IL-Ra protein is purified from the supernatants of cultured monocytes on adherent IgG (Arend and Leung, 1994). A combination of reverse phase chromatography, anion exchange and gel filtration in the purification scheme results in over 10,000-fold purification and a 3% yield. IL-Ra is incorporated with S-methionine in cultured monocytes on adherent IgG. Thus, the process of purification is observed by bioactivity assays. The IL-Ra derived from monocytes consists of three purified forms analyzed by the N-terminal sequence; the size of two forms is 22 kDa and that of the third form is 17 kDa (Perrier et al., 2002). N-terminal analysis of the two forms was successful and revealed amino acids identical through 20 residues and the third form of IL-Ra exhibited a blocked N-terminus. Peptides obtained after digestion of the three forms of IL-Ra with endo-proteinases were then sequenced that revealed an assembly after composition analysis. Absorption with N-glycanase indicated that the 22 kDa form of IL-Ra is glycosylated, while the digested molecules constitute the size of 17 kDa. After N-glycanase digestion, the size of the 17 kDa form remains unchanged. In summary, the IgG-stimulated monocyte supernatants possess three IL-Ra peptide forms: two glycosylated 22 kDa forms and a non-glycosylated 17 kDa form. The presence of N-linked carbohydrate is not necessary to maintain organic activity as the three forms showed the same level of activity in the two bioassays, i.e., production of IL-1 induced PGE2 by fibroblasts and nitrogen induced proliferation of IL-1 augmented murine thymocytes. Multiple IL-Ra forms exist in the 22–25 kDa size range and reflect varying degrees of glycosylated peptide (Arend, 1990).

Based on the analysis of protein sequence, cDNA for IL-Ra is cloned successfully (Howard et al., 1998). From the cultured monocytes, RNA is harvested on IgG for 17 h and is used in πg10 for the preparation of cDNA library. This library is secreted using two synthesized probes of oligonucleotide and a single clone is identified which is hybridized with both probes. Digestion of EcoRI releases a DNA of 1.8 kb insert and hybridized to a total of 5 probes. CDNA is cloned into the vector of expression in M13wp19 which is a sequence of nucleotide.

An active reading frame that encodes for a peptide of 177 amino acids and a 3-untranslated long stretch with 5-untranslated stretch of 14 nucleotides was formed by the cDNA for the inhibition of interleukin-1 receptors. The residues of 25 N-terminal of a protein powerfully suggest a sequence of leader peptides. The established peptide consists of 152 amino acids and is created by immediate cleavage after the 25th residue exhibits the sequence of N-terminal which is identified to native disinfected IL-1Ra. Its isoelectric point is 5.2 which is identical to natural 17 kDa IL-1Ra. This cDNA is seen in Escherichia coli having successful 17 kDa recombinant protein production which inhibited the bioactivity of IL-1 (Arend, 1990). IL-Ra-derived monocyte has a unique structure and is similar to IL-1. Amino acid sequence comparison revealed the homology of 18% of IL-1α and 30% of IL-1β. At the level of amino acid, human IL-1β and IL-1α are homologous (26%) to each other. Many of the homologies sequenced between IL-Ra and IL-1β are located in the C-terminal short segment of the molecule (Rivers-Auty et al., 2018).

An identical protein is purified from the cell line of human myelomonocytic supernatants after stimulation with GM-CSF and differentiation in phorbol myristate acetate. The protein is sequenced partially, and a cDNA is cloned, expressed, and reveals an identical sequence of nucleotide to monocyte-derived cDNA. Equal forms of IL-Ra proteins are cleansed from the human urine and THP-1cells of PMA supernatants (Silva et al., 2019).

The size of the non-glycosylated IL-Ra form is reportedly larger or between 17–21 kDa (Hamilton et al., 2008). Even yet, this molecule's predicted MW is 115, 17 kDa, and it has been sequenced. The genomic DNA structure of IL-Ra proves that the molecule is an IL-1 family member. The human IL-Ra gene possesses 3 introns and 4 exons that are similar to the gene structure for IL-1β and IL-1α. The difference occurs in the first axon in which the IL-Ra gene encodes for a peptide that is not present in IL-1. Gene family is originated by the duplication of genes from the primordial precursor. IL-1α, IL-1β and IL-Ra genes are situated on the human chromosome (Arend, 1990).

The production of IL-Ra occurs through the monocytes of humans, depending on the specific generalization of protein through the culture placed on the substrate of immune complexes which are adherent or sometimes adherent IgG. The inhibited bioactivity was not evaluated in the monocytes which belong to the supernatants and were cultured with complexes of soluble immune molecules, especially with red blood cells that are coated with IgG (Arend, 1990).

Monocytes of human blood express the gene for IL-Ra on stimulation (Ruiz de Souza et al., 1995). IL-Ra protein is visualized during the first 4–6 h. In cells, the primary transcript is IL-Ra which has no peptide and diffused stains are present in cytosol although, the remaining is present intracellularly. The production of IL-Ra occurs in keratinocytes while the attachment of an IL-Ra to the nuclear DNA is blocked by epithelial cells (Espat et al., 1994).

Human monocytes regulate the production of IL-Ra that is produced at the mRNA level (Tron et al., 1988). Adherent human monocytes produce equal amounts of IL-Ra proteins for 24 hours in a culture media. The half-life of mRNA is the same as that of IL-1β and IL-Ra in the cells which are induced with LPS (Dinarello, 2018). Monocytes which are induced with IgG have a high and sustained rate of transcription in IL-Ra and have a significant prolongation in the stability of mRNA. LPS is responsible for the synthesis of IL-Ra in human monocytes. Numerous other findings have demonstrated the various mechanisms by which IL-Ra production takes place. The mononuclear cells of the blood in peripheries were placed in the suspended culture at the lower end of polypropylene tubes. Production of IL-Ra was increased by adding 1% human AB serum to the suspended culture over 24 h (Granowitz et al., 1992). The soluble IgG also increased the synthesis of IL-1Ra. IgG has low induction of IL-1Ra. The production of IL-Ra was persuaded by growth factors, especially after a very short time of serum starvation (Raja et al., 2018). The IgG mediates effects on the production of IL-Ra in human monocytes by the receptors on Fc portion of IgG (Arend, 1993).

The surface of IL-1 consists of three binding sites that separately interact with the extracellular portion having three Ig domains of the IL-1 receptor (Krumm et al., 2014). Contact points for the binding of receptors are different among IL-1Ra, IL-1α, and IL-1β. IL-1 performs functions in vivo on immune and inflammatory cells. IL-1β peptide studies separate the molecular regions that are responsible for mediating the functions. Synthetic nona-peptide exhibits in vivo immunomodulatory properties associated with inflammation during the absence of metabolic changes. A monoclonal antibody in a region of IL-1β inhibits in vivo immuno-stimulation effect in mice without changing the pyrogenic activity. Peptides contained in human pro-IL-1ß residues 208 to 240 mediate the inflammatory effects and pyrogenicity of the molecule (Borthwick, 2016). Inducing inflammatory events, IL-1β regions mediate the action on immune cells by binding to IL-1RI and IL-1RII.

The structural feature of IL-1Ra binding depicts the absence of an agonist effect. In mature IL-1β, mutation at position 11 in arginine to glycine reduces the bioactivity of the molecule towards T-cells (100-fold) and reduces the binding of receptors by 25%. Without any change in receptor binding affinity, in mature IL-1β the mutation occurs at position 145 from aspartate to lysine and leads to a reduction of the biological activity (90%) towards T-cells (Dinarello, 1996). Instead of indicating the presence or absence of active sites, mutations affect IL-1 activities by changing the conformation. IL-1β and IL-1α contain large loops and have β-pleated sheets in an anti-parallel manner. IL-1Ra and IL-1β have similar secondary and tertiary structures, but different regions in the primary sequence contain β-strands. IL-1Ra and IL-1β exhibit a difference in the location of the charged side chain which is important in the binding of receptors and activation (Dinarello and Thompson, 1991).

At position 11 of IL-1β, arginine to glycine induces gene expression earlier in fibroblasts rather than stimulating the late gene expression. There is an equal binding of IL-1Ra to IL-1RI but it triggers at the level of cellular response. The occurrence of a late process of transcription involves the interaction between the ligand and receptor but is absent in IL-1β or IL-1Ra. Reduction in biological activities occurs due to mutation in arginine to glycine at position 11 of IL-1β and amino acid substitution at the same site does not produce similar changes in the function. The analysis of molecular modeling indicates that mutation in arginine to glycine results in the disruption of the structural integrity of 1/12 pair of anti-parallel β-strand. The β-strand 1 collapses into the hydrated space between strands 4, 2, and 1, and structurally alters the cleft in IL-1β which contains conserved amino acid clusters. Aspartate 145 amino acid shows delayed biological activity of IL-1. An initial trigger for biological activities of IL-1 resides in the β-bulge domain that is located between IL-1β 4 and 5 strands and IL-1β 11 mutein. These structural features that are not involved in mediating the activities of IL-1β are present in IL-1Ra. In IL-1Ra, mutation at position 145 in lysine to aspartate led to a reduction of the agonist activities towards both the fibroblasts and T-cells (Arend, 1993).

The binding of epitope receptor for IL-1Ra and IL-1β are formed by rim’s charged surface of the band structure of β-pleated and hydrophobic core which is represented by two side chains of phenylalanine of mature IL-1β at positions 150 and 46 (Evans et al., 1995). Two structural features that mediate IL-1β biological activities are absent in IL-1Ra; a region around residue 145 of aspartic acid and β-bulge between strands 5 and 4. As IL-1α and IL-1β do not possess similar structures, activation and binding of the receptor by IL-1α involves distinct structural determinants (Dinarello and Thompson, 1991).

IL-1Ra is produced in response to various inflammatory or contagious diseases in humans and septic animals (Arend, 2002). IL-1 secretion, amount, and receptor antagonist are balanced in most diseases. Protein synthesis and gene expression of IL-1Ra and IL-1 are differentially regulated. For example, before IL-1Ra, IL-1β is synthesized and transcribed in cells. Cozzolino and coworkers, and Rambaldi, argued that dysregulation in agonist-antagonist synthesis occurs in different disorders when they studied the gene expression of IL-1Ra and IL-1β in fresh cells from individuals suffering from acute myelogenous leukemia (Rambaldi et al., 1993). The cells of leukemia from one of the 11 patients expressed IL-1Ra after the stimulation of IL-1β. Large amounts of circulating IL-1Ra are found in systemic rheumatoid arthritis during sepsis in humans and experimental endotoxemia (Schulte et al., 2013). Circulating IL-1β levels exceed 500 pgml-1 after 3 to 4 h, which then rapidly fall; whereas in the same individuals, peak IL-1Ra level occurs after 4 h, reaches 100-fold concentration of IL-1β and is retained for 12 h. The peak level of IL-1Ra in baboons occurs after 8–10 h during an E. coli sepsis. Thus, the huge and small amounts of IL-1Ra and IL-1β production respectively show a real response in most clinical situations. Patients’ synovial fluid with a disease of rheumatoid arthritis is measured by comparing the levels of IL-1β and IL-1Ra (Dinarello and Thompson, 1991). IL-1Ra produced endogenously contributes to limiting disease severity but is inadequate in overwhelming an acute inflammation or infection (Dinarello et al., 2012a). Exogenous IL-1Ra provides beneficial effects in animal models.

IL-1Ra is secreted into an extracellular compartment and consists of the classical signal peptide, although less than 10% IL-1α and 50% IL-1β are secreted in cell culture (Iyer and Cheng, 2012). 50% IL-1Ra is associated with the cells in adherent monocytes. IL-1Ra intracellular form is described in keratinocytes without a signal peptide and suggests that in these cells, intracellular IL-1Ra encounters the biological activity of IL-1α that remains in the keratinocyte’s cytosolic compartment. Although it does not happen in human monocytes exposed to endotoxin, intracellular IL-1 is 10% less than the amount of IL-1Ra that is still present in the cells. For normal physiological functions in both extracellular and intracellular components, a balance between IL-1Ra and IL-1 is important.

There are numerous types of IL-1 receptors as described in Table 1. But there are two major types, i.e., type I and type II. Type I receptors transduce the signals while type II receptors attach to IL-1 but cannot transduce the signals (Krakauer and Oppenheim, 1998). Fusion and secretion of IL-1Ra occur in the cell (Khayata et al., 2020).

Epithelial cells along with keratinocytes also produce IL-1Ra variant forms. For the IL-1Ra protein, mRNA encodes the developed portion that is the same as the IL-1Ra monocyte, but it varies in the structure of the leader peptide. Monocyte IL-1Ra holds signals sequence 25 amino acid; IL-1Ra keratinocyte contains a truncated sequence of 7-amino acid. The N-terminal consist of the very first 4 amino acids same as with the IL-1Ra leader peptide monocyte and the remaining residues are different. IL-1Ra keratinocyte lacks the sequence of a full signal which is not secreted and remains in the cell. IL-1Ra is found in the skin in large quantities which are limited to the stratum corneum. Production of IL-1Ra increases with an in vivo differentiation of keratinocytes and IL-1Ra forms in the assay of murine thymocytes exhibited an equivalent biological potency (Lee et al., 2018).

The biological relevance of IL-1Ra intracellular variants in epithelial cells has been explored. IL-1Ra serves a role of intracellular signaling which is shown by the finding of an anti-sense IL-1α oligo-deoxy nucleotide when transferred into an endothelial cell that prevents senescence and expands the cell’s proliferative life span. The IL-1Ra molecule participates with an IL-1 for the intracellular responses and controls the effects of IL-1 in differentiation and the growth of the cell (Arend, 1990).

The family of IL-1 consists of IL-1α, IL-1β, and IL-1Ra. From previous studies, it is known that it took 350 million years to evolve IL-1α and IL-1β as a peptide, but only IL-1Ra evolved as a signal peptide. Early members have a molecular mass of 31 kDa when they are first formed and are precursor proteins. The pro and mature IL-1α are both biologically active. Many other growth factors of cytokines like neurotropic factors that have low leading sequences are active in the form of precursors. As compared to these, IL-1β is inactive and also needs cleavage to 17 kDa peptide to maintain biological activity. The precursor of IL-1Ra contains a leader sequence and is cleaved in the mature form and secreted in form of proteins. Another form of IL-1Ra is secreted in the cells i.e., intracellularly. The intracellular secretion of IL-1Ra is the result of replacing the first exon coding on the signal peptide. Both forms are identical. IL-1Ra blocks the cell surface (Symons et al., 1995). IL-1Ra appears as an intracellular protein in the cells of the epithelial layer where it works to block intracellular IL-1Ra. In keratinocytes of the skin, the terminal differentiation occurs by balancing between IL-1Ra and IL-1α (Zheng et al., 2019). In human beings, IL-1α, IL-1β, and IL-1Ra are present at the arm of chromosome 2. Polymorphism of IL-1 gene in human diseases is linked with autoimmune diseases (Dinarello, 1994).

Thymocytes proliferation of augmented IL-1, synthesis of chondrocyte collagenase, endothelial cell adhesiveness induced IL-1 for neutrophils, and synthesis of PGE2 by IL-1 that induces synovial cell is blocked by recombinant IL-1Ra (Chow and Chin, 2020). The protein kinase activation ability of IL-1 in fibroblasts is also blocked by IL-1Ra that follows IL-1 binding to cells. 10-1000-fold IL-1Ra inhibit 50% IL-1 induced in vitro responses; due to the presence of IL-1 receptor cells in excess as compared to the number required to trigger the IL-1 biological response. In IL-6 from human monocytes, TNF and IL-1 production is blocked by IL-1Ra and at equimolar ratios, inhibition of 50% is observed (Jang et al., 2006), although total inhibition of IL-1Ra to the IL-1 is observed at 10-fold in molar excess. This indicates that the effect of ‘spare receptor’ in cells that express the receptor of type I is not important in cells that express the type II receptor.

IL-1 activity injected into animals is also blocked by the recombinant IL-1Ra but, the biological effect of cytokine is blocked in vitro by an excess of IL-1Ra as shown in Table 2. For example, human IL-1ß having potency (100 ng kg⁻¹) for intravenous injection into the rabbits induces a high-grade fever. By injecting 10 µg kg⁻¹ of IL-1Ra, 50% response is blocked, and complete blockage is observed with further injected IL-1Ra (100 µg kg⁻¹). In rabbits, hypotension is induced by IL-1 due to the same dose relationship. In mice, IL-1Ra blocks the induced inflammation of IL-1. When IL-1 is to be injected into a peritoneum, the local accumulation of neutrophils is blocked by IL-1Ra. Circulating IL-6 levels by induction of IL-1 is also blocked by IL-1Ra and IL-1-induced neutrophilia is blocked by IL-1Ra (Dinarello and Thompson, 1991).

IL-1Ra has the ability to bind the IL-1 with the receptor’s cell surface without inducing any intracellular agonist effect (Volarevic et al., 2010). The agonist effect is absent in vitro and is observed in multiple cells, i.e., human fibroblasts, murine thymocytes, human monocytes, rabbit chondrocytes, human endothelial cells, neutrophils and macrophages (Dumas et al., 2012). The presence of hormone-like molecules or a naturally occurring cytokine which is a receptor antagonist is unique in biology. IL-1Ra shows a slight agonist effect at high concentrations in different assays (Dinarello and Thompson, 1991).

Low molecular weight drugs containing proteins are rapidly cleared through the process of deprivation and filtration by the kidneys. The common processes for the quick removal of protein-containing drugs from the flow are degradation by enzymes and mechanisms of biochemical and biophysical clearance. IL-1 receptor antagonist has a low molecular weight and is removed from the kidneys quickly through the mechanism of biochemical and biophysical clearance. The different mechanisms for the clearance of IL-1 receptor antagonist body involve quick distribution in organs of the body, liver metabolism,s and quick removal from the kidneys, all of which lead to changes in the healing dosage of therapeutic agents (Akash et al., 2013a).

Broadly, the materials are categorized into three groups: 1- fusion with polymers and nanoparticles for extension of serum half-life; 2-by the dissolution of loaded particles of IL-1Ra for local or sustained delivery; 3-by gels and injectable coacervate for local or sustained IL-1Ra delivery as described in Table 4. Similarly, to improve the therapeutic efficacy as shown in Table 5 of IL-1Ra, injectable materials are used to extend IL-1Ra half-life and to sustain or localize IL-1Ra delivery as shown in Figs. 1 and 2.

IL-1Ra takes part in normal physiology by using specific antibodies and acts as a natural anti-inflammatory agent. Thus, IL-1Ra possesses an anti-inflammatory potential and can be used as a therapeutic agent against inflammatory disorders, including rheumatoid arthritis. Endogenous IL-1Ra expression in diseases of humans is also described, as the molecule is the most important part of host defense in human beings.

In the following sections, we briefly describe the potential role of IL-1Ra in various diseases.

The preventive impacts of soluble IL-1 receptors (sIL1R), recombinant IL-1Ra, as well as neutralizing antibodies to IL-1ß and IL-1α have suggested the role of IL-1 in a number of animal illness models. Such drugs reduced the magnitude of graft vs. host disorder as well as overall survival of cardiac allografts in rats, increased longevity in rabbits and rats given endotoxin injections, significantly reduced shock in baboons as well as rabbits with bacteremia, declined the prevalence and mortality of inflammatory arthritis in rabbits, mice, and rats, significantly reduced colonic inflammation in rabbits as well as rats, and hindered innovative autoimmune encephalomyelitis in mice (Arend et al., 1998; Dinarello and Wolff, 1993). The collagen-induced arthritis concept in mice provides an illustration of the therapeutic advantage of inhibiting IL-1; however, a discussion of many of these experiments is outside the purview of this paper. When neutralizing antibodies to IL-1β and IL-1α were given while arthritis appeared, the condition was either slowed or stopped; or in the animals that did, arthritis merely manifested as a minor array of abnormalities (van den Berg et al., 1994). Anti-IL1β and anti-IL-1α treatment also dramatically decreased the synovial invasion, inflammation, as well as cartilage degradation in mice with developed arthritis. The capability of chondrocytes to generate novel cartilage matrix elements was recovered after anti-IL1 therapy.