Assessment of Salivary GM-CSF, MIP, and IL-1β Levels in Subjects With Varying Periodontal Conditions

Periodontal disease is characterized by various microbial infections caused by only certain microbial species out of the many types of bacteria present in the oral cavity, with progressive destruction of the tissues supporting the tooth.

Gingivitis is an inflammation caused by substances secreted from microbial plaque accumulated in or near the gingival sulcus, in the gingival tissue surrounding the teeth, and develops due to bacterial plaque. Gingivitis, the mildest form of periodontal disease, is a common condition and can usually be easily treated with good oral hygiene. Gingivitis, as defined, is present in 50% to 90% of adults worldwide. It has been observed that untreated gingivitis lesions are often at risk of developing into periodontitis in individuals. Periodontal disease is a disorder that causes loss of attachment in the tissues surrounding the tooth and destruction of alveolar bone. Periodontitis, which is the main cause of tooth loss in adults, is a common health problem. The initial cause of periodontitis lesions is microbial dental plaque, which is organized on the teeth and contains many pathogenic bacteria. Within the microbial dental plaque, Porphyromonas gingivalis (Pg), Tannerella forsythensis (Tf) and Treponema denticola (Td) play a major role in the formation of active periodontitis lesions.

Although the presence of pathogens is necessary for the onset and progression of periodontal disease, this alone is not sufficient. The host immune response is the main driver of the pathologic process. A dysregulated host immune response or imbalance within the microbial community (dysbiosis) may lead to disruption of homeostasis in the periodontium, leading to destruction of the extracellular matrix and alveolar bone loss. In immune-inflammatory processes triggered by the interaction of host and bacteria, excessive release of proinflammatory cytokines such as IL-1, IL-6, IL-8, prostaglandin E2 (PGE2) and tumor necrosis factor-α (TNF-α) as well as endopeptidases such as matrix metalloproteinases (MMPs) have been found to cause periodontal destruction.

Inflammation in periodontitis and similar local inflammatory lesions and systemic connective tissue diseases causes an increase in biological mediators such as cytokines, prostaglandins and matrix metalloproteinases (MMPs) in the tissues involved and in the systemic circulation.

Members of the colony-stimulating factor (CSF) superfamily are involved in mammalian myelopoiesis, i.e. the formation of monocytes, macrophages, dendritic cells (DCs) and polymorphonuclear phagocytes (neutrophils and eosinophils). There are three main members of this family: Macrophage (M)-CSF (or CSF-1), Granulocyte (G)-CSF (or CSF-3) and Granulocyte-Macrophage (GM)-CSF (or CSF-2). They were first identified as growth factors capable of promoting myeloid colony formation from bone marrow precursors in vitro. In these three CSFs, the entire structure of phagocytes can potentially be controlled . GM-CSF, as its name suggests, was initially identified as a hematopoietic growth factor due to its ability to induce the differentiation of granulocytes and macrophages in vitro by inducing the proliferation and differentiation of bone marrow progenitor cells. However, it was later seen as a cytokine that acts through a specific receptor on myeloid cell populations such as monocytes/macrophages, neutrophils and eosinophils, activating/differentiating them and having a potential role in inflammation.Data from models of autoimmunity and chronic inflammation using mice lacking the GM-CSF gene or neutralizing monoclonal antibodies (mAbs) suggest that GM-CSF may be an important driver of tissue inflammation and associated pain. Examples include arthritis, EAE (Induced Experimental Autoimmune Encephalomyelitis), cardiovascular disease and lung disease. GM-CSF was one of the first cytokines detected in inflamed human synovial fluid and its biological effects outside the bone marrow vary depending on its location and concentration.

The role of this cytokine in regulating inflammation has made it attractive targets for therapeutic interventions, especially in the treatment of chronic inflammation and cancer. Many of these strategies target GM-CSF for its inflammatory properties. Understanding the cellular and molecular mechanisms by which GM-CSF exerts these diverse effects is essential for the development of better therapies for chronic inflammatory diseases. Antibodies targeting GM-CSF or its receptor GMRa are currently in early-stage clinical development for the treatment of rheumatoid arthritis and multiple sclerosis.

In a study by Zhu et al, salivary levels of GM-CSF were found to be specifically elevated in the erosive oral lichen planus group. GM-CSF has been shown to increase matrix metalloproteinase expression in monocyte-macrophages and in some cases may lead to the development of bone resorbing osteoclast precursors.

Macrophage Inflammatory Protein (MIP)-1α was first identified in 1988 as MIP-1α, an 8-kd protein pair partially purified from endotoxin-stimulated macrophages. MIP-1α is a C-C family chemokine that is a potent chemoattractant for monocytes and can be stimulated by lipopolysaccharide (LPS). It was named macrophage inflammatory protein because it induces neutrophil accumulation when injected into the footpads of mice.

MIP-1α is produced by cells during infection or inflammation. MIP-1α expression can be induced in various cell types including monocytes, macrophage cell lines, mast cell lines, Langerhans cells, fibroblasts and T lymphocytes.

MIP-1α plays an important role in the pathogenesis of various inflammatory diseases and conditions such as periodontitis, multiple myeloma, Sjögren's syndrome and rheumatoid arthritis . They fulfill various biological functions such as recruitment of inflammatory cells, wound healing, inhibition of stem cells and maintenance of effector immune response. MIP-1α promotes the orientation and movement of osteoclasts. It initiates the process of osteoclastogenesis by directly activating osteoclast cells.

In bone marrow samples from patients with Multiple Myeloma, MIP-1α/CCL3 mRNA expression is higher compared to healthy individuals. There is a strong positive correlation between bone resorption markers such as serum bone-specific alkaline phosphatase (S-BALP) and serum MIP-1α levels in patients with multiple lytic bone lesions.

MIP-1α has recently been shown to play a role in the regulation of tumor metastasis.

Oral squamous cell carcinoma (OSCC) patients with metastatic lymph nodes had increased MIP-1α mRNA expression compared to those with non-metastatic lymph nodes. This suggests a possible role of MIP-1α in tumor migration.

In a study conducted by Petkovic et al. in which peri-implant groove fluid was evaluated, MIP-1α values were found to be higher in peri-implant mucositis groups compared to healthy control group patients. In the same study, the mean peri-implant groove fluid values of MIP-1α were found to be statistically significantly higher in the group with advanced mucositis compared to the group with early mucositis.

IL-1, the first interleukin identified, has many biological activities. In fact, before the molecular identification of IL-1 in the mid-1980s, various biological functions of this cytokine had been studied for many years under different names such as leukocyte endogenous mediator, hematopoietin 1, endogenous pyrogen, catabolin and osteoclast activating factor. In the last decade, other members of the IL-1 family have been identified and their important roles in innate and adaptive immune responses have been established.

Many aspects of the diverse biological roles of IL-1α and its functional homolog but structurally distinct relative IL-1β have been elucidated by the Dinarello laboratory. IL-1α and IL-1β are key members of the IL-1 family, which currently consists of eleven members. IL-1α and IL-1β are key proinflammatory cytokines that exert pleiotrophic effects in various cells and play critical roles in both acute and chronic inflammatory and autoimmune disorders. These cytokines interact with two types of receptors, IL-1 type 1 receptor (IL-1RI) and IL-1 type 2 receptor (IL-1 RII).

IL-1α and IL-1β carry out signal transduction through IL-1RI. IL-1β has important homeostatic functions in the normal organism such as regulation of nutrition, sleep and temperature. However, overproduction of IL-1β is involved in pathophysiological changes that occur in different disease states such as rheumatoid arthritis, neuropathic pain, inflammatory bowel disease, osteoarthritis, vascular disease, multiple sclerosis and Alzheimer's disease. IL-1β can be released from immune cells such as keratinocytes, fibroblasts, synoviocytes, endothelial, neuronal, macrophages and mast cells and glial cells such as Schwann cells, microglia and astrocytes.

IL-1β plays a key role in the regulation of the innate immune response and underlies autoinflammatory diseases such as cryopyrin-associated periodic syndromes (CAPS) and familial Mediterranean fever (FMF).

In a study by Fonseca et al. in which peri-implant groove fluid was evaluated, the IL-1β value in peri-implant groove fluid of peri-implantitis regions was higher than that of peri-implant mucositis regions. Curtis et al. showed that IL-1β measurement can make an important contribution to clinical findings in the diagnosis of peri-implantitis.

Relvas et al. observed that salivary IL-1β levels were higher in periodontitis stage III/IV patients than in periodontally healthy control patients and periodontitis stage I/II patients. In addition, IL-1β was found to be especially prominent in patients with grade C periodontitis and positively correlated with bone loss.

Saliva contains electrolytes such as sodium, potassium, calcium, magnesium, bicarbonate and phosphate. In addition, saliva contains immunoglobulins, proteins, enzymes, mucins and the nitrogenous compounds urea and ammonia. In recent years, saliva has been increasingly recognized as a useful tool in ancillary diagnostic methods. Sialometry and sialochemistry techniques are used in the diagnosis of systemic diseases, in the follow-up of general health status and as a risk indicator for diseases that determine the relationship between oral health and systemic health.

Saliva has been identified as an effective tool for screening and monitoring periodontitis because it is easily collectable and can reflect the inflammatory status of the whole mouth. Indeed, various cytokines and immune mediators present in saliva have been associated with inflammatory processes that progress with unstable responses.

To the best of the author's knowledge, no studies have evaluated salivary GM-CSF and MIP-1α levels in relation to periodontal disease in the existing literature. This study aimed to investigate the changes of these cytokines in the presence of periodontal disease by comparatively examining the levels of GM-CSF, MIP-1α and IL-1β in saliva samples obtained from healthy individuals with gingivitis and periodontitis, to show their potential to be used in the diagnosis of the disease or to provide preliminary information for future treatments that may be performed through these cytokine pathways.