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Studies were conducted to determine the reactivity of six monoclonal antibodies specific for major histocompatibility complex class II molecules expressed on a bovine B cell line homozygous for BoLA-DR and BoLA-DQ alleles. Direct immunoprecipitation, serial immunodepletion experiments, and two-dimensional gel electrophoresis revealed that these antibodies reacted with three different molecules. Bovine orthologues of HLA-DR were recognized by three monoclonal antibodies--H34A, TH12A, and TH14B. Orthologues of HLA-DQ were characterized by two other monoclonal antibodies, TH22A and TH81A. A third BoLA class II antigen, neither DR nor DQ was revealed by the last monoclonal antibody H42A. The relation of this molecule to known molecules in humans and other species remains to be established. However, cumulative data suggest that the determinant is expressed on a molecule related to HLA-DP.

We have developed a simple and reliable method, PCR-PHFA (polymerase chain reaction dependent preferential homoduplex formation assay), for detection of single base substitutions within PCR amplicons. This technique is based upon strand competition during hybridization between a double labeled amplicon, prepared from biotin and DNP labeled primers, and an unlabeled amplicon. Under the precisely controlled temperature gradient, the preferential formation of a homoduplex over a heteroduplex occurs. After annealing, the identical sequence of the double labeled and unlabeled amplicon resulted in a low population of regenerated double labeled dsDNA due to strand exchange between them. Even when the two differed by only a single base substitution, double labeled molecule was regenerated efficiently because of preferential homoduplex formation. The regenerated double labeled molecule was captured onto a streptavidin coated microtiter plate and quantified enzymatically with a chromogenic substrate. The technique has been successfully applied in HLA-DPB1 typing. Furthermore, we detected a mutated gene even in the presence of a large excess of the corresponding normal gene.

HLA types in Japanese patients with primary hypothyroidism were analyzed to see whether those with blocking-type TSH receptor antibody (TSH-R BAb M) differed genetically from those with idiopathic myxedema (IM). HLA typings of -A, -B, -C, -DR, and -DQ (73 antigens) were performed serologically, and those of -D and -DP (29 antigens) were analyzed by the restriction fragment length polymorphism method. Thirty patients were studied with TSH-R BAb M, and 28 with IM. The data were analyzed and compared with our previous results from 88 Graves' patients, 46 Hashimoto patients, and 186 control subjects. Overall, 192 patients with 4 autoimmune thyroid disorders showed a decrease in -Aw19 and an increase in -DQw4 (corrected P < 0.05) and significant associations of -Aw33, -Bw46, -Cw3, -DRw8, -DR9, and -DQw3. In TSH-R BAb M patients, increases in -B35, -Bw60, and -Dw8 and decreases in -DR4 and -DPw2 were seen, whereas IM patients showed increased -DPw2, -Bw61, and -Dw23. In comparisons between TSH-R BAb M and IM, the difference in -DPw2 was highly significant. HLA-B35 differed significantly in these 2 types of hypothyroidism. In conclusion, TSH-R BAb M patients have decreased frequency of -DPw2 and are genetically similar to Graves' disease, whereas IM patients are characterized by high frequency of -DPw2 and are genetically similar to Hashimoto's thyroiditis.

Skin biopsies were taken from 11 patients with morphoea, nine with acrosclerosis and 10 with diffuse systemic sclerosis and processed for immunohistochemical studies using a panel of monoclonal antibodies including antibodies to MHC class II antigens. A significantly higher percentage of HLA-DR positive dermal cells were observed in the reticular dermis in biopsies from patients with morphoea (44.1 +/- 16.2%), acrosclerosis (15.9 +/- 5.4%) and systemic sclerosis (39.5 +/- 2.3%) when compared with the controls (6.6 +/- 2%). A smaller percentage of dermal cells also expressed HLA-DP and -DQ. The degree of monocnuclear cell infiltrate in the biopsies, however, did not correlate with the percentage of HLA class II positive fibroblasts. In organ culture, the expression of the HLA class II antigens was almost totally lost after 3 days and was no longer detected on fibroblasts after 3 weeks of culture.

A possible role for retinal pigment epithelial cells (RPE) as local antigen presenting cells in immune inflammatory eye disease was investigated by studying the in vitro response of human RPE cells to stimulation with purified IFN-gamma or Con A induced lymphokine. RPE cells cultured with a single dose of 50-1000 u/ml IFN-gamma for up to 8 days to allow maximal Class II gene transcription, expressed HLA DP, DR and DQ antigens in a dose-dependent manner with 80% or more of cells positive for each antigen at the higher concentration. After removal of a suboptimal IFN-gamma stimulus, HLA-DR antigen expression persisted for at least 15 days. HLA-DP and DQ antigens persisted only after maximal IFN-gamma stimulation. Lymphokine from Con A stimulated lymphocytes induced higher levels of DR and DQ expression (80%) over DP (15%) implying complex interactions with other mediators present in the lymphocyte culture supernatant. Since RPE cells phagocytose and recycle autoantigen-rich retinal rod outer segments and co-express HLA DR and DQ Class II antigens in response to IFN-gamma stimulation, an immunoregulatory role in conditions in which retinal autoimmunity is implicated, such as chronic idiopathic posterior uveitis and retinal vasculitis is postulated for these cells.

The expression of human histocompatibility leukocyte antigen class II antigens on gastric epithelia and gastric carcinoma (GaCa) was studied with the use of murine monoclonal antibodies to DR, DQ, and DP antigens. DR and DP antigens but not DQ antigens were demonstrated in fundic glands of normal gastric epithelia, and DP+ cells were located in part of the DR+ epithelia. Of 15 GaCas examined, 11 expressed DR antigen, and the degree of the expression varied considerably among the specimens. DP antigen was found in 3 of the 11 DR+ carcinomas, and the DQ antigen was found in one of the 3 DR+ DP+ specimens. Thus the expression of class II antigens in normal gastric epithelia and GaCas appears to be in the order of DR, DP, and DQ. Studies on 3 GaCa cell lines (Kato III, MKN28, and MKN45) demonstrated that 1 line (Kato III) expressed DR antigen only, and the remaining lines were negative. Interferon (IFN)-gamma treatment enhanced the expression of DR antigen on Kato III cells and induced expression of DQ and DP antigens. The IFN-gamma treatment also induced expression of DR antigen but not DQ or DP antigens in 1 of the 2 negative cell lines. The induction of the class II antigens by IFN-gamma was shown to be dose dependent. However, maximal induction of DQ and DP antigens on the Kato III cells and DR antigens on MKN45 cells required 10 times more IFN-gamma than that needed for the maximal expression of DR antigen on Kato III. Northern blot analyses of cytoplasmic RNA from these cells were in agreement with and affirmed the above-described expression of the class II antigens on the cell lines. The DR antigen on the Kato III cells was capable of stimulating allogeneic lymphocytes in MLR, and its stimulatory activity was significantly enhanced by IFN-gamma. These results demonstrated a differential expression of class II antigens in the "DR, DP, and DQ order" in normal gastric epithelia, GaCa cells, and GaCa cell lines, suggesting different mechanisms acting discordantly on the expression of each of these antigens and that the DR antigen on the GaCa cell lines possesses MLR-stimulatory ability.

Frozen sections of jejunal mucosa from control subjects and patients with both treated and untreated coeliac disease were examined for HLA class II DR, DP and DQ expression. Different staining patterns with monoclonal antibodies to the different class II subgroups were observed with the control subjects. There was some inter-subject variation but in general DR greater than DP greater than DQ staining was observed with the villous enterocytes staining most strongly with the staining decreasing towards the crypt bases. The patients with treated coeliac disease gave a similar pattern to the controls. The patients with untreated coeliac disease generally gave a more intense and relatively uniform staining of both surface and crypt enterocytes for all class II subgroups.

Dendritic cells were enriched from synovial fluids (SF) of patients with inflammatory arthritis and studied by immunogold labelling and electron microscopy for expression of histocompatability antigens of the HLA-D locus. Dendritic cells from SF were larger than most of these from peripheral blood with a more extensive Golgi region and more lysosomes and microfilaments. Class II histocompatability antigens HLA-DR, -DP, -DQ and that labelled by the antibody RFDI were abundant on the dendritic cells. The macrophages in the enriched cells showed labelling for DR but little labelling with the other antibodies. DR, DP and RFDI were often concentrated at areas of contact between dendritic and other cells (other dendritic cells, macrophages or lymphocytes). On incubating labelled cells at 37 degrees C for 30 min many macrophages lost their DR label but dendritic cells always retained some surface label. Some gold labelling DR and DP was found in characteristic channels between the veils and became internalized in membrane-bound structures. A small proportion of the RFDI label internalized in areas resembling coated pits. Less DQ label internalized and appeared on vesicles inside vacuoles. Material bound to different class II molecules may thus be internalized or processed differently by dendritic cells. The presence in inflammatory lesions of large activated dendritic cells with high expression of class II antigens suggests that these cells could be presenting antigen to lymphocytes within the joints.

The expression of HLA-DR, HLA-DP and HLA-DQ antigens and of the associated invariant chain (Ii) was studied immunohistologically in sessile cells of normal ileum and ileum affected by Crohn's disease which was taken as a model for chronic inflammation. Corresponding to the local inflammatory cell density, a considerable neo-expression of MHC class II antigens and Ii was observed in epithelial cells, vascular endothelial cells, and Schwann cells of the enteric nerve plexus. While HLA-DP and HLA-DQ antigens were undetectable in sessile cells of normal ileum, class II antigens expression in ileitis followed the order HLA-DR greater than or equal to HLA-DP greater than or equal to HLA-DQ. In various cell types a differential expression of Ii and class II antigens was noted. In normal crypt enterocytes and in arterial endothelial cells of inflamed ileum, Ii was found in the absence of class II antigens. On the other hand, most Schwann cells and internodal nerve strands of the submucous and myenteric plexus exhibited HLA-DR (-DP, -DQ) antigens in the absence of detectable Ii. Moreover, HLA-DR+ endothelial cells in normal tissue specimens were Ii-, and in inflamed intestine HLA-DR expression of venous/venular and capillary endothelium greatly exceeded Ii expression. The observation of both Ii+/HLA-DR- and Ii-/HLA-DR+ (HLA-DP+, HLA-DQ+) cells questions the previously assumed close association of Ii and class II antigen expression.

Human thyroid epithelial cells (thyrocytes) express HLA Class II molecules in autoimmune thyroid diseases (ATD). Normal thyrocytes do not express Class II, but can be induced to do so by culture with interferon-gamma (gamma-IFN). We have examined HLA-D subregion expression in sections and monolayers of thyroid by indirect immunofluorescence using appropriate monoclonal antibodies. The results indicate that, in ATD, the incidence and intensity of Class II subregion expression by thyrocytes varies between patients, and follows the pattern DR greater than DP greater than DQ. The same hierarchy is observed in cultured normal thyrocytes treated with gamma-IFN: strong induction of Class II, and of DP and DQ in particular, requires relatively high concentrations of gamma-IFN or additional factors such as thyroid stimulating hormone. These findings suggest that HLA-D subregion expression by thyrocytes in on-going ATD is determined by the levels of disease related factors in the affected tissue.

Monocytes that bear HLA Class II antigens, such as HLA-DR, HLA-DQ, or HLA-DP, are obligatory for many cell-mediated immunological processes. Patients with thermal injury suffer from hypoimmunity and are at risk for developing life-threatening septic episodes. To determine whether an alteration in expression of HLA Class II antigens is involved in the defect, monocytes from the peripheral blood of burn patients and controls were double-stained with anti-Leu-M3 and either anti-HLA-DR, HLA-DQ, or HLA-DP monoclonal antibodies. As analysed by flow cytometry the percentage of Leu-M3+ monocytes from the peripheral blood from patients and controls was the same. The percentage of Leu-M3+ monocytes bearing the HLA Class II antigens and the density of antigen on the monocytes, however, was significantly reduced post-burn compared with controls. In nearly all cases these changes were detected as early as 24 h post-burn before any drug therapy was implemented. In-vivo re-expression of normal levels of HLA Class II coincided with patient recovery. In-vitro exposure of post-burn Leu-M3+ cells to IFN-gamma for 72 h restored HLA Class II expression to control levels. It is possible that the reductions in HLA Class II expression may be involved in the general immunosuppression that follows thermal injury.