of freshly isolated PBMCs from ragweed-sensitive atopic subjects and nonatopic subjects were stimulated with ragweed or PHA in the presence or absence of rhIL-16. Supernatant levels of IL-4, IL-5, and IFN-gamma were determined by means of ELISA at different time points between 2 and 6 days. Effects of IL-16 on antigen-induced cellular proliferative responses were determined. RESULTS: No IL-4 protein was detected after antigen stimulation of PBMCs from atopic subjects, whereas significant levels of IL-5 were measured on day 6 (median, 534.9 pg/mL). IL-5 secretion was abolished in PBMC cultures depleted of CD4+ cells. The addition of rhIL-16 in antigen-stimulated PBMC cultures significantly reduced the amount of IL-5 released (median, 99.8 pg/mL; P <.001). Detectable levels of IFN-gamma (median, 53.3 pg/mL) were identified after antigen stimulation. The addition of rhIL-16 in antigen-stimulated PBMC cultures significantly increased IFN-gamma levels (median, 255.6 pg/mL; P <.05). Effects of rhIL-16 appear to be specific for antigen-stimulated PBMCs in atopic subjects because rhIL-16 did not alter IL-5 or IFN-gamma production in response to PHA nor did rhIL-16 alter cytokine production in nonatopic normal subjects. CONCLUSION: These studies suggest that IL-16 can play a role in regulating the production of cytokines seen in allergic states in response to antigen.     

Aberrant interleukin (IL)-4 and IL-5 production in vitro by CD4+ helper T cells from atopic subjects.

The cytokine secretion profiles of T cell lines (TCL) specific for purified protein derivative (PPD) or streptokinase (SK), contemporarily derived from nine atopic and nine nonatopic individuals, were compared. Upon stimulation with phorbol myristate acetate (PMA) plus anti-CD3 monoclonal antibody (mAb), all TCL from both atopics and nonatopics produced interleukin (IL)-2 and interferon (IFN)-gamma. The mean IL-2 production by PPD- or SK-specific TCL from both atopics and nonatopics was similar, whereas the mean IFN-gamma production by TCL derived from atopics was significantly lower. In addition, both PPD- and SK-specific TCL from atopics produced detectable amounts of IL-4 and IL-5, whereas the corresponding TCL derived from nonatopics did not. A total number of 107 and 99 PPD-specific CD4+ T cell clones (TCC) were then derived from TCL of 4 atopic and 4 nonatopic donors and assessed for their profile of cytokine production in response to stimulation with either PMA plus anti-CD3 mAb or the specific antigen. Under both these experimental conditions, virtually all PPD-specific TCC from both atopic and nonatopic individuals produced IL-2 and IFN-gamma. In contrast, the great majority of PPD-specific TCC derived from nonatopic individuals did not produce IL-4 and IL-5, whereas high proportions of PPD-specific TCC derived from atopic donors displayed the ability to produce noticeable amounts of IL-4 and IL-5 besides IL-2 and IFN-gamma. These data indicate that CD4+ T cells from atopic individuals are able to produce IL-4 and IL-5 in response to bacterial antigens, such as PPD and SK, that usually evoke responses with a restricted type-1 T helper (Th1)-like cytokine profile in nonatopic individuals. Aberrant IL-4 production by Th cells may represent one of the immune alterations responsible for enhanced IgE antibody production in atopic people.     

Relationship of in vivo and ex vivo levels of TH1 and TH2 cytokines with viremia in HAART patients with and without opportunistic infections

TH1/TH2 cytokines' imbalance is critical to HIV-1 progression and pathogenesis. Opportunistic infections-related cytokine perturbations in the setting of highly active antiretroviral therapy (HAART) are unclear. The objective of this cross-sectional study was to identify the relationship between TH1/TH2 cytokines and viremia in HAART patients with/without opportunistic infections. Sera from 17 HAART patients with and 43 without opportunistic infections, and 20 HIV-seronegative controls were used to measure the levels of IL-2, IFN-gamma, IL-4, and IL-10 proteins and mRNAs by ELISA and RNase protection assays, respectively. Ex vivo cytokine production by the CD4+/CD8+ T cells from four low and four high viremia patients randomly selected from non-opportunistic infection group was also evaluated. Serum IL-2 and IFN-gamma levels were lower (P < 0.05) in patients than controls; this reduction was more pronounced for IFN-gamma in non-opportunistic infection patients. IL-4 and IL-10 were higher in patients than controls; this elevation was more remarkable in patients with opportunistic infections. Serum TH1/TH2 cytokine levels correlated with viremia. In vitro cytokine production assays showed that CD4+ T cells from low viremia patients mainly produced IL-2 and IFN-gamma, CD8+ T cells from high viremia patients produced IL-4, and both subsets comparably produced IL-10 in patients with similar viremia. Positive correlations between sera/supernatant proteins and cellular mRNAs were also found statistically significant (P < 0.05). It was therefore concluded that in vivo TH1/TH2 cytokine levels in HAART patients and their ex vivo production by the CD4+/CD8+ T cells correlated with viremia and were also modulated by the presence of opportunistic infections in these patients.     

Limiting dilution analysis of CD4 T-cell cytokine production in mice administered native versus polymerized ovalbumin: directed induction of T-helper type-1-like activation.

Polarized expression of T-helper type-1 (Th1)- or Th2-like patterns of cytokine production frequently correlates with disease outcome. Previously, we have described the long-lived reciprocal regulation of ovalbumin (OVA)-specific IgE (> 95% inhibition) and IgG2a (300-800-fold increased) production following administration of high MW OVA polymers (OVA-POL), in both de novo and ongoing OVA (alum)-induced responses. Here, limiting dilution analysis (LDA) was used to compare precursor frequencies of CD4 T cells producing interferon-gamma (IFN-gamma), interleukin-4 (IL-4) or IL-10 following OVA versus OVA-POL exposure in vivo. Adjuvants were not used, so as to circumvent their impact on measurement of precursor frequencies. We found that the two forms of antigen elicited T-cell activation of comparable intensity, as indicated by equivalent precursor frequencies of clonogenic antigen-specific CD4 T cells. However, they elicited qualitatively different cytokine responses. OVA-POL treatment led to 10-fold higher (mean of six independent LDA experiments) frequencies of IFN-gamma-producing cells, and a mean fivefold lower frequency of IL-10-producing cells, than was observed following in vivo administration of unmodified OVA. Thus, the high MW polymerized form of antigen acted to steer commitment of naive (for this antigen) CD4 T-cell activation from a situation in which IL-10 producers outnumbered IFN-gamma-producing cells by a factor of 4:1 (found in mice administered OVA), to one where IFN-gamma producers dominated by a factor of 11:1 (in mice given OVA-POL), i.e. a qualitative shift in the nature of the OVA-specific response induced from Th2-like to Th1-like. In vivo co-administration of anti-IFN-gamma monoclonal antibody (mAb) abolished the capacity of OVA-POL to preferentially elicit Th1-like dominance. Interestingly, although the ratios of IFN-gamma:IL-4 and IFN-gamma:IL-10 OVA-specific precursor frequencies were strongly increased following OVA-POL exposure (mean 18- and 47-fold higher), the frequency of IL-4-producing CD4 T cells did not differ significantly. The data suggest that this modified antigen promotes in vivo commitment of naive T cells towards a Th1-like response, with consequent inhibition of IgE and enhancement of IgG2a responses, not through direct effects on IL-4 production, but via decreased frequencies of IL-10 and increased frequencies of IFN-gamma-producing OVA-specific CD4 cells. Collectively, the data (1) demonstrate the ability to manipulate commitment of antigen-driven CD4 T-cell populations in naive mice to specific patterns of cytokine gene expression, and (2) provide in vivo evidence of the regulatory role played by IFN-gamma in limiting induction and/or expansion of IL-4- and IL-10-producing CD4 cells to protein allergens.     

CXCR3-mediated opposite effects of CXCL10 and CXCL4 on TH1 or TH2 cytokine production

BACKGROUND: Two variants of the CXCR3 receptor exist, one (CXCR3-A) reactive with CXCL9, CXCL10, and CXCL11 and the other (CXCR3-B) also reactive with CXCL4. Both variants are contemporarily expressed by human T cells. OBJECTIVE: We sought to investigate the in vitro effects of CXCL10 and CXCL4 on the production of TH1 or TH2 cytokines. METHODS: The cytokine profile of antigen-specific human CD4+ T-cell lines obtained in the absence or presence of CXCL10 or CXCL4 was evaluated by means of quantitative RT-PCR, flow cytometry, and ELISA. RESULTS: CXCL10 upregulated IFN-gamma and downregulated IL-4, IL-5, and IL-13 production, whereas CXCL4 downregulated IFN-gamma and upregulated TH2 cytokines. Similar effects were also observed on polyclonally activated pure naive CD4+ T cells. The opposite effects of CXCL10 and CXCL4 on TH1 and TH2 cytokine production were inhibited by an anti-CXCR3 antibody able to neutralize both CXCR3-A and CXCR3-B and were apparently related to the activation of distinct signal transduction pathways. Moreover, CXCL10 upregulated mRNA levels of T-box expressed in T cells and downregulated GATA-3 expression, whereas CXCL4 downregulated T-box expressed in T cells and upregulated GATA-3. Finally, CXCL4, but not CXCL10, induced direct activation of IL-5 and IL-13 promoters. CONCLUSION: CXCL10 and CXCL4 exert opposite effects on the production of human TH1 and TH2 cytokines, likely through their respective interaction with CXCR3-A or CXCR3-B and the consequent activation of different signal transduction pathways. This might represent an internal regulatory pathway of TH cell responses and might contribute to the modulation of chronic inflammatory reactions, including allergy.     

Polyclonal and clonal analysis of human CD4+ T-lymphocyte responses to nut extracts.

The induction of IgE antibodies to aeroallergens depends upon antigen-specific CD4+ helper T cells of an ''interleukin-4 (IL-4)-dominant'' phenotype. Nuts also drive IgE-mediated hypersensitivity and are the most dangerous of the orally encountered allergens. We have studied the polyclonal T-cell responses of atopic and non-atopic individuals to extracts of peanut, brazilnut and hazelnut. Strong proliferative responses were observed in all patients but specific IgE was only present in the nut-allergic patients suggesting a similar pathogenic mechanism to aeroallergen-mediated hypersensitivity. To investigate this hypothesis a panel of peanut-reactive T-cell clones was raised from a peanut- and brazilnut-allergic individual without hazelnut allergy. The antigen specificity, major histocompatibility complex (MHC) class II restriction and cytokine profiles of the T-cell clones were determined. With the exception of one T-cell clone, which proliferated in response to both peanut and hazelnut extract, the peanut T-cell clones were not cross-reactive with hazelnut or brazilnut. The T-cell clones recognized antigen in association with HLA-DR and HLA-DP but not HLA-DQ class II molecules. The peanut-specific clones produced high levels of IL-4 and low levels of interferon-gamma (IFN-gamma), exhibiting the ''TH2-like'' profile which dominates the aeroallergen response. In contrast, the T-cell clone that was cross-reactive on both peanut and hazelnut allergen had a Th0-like phenotype, consistent with the lack of specific serum IgE to hazelnut. These results support the importance of functionally distinct T-cell populations that recognize oral allergens. The relative production of IL-4 and IFN-gamma of the cloned T cells in the peanut-allergic patients plays a role in determining whether or not IgE antibody responses are induced with the associated potential to develop anaphylactic reactions.