Published papers

In this study, we have investigated the cellular and molecular basis for immunoregulatory function of T4+ cells after autologous mixed lymphocyte reaction (AMLR) activation. We demonstrated that the T4+2H4+ subset but not the T4+2H4- subset can proliferate maximally in response to autologous non-T cells. T4+ cells activated by AMLR exerted suppressor activity on pokeweed mitogen-driven IgG synthesis of autologous peripheral blood lymphocytes. The suppressor activity by AMLR-activated T4+ cells required the presence of fresh T8+ cells in the secondary culture, indicating that AMLR-activated T4+ cells functioned as a suppressor inducer rather than as a suppressor effector population. Following activation of T4+ cells in AMLR, it is the T4+2H4+ subset which induces suppression through the T8 population. Moreover, the treatment of AMLR-activated T4+2H4+ cells with anti-2H4 antibody, but not other antibodies, resulted in the abolishment of suppressor inducer function of such cells, suggesting that the 2H4 molecule itself may be involved in the suppressor induced function. The 2H4 antigen on such cells was shown to be comprised of 220-kDa and 200-kDa glycoproteins. These results support the notion that the AMLR may play an important role in generating suppressor inducer signals and in down-regulating the immune response following self major histocompatibility complex recognition. More importantly, the present studies indicate that the 2H4 antigen on T4 cells serves not only as a phenotypic marker of suppressor inducer cells, but may have a functionally important role itself in inducing suppression.

It has been demonstrated that the T4+2H4+ subset functioned as a suppressor inducer cell, whereas the reciprocal T4+2H4- subset provided help for B cell Ig production. In the present studies, a series of monoclonal antibodies to cell surface structures expressed on subsets of cells were examined for their effects on the proliferative and immunoregulatory functions generated in AMLR. We demonstrated that anti-T4 antibody preferentialy inhibited the proliferative response of the T4+2H4+ but not T4+2H4- cells against self-MHC antigens. In contrast, anti-T3 and anti-Ia antibodies inhibited the response of both subsets of cells. This subset preference of anti-T4 antibody was not attributable to either the isolation procedures used or a shift in the kinetics of proliferation to autologous self-MHC antigens. Moreover, both IL 2 production and the immunoregulatory function of the T4+2H4+ subset was profoundly inhibited by anti-T4 antibody, whereas the T4+2H4- subset was minimally influenced. In the absence of Ia molecules, T4+2H4+ but not T4+2H4- cell proliferation was inhibited with anti-T4 antibody. Together, these results suggest that the T4 molecule plays a distinct functional role in the differential triggering of subsets of T4+ cells.

The present study was undertaken to determine the nature of the immunoregulatory T-cell defect after autologous mixed lymphocyte reaction (AMLR) activation in patients with systemic lupus erythematosus (SLE). Although AMLR was decreased in patients with SLE compared with normals, there was no difference in major proliferative cells (T4 cells and T4+JRA+ subset) in response to AMLR. Functional activity of AMLR-stimulated T4 subsets in patients with SLE and normals was examined in helper and suppressor/inducer assay, using pokeweed mitogen (PWM)-derived IgG synthesis. The T4+JRA- (helper) subset from SLE patients showed no greater activity than normals. However, the T4+JRA- (helper) subset from SLE patients showed no greater activity than normals. However the T4+JRA+ (suppressor/inducer) subset from SLE patients showed decreased suppression induction compared with normals. This defect in the suppressor/inducer function was demonstrated even in patients with inactive SLR or in remission.

The aim of the study was to elucidate the involvement of Fas antigen in human autoimmune disease, by analysing serum levels of soluble Fas/APO1 protein in patients with various autoimmune diseases, including system lupus erythematosus (SLE), rheumatoid arthritis (RA), systemic sclerosis (SSc), polymyositis/dermatomyositis (PM/DM), Behcet's syndrome and Sjogren's syndrome (SjS). The levels of soluble Fas/APO-1 in sera were quantitated by a sandwich enzyme-linked immunosorbent assay. Soluble Fas/APO-1 levels were significantly increased in serum from patients with primary Sjogren's syndrome (1°SjS) compared with control subjects. However, no significant differences in soluble Fas/APO-1 levels were noted in patients with secondary Sjogren's syndrome (2°SjS) nor in patients with any of the other autoimmune diseases. The soluble Fas/APO-1 level in 1°SjS patients with extraglandular diseases was significantly higher than that in patients without extraglandular diseases. These results suggest that soluble Fas/APO-1 protein may play an important role in the pathogenesis of 1°SS.

A 25-year-old chronically immunosuppressed woman with systemic lupus erythematosus (SLE) died after developing subacute granulomatous encephalitis caused by Acanthamoeba. Amoebic trophozoites were also found in the lung, suggesting a primary pulmonary focus of infection. The infectious encephalitis was difficult to differentiate from a flare-up of central nervous system lupus. This case illustrates that Acanthamoeba can cause fatal encephalitis in lupus patients, as well as in patients with acquired immunodeficiency syndrome as previously reported. To our knowledge, this is the first reported case of granulomatous amoebic encephalitis due to Acanthamoeba in a patient with SLE.

Purpose. We previously reported allergic conjunctivitis was associated with a local pattern of cytokines and identified respiratory syncytial virus (RSV) in the conjunctival epithelium, in patients with allergic conjunctivitis. We hypothesized that RSV infected the normal conjunctival epithelium and produced interleukin-4 (IL-4), causing an allergic reaction. To determine this event, we studied production of IL-4 by human conjunctival epithelial cells, after infection with RSV. Methods. Conjunctival samples were collected with a special brush and were challenged with 5 x 106 pfu/ml of live RSV. RSV infection was detected by immunocytochemical study and RT-PCR. HLA-DR antigen was expressed by flow cytometry. IL-4 was assayed by a sandwich ELISA. Results. Immunocytochemical study and RT-PCR showed that RSV infected the conjunctival epithelial cells. HLA-DR was demonstrated in the RSV-infected conjunctival epithelial cells. Average concentrations of IL-4 in culture supernatants in infected cells were 3.8 ± 1.8, 3.6 ± 1.1, and 5.4 ± 1.8 pg/ml at 24, 48, and 72 h, respectively. Levels in non-infected cells were 0.33 ± 0.0, 0.59 ± 0.69, and 0.4 ± 0.22 pg/ml, respectively (p > 0.001). Conclusion. Findings suggest that the expression of IL-4 is associated with intracellular infection by RSV in conjunctiva. With our former reports, RSV infection in conjunctival epithelial cells can be suggested as one of the possible environmental pathogens of allergic conjunctivitis.

We report a case of fatal measles viral pneumonia in a patient with Hodgkin's disease who had no rash. The measles viral cDNA was detected in autopsy tissue from the lung by reverse transcription-polymerase chain reaction. This method was then applied successfully to stored serum. The diagnosis of measles viral pneumonia may be improved by the application of RT-PCR using peripheral blood. Sequence analysis of amplified cDNA suggested the virus infecting this patient was a recent strain, predominantly isolated after 1980. The fatal outcome may have been due to a lack of immune response to the newer strain.

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease of unknown aetiology. Although it has been reported that T cells might be responsible for the pathogenesis of SLE, it remains unclear whether immune aberrations of SLE T cells are the primary event in this pathological process. We have recently reported that tyrosine phosphorylation and expression of the T cell receptor zeta chain (TCR ζ) was significantly decreased in SLE T cells and that two SLE patients exhibited a 36 bp, exon 7 deletion of the TCR ζ mRNA. To investigate further common mutations in TCR ζ mRNA among SLE patients, mRNA was isolated from the peripheral blood T cells of two normal controls, two systemic sclerosis (SSc) patients, and eight SLE patients. TCR ζ cDNA was amplified by RT-PCR. Five out of the eight SLE patients exhibited abnormal migration patterns of the TCR ζ cDNA in PCR single stranded conformational polymorphism analysis. PCR products were ligated into pUC18 and five clones obtained were sequenced. Analysis of the nucleotide sequences revealed that all of the five pUC18 clones from the normal controls and SSc patients had the normal nucleotide sequence, whereas all eight SLE patients had mutations in TCR ζ cDNA accompanied by predicted amino acid substitutions. Mutations found in six of these patients corresponded to those of the third immunoreceptor tyrosine-based activation motif (ITAM) domain or the GTP/GDP binding site in TCR ζ. Thus, these mutations in TCR ζ mRNA could be responsible for the decreased expression of the TCR ζ protein in SLE T cells.

Objective. To determine the possible role of a novel integrin, αEβ7, in the pathogenesis of systemic lupus erythematosus (SLE). Methods. Expression of αEβ7 was examined on peripheral blood lymphocytes (PBL) from normal subjects (n = 25) and patients with SLE (n = 31), primary Sjogren's syndrome (n = 7), or polymyositis/dermatomyositis (n = 8) by cytofluorometry and/or immunoprecipitation. Adhesion of αEβ7+ T cells to HSG epithelial cells was investigated using a confocal image analyzer. Results. After phytohemagglutinin stimulation, expression of αEβ7 on PBL, especially on CD8+ T cells, was significantly higher in SLE patients than in normal subjects (P < 0.01). Elevated αEβ7 expression was associated with the presence of oral ulcers or serositis (P < 0.05). Activated SLE T cells with enhanced αEβ7 expression strongly adhered to HSG; this adhesion was partially blocked by anti-αEβ7. Conclusion. Expression and adhesion of αEβ7 on activated PBL was significantly increased in patients with SLE with epithelial involvement. This suggests a role of this novel integrin in tissue-specific retention of activated PBL, due to increased αEβ7-E- cadherin interaction, which may contribute to epithelial inflammation.

Objective. To examine the expression of surface structures important in natural killer (NK) cell function and the roles of serum factors affecting the expression of surface antigens on these cells in patients with Sjogren's syndrome (SS). Methods. Peripheral blood mononuclear cells (PBMC) of 18 patients with SS were analyzed by immunofluorescence on a flow cytometer. The antigen recognized by autoantibodies in their sera was analyzed by immunofluorescence and by immunoprecipitation. Results. Expression of Fcγ receptor III (CDI 6), an important indicator of NK cytolytic activity, on PBMC was significantly decreased in patients with SS who have extraglandular disease, while the expression of Fcγ RI and II was normal. Moreover, F(ab')2 of IgG in these patients' sera was shown to bind to CD16+ cells. Immunoprecipitation study showed that it bound to CD16 itself. Conclusion. The depressed expression of CD16 on PBMC and the presence of antibody binding to CD16 in the patients' sera may be, at least in part, responsible for the altered function of NK cells observed in SS.

PURPOSE. To evaluate the effects of a lipophilic analog of superoxide dismutase (SOD) in the prevention of polymorphonuclear leukocyte (PMN)- induced damage to corneal epithelial cells in vitro and in bacterial corneal ulcers in vivo. METHODS. Immortalized human corneal epithelial cells (T- HCEC) were cocultured with human PMNs activated with N-formyl-methionyl- leucyl-phenylalanine for 18 hours, after which lactate dehydrogenase (LDH) activity of the supernatant was measured as a marker of cellular damage. The inhibitory effects of lecithin-bound SOD (PC-SOD) and unmodified SOD, as well as PMNs pretreated with anti-CD 18 monoclonal antibody, were compared with untreated control. The retention of each drug on the ocular surface of healthy volunteers was measured by flow cytometry using brush cytology samples. The protective effects of a 0.1% solution of PC-SOD on Pseudomonas aeruginosa corneal infection in guinea pigs were assessed by inflammatory grading scores and histology. RESULTS. Both PC-SOD and SOD effectively suppressed PMN-induced LDH release in T-HCEC in a dose-dependent manner. LDH release was also attenuated when PMNs were pretreated with anti-CD 18 antibodies, suggesting that adhesion molecules were involved in the process. Brush cytology of conjunctival samples showed that PC-SOD was retained longer on the ocular surface compared with unmodified SOD. PC-SOD significantly prevented excessive tissue damage by infiltrating PMNs in P. aeruginosa corneal infection, whereas in control eyes, perforation of the cornea occurred by 6 days. CONCLUSIONS. PC-SOD was effective in attenuating PMN- related tissue damage to corneal tissue both in vitro and in P. aeruginosa infection in guinea pigs.

Background - Recent 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 anti 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.

Purpose: We measured the secretion volume of tear and sauva in MRL and BALB/c mice to confirm that lacrimationand salivation is related to lymphocyte infiltration in these glands. Methods: To quantify the tear secretion volume. Schirmer test with cotton thread test was performed in MRL/ipr, MRL/+ and BALB/c mice. Saliva secretion volume was measured with capillary tube. Lymphocytic infiltration into the exocrine glands was graded, and infiltrated lymphocytes were characterized by immunohislochemistry. The apoptotic cells in the exocrine glands were detected by TUNEL method. Results: There were no significant differences in tear secretion between BALB/c and MRL/lpr or MRL/+ mice. In MRL/lpr the saliva secretion was reduced in the comparison with BALB/c mice. Immunohistochemical analysis revealed that the CD4 positive T lymphocytes were located in the focus region in both of the glands in MRL/lpr and MRL/+ mice. In the salivary gland, the CDS positive T lymphocytes were observed in the focus region and around acini and duct in MRL/lpr mice. In the salivary glands the apoptotic cells were detected at the ductal and acinar cells only in MRL/lpr mice. Conclusion: In this study, we found that the CD4 positive T lymphocyte dose not necessarily result in the decrease of the secretion. In salivary gland. CDS positive lymphocytes around acini and duct may cause the apoptosis of the acinar and ductal epithelial ceils and the dysfunction of the salivary secretion.

Cytology using the brush technique is readily available and is a rapid means of establishing a presumptive diagnosis of ocular surface changes. However, those techniques have some limitations when obtaining cells from certain localized areas particularly if using the Cytobrush-S®. We have described here a new type of brush (Accellon-M®), which can collect the local cells using its spherical tip, and evaluated them by comparing with Cytobrush-S®. Furthermore, we differentiated epithelial cells from non- epithelial cells that were collected by brushing, and by a combination of brush cytology and flow cytometry using an anti-keratin antibody, AE-3, which reacts with all basic epithelial keratins. Accellon-M® could collect the epithelial cells from conjunctiva as effectively as with the Cytobrush-S®, and there were no statistical differences between both groups. AE-3, which is reported as the marker of epithelial cells, were detected quantitatively by a combination of brush cytology and flow cytometry techniques. The result of the present study emphasizes that both the Cytobrush-S® and the Accellon- M® are valuable for conjunctival brush cytology. An additional positive feature of the Accellon-M® may be improved cell collection from the conjunctival epithelium especially when the target cells are in a limited area. The combination of the brush cytology and the flow cytometry technique appears to be a useful adjunct as an additional diagnostic or research tool for use in the detection of various antigens in the conjunctival epithelium.

Purpose. The host response to allergens appears to be regulated by specific patterns of local cytokine production. More than 20,000 conjunctival superficial cells were collected with a special brush, a smaller version of the Cytobrush used in cervical cytology, from the upper palpebral conjunctiva. Methods. Samples were obtained by cytology brush from seven patients with allergic conjunctivitis and from seven healthy volunteers. Giemsa staining, immunocytochemistry, and flow cytometric analysis were performed. Cytokine gene expression was assayed by the reverse- transcription-polymerase chain reaction method. Results. Giemsa staining of cytocentrifuged preparations from patients with allergic conjunctivitis showed conjunctival epithelial cells with lymphocytes, mast cells, and eosinophils. In an immunohistochemically study, a few CD3- and CD4- bearing cells, but not CD20- and CD14- bearing cells, were seen in patients. In 82.6 ± 17% of the samples obtained from allergic patients, HLA-DR was present, but it was present in only 34.2 ± 17.8% of samples from control subjects (P = 0.0001) using flow cytometric analysis. Steady state transcripts of mRNA for cytokines were analyzed with RT-PCR in conjunctival cell samples, and results showed that samples from allergic conjunctivitis expressed increased transcripts of interleukin 4 and interleukin 13 but virtually no interleukin 2 or interferon-γ; six samples from seven healthy subjects expressed no interleukin 2, interleukin 4, interleukin 13, or interferon-γ transcripts. Conclusions. These results suggest that the clinical features of allergic conjunctivitis in humans are associated with a specific local pattern of proinflammatory cytokine expression.

Purpose. Sjogren's syndrome (SS) is a systemic autoimmune disease characterized by decreased lacrimal and salivary gland function causing dry eye and dry mouth. We examined several parameters as measures of lacrimal function and compared the serum soluble interleukin-2 receptor (sIL-2R) and serum autoantibodies as measures of systemic autoimmune condition. Methods. Serum levels of sIL-2R, antinuclear antibody (ANA), and rheumatoid factor (RF), in 128 patients (16 men, 112 women; average age, 54.3±12.5 years) were measured. The lacrimal function was evaluated clinically. Results. There was no correlation between systemic condition and the cotton-thread test, Schirmer test with or without anesthesia, tear clearance test, tear-function index, or the BUT. However, the Schirmer test with nasal stimulation and rose bengal and fluorescein staining was positively correlated with systemic parameters. Conclusion. We show the correlation of serum sIL-2R, ANA, and RF to lacrimal function, which is good evidence that systemic immune conditions directly affect local lacrimal gland function.

We analyzed the levels of expression of different surface adhesion molecules on normal human conjunctival epithelial cells with cytology and flow cytometry. The levels of β1 (CD29), VLA-1 (CD49a), VLA-2 (CD49b), and VLA-3 (CD49c) integrins were high, whereas those of VLA-5 (CD49e) and VLA-6 (CD49f) were faint, and that of VLA-4 (CD49d) was undetectable. Among the β2 integrins, the levels of Mac-1 (CD11b) and p150.25 (CD11c) were positive, while LFA-1 (CD11a) was not detectable. HLA class I and class II were also expressed. However, CR2 (CD21), CD57, CD44 and ICAM-1 (CD54) were not detected. Our study suggests that these techniques may aid in identifying changes in the expression levels of various surface antigens on conjunctival epithelial cells that could occur in ocular surface disease.