Absence of in vitro responses to type II collagen by splenocytes from arthritic BALB/c mice is possibly caused by intrinsic CD25+ regulatory cells.

Collagen-induced arthritis-resistant BALB/c mice develop arthritis if a foreign protein is added to an emulsion of type II collagen (CII) and adjuvant. The IgG autoantibody activity to CII is increased, whereas no CII autoreactive T cells in vitro can be recorded. In this study, we have explored whether CD25+ cells inhibit T-cell autoreactivity to CII. We also followed the IgG anti-CII autoantibody activity and the IL-6 level in serum during the development of arthritis. BALB/c mice were coimmunized with bovine CII (BCII) and keyhole limpet haemocyanin (KLH) in complete Freund's adjuvant and boostered 3 weeks later. Control animals were immunized with either BCII or KLH. Sera were collected prior to and during the development of arthritis and examined for IgG anti-CII antibody activity and IL-6 content. When all BCII-KLH immunized mice had developed arthritis, splenocytes were prepared, with and without CD25+ cells, and tested for BCII reactivity in vitro. The serum IgG, IgG1 and IgG2a anti-CII antibody activities and the IL-6 level were significantly higher in BCII-KLH immunized mice than in BCII-immunized animals that failed to develop arthritis. The BCII-specific IL-2 secretion in vitro was significantly increased in CD25-depleted splenocyte cultures prepared from arthritic BCII-KLH-immunized mice. Development of arthritis in BALB/c mice induced by coimmunization with BCII/KLH results in increased levels of circulating IL-6 and IgG autoantibodies to CII. The arthritogenic BCII-KLH immunization potentiates BCII-specific IL-2 secretion by CD25-depleted splenocytes, but CD25+ cells hamper the outcome of their action, at least in vitro.

Induction of experimental arthritis in BALB/c mice by inclusion of a foreign protein in the collagen inoculum.

The susceptibility of mice to collagen-induced arthritis (CIA) has, among other things, been linked to the major histocompatibility complex class II genes as well as other genes. This study was designed to examine the possibilities to establish CIA in low susceptible I-Ad (Balb/C) mice. Balb/C mice were immunized twice with bovine type II collagen (BCII) in complete Freund's adjuvant (CFA) containing the amount of Mycobacterium tuberculosis needed to induce CIA in low susceptible I-Ab (C57BL/6) mice. Some mice received the conceivable arthritogenic inoculum mixed with ovalbumin (OVA). Clinical arthritis was monitored. Antibody activity and T-cell reactivity to BCII were determined. Unexpectedly, only mice that were immunized with the BCII-OVA mixture developed arthritis. Combining BCII with another foreign protein, keyhole limpet hemocyanin, but not the self-protein mouse serum albumin, also triggered arthritis. Prior to the appearance of arthritis the serum levels of IgG autoantibodies to BCII were higher in the coimmunized mice than in the mice that were immunized with BCII alone. Yet, splenocytes stimulated in vitro with BCII did not proliferate or produce interferon-gamma. Immunization of Balb/c mice with an emulsion-containing CFA and BCII mixed with a foreign body, but not a self-protein, elevates the level of circulating autoantibodies to CII and subsequently induces arthritis.

Low HEMA conjugation induces high autoantibody titer in mice.

2-hydroxyethylmethacrylate (HEMA) is a known causal agent of hypersensitivity to resin composites. We have reported that immunization with HEMA conjugated to mouse serum albumin (MSA) induces an autoantibody response in mice. In this study, we investigated both the activity and the avidity of autoantibodies induced by immunization with various HEMA conjugations to MSA. Female Balb/c mice were given MSA carrying 3, 7, 15, or 22 HEMA molecules. Antigen-specific IgG and IgE antibodies were determined by ELISA, and average antibody avidity by thiocyanate dissociation. Immunization with MSA carrying the lowest number of HEMA molecules induced a significantly higher IgG and IgE anti-MSA autoantibody response, with significantly higher IgG antibody avidity, than did the more heavily conjugated preparations. The results suggest that the lower the degree of HEMA conjugation to self-protein, the higher the risk for autoantibody production to the carrier protein. These findings suggest a mechanism of potential relevance in humans.

Mucosal immunity.

Mucosal defense is provided by a number of host factors countering the specific virulence factors of the many microorganisms infecting the mucous membranes. Secretory IgA antibodies presumably play an important role. Increase of the sIgA antibodies may most advantageously be attained by parenteral immunization, following mucosal priming. This was demonstrated in a rat model, where it was also noted that antigen injection into PP induced high milk IgA antibody levels. In man, parenteral vaccination against polio increased the sIgA antibody levels in the milk of mothers previously exposed naturally to the poliovirus. The response was relatively short-lived. In the previously unexposed, there was little or no response. By contrast peroral immunization with live poliovirus vaccine did not increase, or even decrease, the milk sIgA poliovirus antibody levels. Although salivary sIgA antibodies against antigens of colonizing E. coli appear during the first days of life, they are slow to increase. This deficiency is richly compensated for by all the sIgA antibodies that are provided the baby through the milk. No transfer of dimeric IgA into the milk could be shown in lactating rats, in contrast to what has been reported in mice. There is no evidence for a contribution to milk sIgA from serum in man. Close to parturition, human milk often contains some 7S IgA and various sizes of free SC, in addition to the dominating 11S sIgA. A few days later there is almost exclusively monomeric SC and 11S sIgA. IgG antibodies also play a role at the mucosal level. IgG2 antibodies against the bacterial polysaccharide capsule are as slow to appear as sIgA in ontogeny, possibly explaining the prevalence of infections with encapsulated bacteria and the poor response to polysaccharide vaccines in early childhood. Other defense factors preventing infections by way of mucous membranes may be important. Thus, oligosaccharides present in human milk seem to specifically prevent pneumococcal attachment to retropharyngeal cells. This anti-attachment capacity, in addition to that provided by milk and salivary IgA antibodies, may explain why breast-fed babies have less otitis media than formula-fed ones.

Voluntary chronic exercise augments in vivo natural immunity in rats.

The effect of chronic voluntary exercise on the immune response was studied in spontaneously hypertensive rats. Exercise consisted of voluntary running in wheels for 5 wk, and the mean running distance was 4.2 km/24 h. In vivo cytotoxicity was measured as clearance of injected 51Cr-labeled YAC-1 lymphoma cells from the lungs. The clearance of YAC-1 cells in vivo was significantly increased in runners compared with sedentary controls (P < 0.001). The total number of mononuclear cells in the spleen was significantly decreased in runners compared with controls. Analysis of splenic lymphocyte phenotypes revealed a significantly increased fraction of OX52+/CD5- natural killer cells in runners compared with sedentary controls. In contrast to changes in natural immunity, immunoglobulins G and M levels in serum, the antibody response to antigen in vivo, and the proliferation of splenic T cells in vitro were unchanged. Our data suggest that chronic voluntary exercise augments natural cytotoxicity mechanisms in vivo, whereas splenic T-cell proliferation and the antibody-mediated immune response remain unchanged.

Difference in capacity between macrophages and dendritic cells from rat incisor pulp to provide accessory signals to concanavalin-A-stimulated T-lymphocytes.

The present study compared the ability of dendritic cells and macrophages derived from the dental pulp to provide accessory signals to Concanavalin A (Con A)-stimulated T-lymphocytes. Pulpal cells from maxillary and mandibular rat incisors were enzymatically released with collagenase. T-lymphocytes were isolated from rat cervical lymph nodes. In initial experiments, suspensions of unseparated pulpal cells were found to provide co-stimulatory help to Con-A-treated T-lymphocytes. The proliferation rate correlated well with the number of cells in the pulp suspension and followed a time course characteristic of a Con-A-driven proliferation of T-lymphocytes. Depletion of class II molecule-expressing cells from the unpurified suspension of pulpal cells resulted in lost ability to provide accessory signals to Con-A-stimulated T-lymphocytes. In contrast, removal of ED2-positive cells, i.e., macrophages, did not affect the ability of the suspension to give this assistance. Partially purified class II molecule-expressing cells enhanced the proliferative response, while addition of enriched macrophages did not. It was concluded that cells in the normal dental pulp with the characteristics of dendritic cells have the capacity to provide help to Con-A-stimulated T-lymphocytes, while cells with the macrophage phenotype lack this ability.

Reduced in vivo cell-mediated immune responses to mumps, tuberculin, and streptokinase/streptodornase but not to Candida albicans in oral lichen planus.

Oral lichen planus is considered to be a T-cell-mediated disease. The purpose of this study was to investigate the capacity of T-lymphocytes in oral lichen planus patients to respond to a number of commonly encountered environmental antigens in vivo. To do this, we assessed dermal delayed-type hypersensitivity responses to mumps, streptokinase/streptodornase, Candida albicans, and purified protein derivative of tuberculin (PPD) in 17 oral lichen planus patients and in matched controls. Reduced induration in response toward mumps, PPD, and streptokinase/streptodornase was demonstrated in oral lichen planus patients compared with controls. In addition, the total sum of induration diameters was decreased in the patients. However, C. albicans stimulation resulted in similar levels of response in both groups. The differences in induration size between matched patients and controls for mumps and PPD were thus significantly greater than the corresponding differences for the C. albicans antigen. This suggests that a selective difference in the response to these antigens exists in oral lichen planus patients. The results may point to a loss of memory T-helper function to infrequently encountered environmental antigens, represented by mumps, PPD, and streptokinase/streptodornase, contrarily to memory function to common antigens (C. albicans), which seem to be unaffected.

HEMA bound to self-protein promotes auto-antibody production in mice.

While several studies report that acrylic monomers contained in dental materials may cause hypersensitivity reactions, little is known of the associated immune response. Here we address the potential of 2-hydroxyethyl-methacrylate (HEMA) to bind to endogenous protein and elicit auto-antibody production in vivo. Albumin was incubated with HEMA at various times and pH. Following confirmation of the conjugation by inhibition of trinitrophenyl (TNP) binding, female Balb/c mice received HEMA conjugated to mouse serum albumin (MSA) in Freund's incomplete adjuvant or saline subcutaneously. ELISA was used to determine the serum antibody responses to native and modified MSA. IL-2 production in spleen cell cultures stimulated with HEMA-conjugated MSA was measured. HEMA reacted with serum albumin at physiological conditions. HEMA-conjugated MSA induced IL-2 secretion and production of IgG antibodies to native MSA. The results suggest that modification of an endogenous protein like serum albumin with HEMA may defeat the control of immune responses to this self-protein.

Langerhans cells from oral epithelium are more effective in stimulating allogeneic t-cells in vitro than Langerhans cells from skin epithelium.

Dendritic cells, such as Langerhans cells (LC), in different ectodermal compartments may have different functional capabilities. The present study was undertaken to compare oral Langerhans cells (LC) with those of the epidermis in terms of their ability to co-stimulate T-cells in vitro. A Mixed Epithelial Cell Lymphocyte Reaction (MELR) and a mitogen-driven (concanavalin A) T-cell proliferation assay were used. In both assays, LC in a crude cell suspension of freshly isolated oral epithelial cells were found to be five times more effective in mediating T-cell proliferation than freshly isolated epidermal LC. Twenty-four-hour cell culture at 37 degrees C enhanced the T-cell response in the MELR compared with cells cultured at 4 degrees C. This applied to both skin and oral epithelial cells. Oral and skin epithelial cell suspensions depleted of LC lost the capacity to stimulate allogeneic T-cells. Incubation of the epithelial cell suspensions with recombinant Granulocyte/Macrophage-Colony Stimulating Factor (rGM-CSF) did not enhance the co-stimulating capacity of the LC. Titration of different numbers of oral and skin LC to T-cells showed that skin LC were never able to reach more than 44% of the maximal stimulatory capacity of oral LC. Data show that oral LC are more efficient than skin LC in providing co-stimulatory signals to T-cells, suggesting a difference in functional capacity between the two cell populations.

Structural and functional association between substance P- and calcitonin gene-related peptide-immunoreactive nerves and accessory cells in the rat dental pulp.

Defense mechanisms of the dentin/pulp complex involve a variety of biological systems in which immunocompetent cells, the nervous system, and the vascular supply play important roles. In the present study, pulpal accessory cells were examined regarding (i) their structural relationship to nerves and (ii) how the functional capacities of these cells were affected by neuropeptides. Micro-anatomic association was investigated in the normal rat molar pulp with the use of double-immunofluorescence staining and dual-channel confocal laser scanning microscopy. Examinations of confocal laser scanning microscopic images from single focal planes revealed the presence of apparent contacts between thin, varicose nerve fibers and immunocompetent cells, indicating proximity between these two structures. The close associations were most frequently observed in the para-odontoblastic region of the coronal pulp, where more than 70% of class II antigen-expressing (OX6+) cells showed proximity to nerve fibers immunoreactive to calcitonin gene-related peptide. The corresponding figure for substance P was about 50%. ED2+ macrophages closely associated with nerves were less frequently observed. Functional studies conducted in vitro demonstrated that 10(-9) to 10(-7) mol/L of substance P significantly increased (p < 0.05), while 10(-7) to 10(-6) mol/L of calcitonin gene-related peptide suppressed (p < 0.01) proliferation of purified T-lymphocytes stimulated with sub-optimal concentrations of concanavalin A in the presence of rat incisor pulpal cells as accessory cells. These data suggest that pulpal sensory nerve fibers and their products may have an influence upon the immune defense of the dental pulp.

Distribution of interleukin-2, -4, -10, tumour necrosis factor-alpha and transforming growth factor-beta mRNAs in oral lichen planus.

In the present study, MRNA for the cytokines interleukin-2 (IL-2), IL-4, IL-10 tumour necrosis factor-alpha (TNF-alpha) and transforming growth factor beta-1 (TGF-beta-1) were investigated in oral lichen planus (OLP) lesions using in situ hybridization with 35S-labelled oligonucleotide probes on frozen tissue sections. In addition, the expression of interferon-gamma (IFN-gamma), IL-10 and IL-4 mRNAs was analysed in cultured lesional T lymphocytes from oral lichen planus by polymerase chain reaction. Cells expressing mRNA for IL-2, IL-4, IL-10, TNF-alpha and TGF-beta 1 were found in all the biopsies studied. Approximately 1-2% of the total number of infiltrating cells in the lesions were positive for each of the different cytokine mRNAs. Most biopsies contained basement membrane-oriented, mRNA-positive cells. In the cultured T-cell lines, message for IFN-gamma was detected in all the patients, IL-10 in all but one, and IL-4 in just one of the seven patients investigated. The results suggest that mRNA for both pro- and anti-inflammatory cytokines, i.e., mixed T-helper 1 (TH1) and TH2 cytokine profiles, are generated simultaneously by a limited number of cells in chronic lesions of OLP.

HEMA enhances IgG1 production by human B-cells in vitro.

UNLABELLED: We have previously shown that the resin monomer 2-hydroxyethylmethacrylate (HEMA) affects mouse B-lymphocyte activity, leading to increased IgG1 antibody production in vivo. In the present study, we tested, in vitro, the hypothesis that HEMA also affects human B-lymphocyte activity. The in vitro production of IgG1, IgM, and IgA in supernatants from purified human CD19+ B-lymphocyte cultures, containing different concentrations of HEMA, was assayed with ELISA. Proliferation was measured by [methyl-(3)H] thymidine incorporation. Of the different HEMA concentrations used, the lower concentrations caused a significant increase in IgG1 production, but not in IgM or IgA production, in vitro. The lower HEMA concentrations did not significantly change B-cell proliferation. At the highest concentration, HEMA significantly suppressed IgG1 and IgM production, as well as B-cell proliferation, in vitro. In conclusion, HEMA can, at certain concentrations, selectively enhance human B-lymphocyte IgG1 production. ABBREVIATIONS: 2-hydroxyethylmethacrylate (HEMA), Dulbecco's Modified Eagle's Medium supplemented with heat-inactivated fetal bovine serum, gentamycin, penicillin, and streptomycin (D-MEM++++), Enzyme-linked Immuno-sorbent Assay (ELISA), phosphate-buffered saline (PBS), ovalbumin (OVA), pokeweed mitogen (PWM), counts per minute (CPM).