Many autoimmune diseases, including primary Sjögren's syndrome (SS), are characterized by increased numbers of mast cells and increased histamine levels in serum and tissue fluids (1–3). However, in spite of some initial enthusiasm, the tentative role of mast cells and histamine in autoimmunity was not supported by trials with H1 or H2 histamine receptor antagonists (4, 5).
Later it was found that in addition to the professional histamine-synthesizing, -storing, and -releasing cells, such as mast cells and basophils, many other cells, including dendritic cells (6) and T cells (7), can synthesize histamine, albeit at a 100–1,000-fold lower rate. These cells release histamine as they synthesize it, without intermediate storage or stimulated release. At first these observations raised little interest, but when the highly histamine-sensitive H4 histamine receptor was discovered in 2000 (8), the potential role of histamine in autoimmune diseases became a hot topic. In fact, H4 histamine receptor has a >10,000-fold higher affinity for histamine than H1 histamine receptor (pKi 8.3 versus pKi 4.2) (9). Therapeutic concentrations of the traditional antihistamines and H2 histamine receptor antagonists do not affect H4 histamine receptor at all. Further, H4 histamine receptor has been found in many hematopoietic cells, including dendritic cells and lymphocytes, and at lower levels in some peripheral tissue (8).
SS is sometimes referred to as autoimmune epitheliitis because the tubuloacinar epithelial cells are believed to form a source of autoantigens and alarmins and to function as nonprofessional antigen-presenting cells, equipped with class I major histocompatibility complex (MHC) and class II MHC antigens and with costimulatory molecules (10). Due to these exciting new developments, we decided to investigate whether H4 histamine receptor is present in salivary glands (SGs) at the messenger RNA (mRNA), H4 histamine receptor protein, and functional levels and to determine whether it is only constitutively expressed or is perhaps affected by autoimmunity in SS.
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Due to previous observations of increased mast cell numbers in tissue and high histamine levels in body fluids in various autoimmune diseases (1–3), histamine has been thought to play a role in autoimmunity. New findings show that so-called nonprofessional histamine-synthesizing cells produce small quantities of histamine and that histamine in relatively low concentrations can stimulate the more recently discovered high- avidity H4 histamine receptor (6, 7). Therefore, we analyzed the presence of H4 histamine receptor mRNA in healthy HSGs. H4 histamine receptor mRNA was found, which suggests local production by some SG cells in human LSGs.
In the present study, immunohistochemical staining was performed to localize the corresponding H4 histamine receptor protein. This is relevant because bone marrow–derived hematopoietic cells contain H4 histamine receptor (8, 9). Intravascular and tissue leukocytes could account for the H4 histamine receptor mRNA that was detected in HSGs by PCR. However, immunostaining showed that H4 histamine receptor was mostly localized to the resident acinar and ductal epithelial cells. Some H4 histamine receptor immunoreactive leukocytes were seen intravascularly and in the interstitial tissue. Immunostaining, using antibodies specifically produced against human H4 histamine receptor, confirmed and extended the mRNA-level data.
The effect of H4 histamine receptor stimulation on human submandibular gland–derived SG cells was studied using the H4 histamine receptor–specific agonist ST-1006 (12). Cultured HSG cells were stimulated with an effective concentration of ST-1006, and the cell culture supernatant was screened for 39 different cytokines using xMAP technology and multiplex kits. This approach is useful for screening, but its interassay variation is too high for accurate measurements. Therefore, the results were validated by measuring the 2 cytokines that appeared to be the most affected, IL-8 (also known as angiogenic factor or CXCL8) and VEGF (VEGF-A), using ELISA.
Compared to healthy control glands, the proportion of IL-8–immunoreactive cells was increased in the acinar and ductal epithelial cells in glands from SS patients. Salivary IL-8 concentration is higher in SS patients than in healthy controls. VEGF has been determined to play a harmful role in SS due to a strong relationship between pathologic neovascularization and impaired secretory function. In SS patients, histamine derived from the activated mast cells and nonprofessional histamine-producing dendritic cells and lymphocytes may excessively stimulate tubuloacinar cells via H4 histamine receptor to produce IL-8 and VEGF. This may lead to down-regulation of H4 histamine receptor at the mRNA and receptor protein levels in SS (see below).
Next, we compared H4 histamine receptor levels in SGs from healthy controls to those in SGs from patients with SS. Although mRNA levels were slightly higher in healthy controls, in this small sample the difference was not statistically significant. However, immunostaining showed strong H4 histamine receptor staining in healthy glands but very weak H4 histamine receptor staining in glands from SS patients. This is consistent with our hypothesis that the histamine-producing cells stimulate H4 histamine receptor–positive tubuloacinar cells so that their H4 histamine receptor protein expression is down-regulated, e.g., via clathrin-mediated endocytosis and subsequent degradation of H4 histamine receptor in secondary lysosomes. SGs from SS patients were relatively heavily infiltrated by hematopoietic cells, which represent the best recognized cellular source of H4 histamine receptor (8, 9). In spite of this, the H4 histamine receptor levels were lower in glands from SS patients than in glands from healthy controls.
Measurements of histamine in saliva, using optical beam deflection fluorometry after high-performance liquid chromatography and ELISA, show that salivary histamine levels range from 0.31 to 12.4 ng/ml (2.8–112.7 nmoles [1 nmole = 0.11 ng/ml]) (14). These salivary histamine values are all too low to affect H1 histamine receptor (pKi 4.2) or H2 histamine receptor (pKi 4.3), but they do stimulate H4 histamine receptor, because 10−8.1M histamine will half-maximally activate this receptor (H4 histamine receptor pKi 8.1) (9). This suggests that H4 histamine receptor may play some constitutive role in the maintenance of healthy salivary epithelium, a function which, based on changes in local histamine synthesis and/or greatly diminished H4 histamine receptor levels, is disturbed in SS.
H4 histamine receptor exists in both active and inactive forms, which undergo spontaneous isomerization. One of the important consequences is that H4 histamine receptor, even in vivo, is constitutively (physiologically and dynamically)—without any ligand—almost half-maximally activated (15). Histamine agonists bind to the active H4 histamine receptor isomer, stabilizing it and, thus, enhance H4 histamine receptor activity. Many H4 histamine receptor antagonists are inverse agonists, which bind to the inactive H4 histamine receptor isomer, stabilizing it. This decreases H4 histamine receptor activity. Neutral antagonists bind to both the active and inactive H4 histamine receptor isomers, without discrimination, and do not alter H4 histamine receptor activity, but they interfere with the subsequent binding of agonists or inverse agonists. This is consistent with the role of H4 histamine receptor in the maintenance of the healthy tubuloacinar epithelium, with or without its natural histamine ligand in saliva. Further, H4 histamine receptor is internalized after ligand binding or spontaneously (Stegaev V, et al: unpublished observations), leading to desensitization, but also to an ability to rapidly translocate to the cell surface via endocytotic receptor recirculation. This phenomenon may also affect the H4 histamine receptor staining pattern.
Due to the clinical importance and commercial success of the H1 and H2 histamine receptor blockbuster drugs, there is great interest in H4 histamine receptor antagonists, inverse agonists, and agonists in the pharmaceutical industry. A role for mast cells and histamine in autoimmune diseases has long been suspected. The discovery of the nonprofessional histamine-synthesizing cells, and later, at the turn of the millennium, of H4 histamine receptor, has revived these expectations. Many H4 histamine receptor–selective small molecular weight chemical compounds are undergoing intense investigation by major drug companies (15). They might in the future provide alternatives to relatively expensive and widely used biologic agents for the treatment of autoimmune diseases, including SS.
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- PATIENTS AND METHODS
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All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Konttinen had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study conception and design. Stegaev, Sillat, Mackiewicz, Chazot, Konttinen.
Acquisition of data. Stegaev, Sillat, Porola, Hänninen, Mieliauskaite, Rotar, Mackiewicz, Stark, Konttinen.
Analysis and interpretation of data. Stegaev, Sillat, Falus, Buzás, Mackiewicz, Chazot, Konttinen.