Macrophages are critical during the inflammatory process in phagocytosis and the clearance of pathogens. In resting macrophages, calpain-mediated cleavage of selenoprotein K, between Arg⁸¹ and Gly⁸², is essential to maintain the inactive condition of macrophages. Macrophages are activated when cleavage is inhibited by the upregulation of the endogenous inhibitor of calpain, calpastatin, which is induced by Toll-like receptors (9).

Macrophages act as key components in the innate immune system. However, they are the main targets for certain pathogens to avoid host defenses at the same time. Group B streptococcus (GBS) induces macrophage membrane permeability disruption for the cleavage of cytoskeleton proteins by calpain (10). In addition, calpain may degrade the pro-apoptotic factors, BCL2 associated X, apoptosis regulator and BH3 interacting domain death agonist, resulting in the apoptosis of macrophages under the stimuli of GBS (11). Streptococcus pyogenes-induced oncosis of macrophages is associated with calpain, which may mediate the mitochondrial permeability transition (12). Furthermore, calpain activation is involved in endoplasmic reticulum stress-mediated apoptosis during the pathogenesis of Mycobacterium kansasii (13). Leishmania infection may activate calpain-1 via the G-protein signaling pathway; the activated calpain-1 may inhibit the transcription of RNA polymerase III, impairing the function of macrophages, thus helping the pathogen to establish an infection in the host cells (14).

Neutrophils, key participants in the innate system that increase significantly during inflammation, contribute to clearing pathogens by phagocytosis, as well as via other mechanisms. It has been revealed that calpain and calpastatin are critical for the chemotaxis of neutrophils. In contrast to all other cell types, constitutive calpain activity exerts a negative role in polarization and migration in resting neutrophils by regulating the activities of cell division control protein 42 homolog and Rho GTPase binding protein Rac1 (15). Inhibition of calpain activity may promote neutrophil migration associated with the activation of distinct signaling molecules (16).

Following migration to the endothelium, the morphology of neutrophils dramatically changes from spherical to flattened, which may attenuate the shear stress of blood flow, preventing cell rolling. It has been shown that this shape change is triggered by elevated physiological Ca²⁺ (the activator of calpain) influx, which is induced by inositol 1,4,5-triphosphate. Calpain may cleave the 4.1/ezrin/radixin/moesin domain and an actin-binding domain on the cytoskeleton. This enables the wrinkled membrane to flatten out, facilitating leukocyte plasma membrane expansion (17,18).

It has been shown that calpains participate in the apoptosis of neutrophils, which usually survive for only 24 h on average. With the depleted endogenous inhibitor, calpastatin, the enhanced activation of calpain is sufficient to cause apoptosis of neutrophils (19). In addition to apoptosis, calpains participate in the process of apoptotic-to-necrotic transition of neutrophils, as calpain activity may prevent the ingestion of neutrophils, resulting in the release of pathogenic molecules from the death neutrophils, such as protease and hydrolases, that may damage the local tissue (20).

The expression of calpain is particularly low in resting lymphocytes; however, it has been shown that synthesis and secretion of calpain occurs in active lymphocytes, particularly in T cells (21). Inhibition of calpain activity has indicated attenuation of T cell proliferation, suggesting that calpain is involved in the regulation of lymphocyte proliferation (22). In addition, calpain may be implicated in the differentiation of T cells, as the calpain:calpastatin ratio varied during T helper (Th)1, Th2 and Th17 development (23). As with macrophages and neutrophils, the migration of lymphocytes may be regulated by the activation of calpain. The regulation process might be associated with an integrin on the lymphocyte surface, termed lymphocyte function-associated antigen-1 (LFA-1). In a previous study, calpain promoted LFA-1 adhesion to intercellular adhesion molecule −1 (ICAM-1), causing the T cells to remain in the lymph node or the infection area (24). Recently, however, a novel study showed that calpain-2 may contribute to turnover of LFA-1 adhesion on migrating T cells (25), which may be essential to maintaining the active state of T cells in peripheral blood. In addition, calpain in endothelia is essential to lymphocyte transendothelial migration (26). Furthermore, inhibition of calpain may reduce the Th1/Th17 inflammatory cytokines, including IL-12, IL-17, TNF-α and granulocyte colony-stimulating factor (22).

A study on the expression of calpain-1 and calpain-2 in peripheral blood lymphocytes from different age groups showed that the expression levels of calpains were decreased in the elderly group (27). As mentioned above, calpains participate in the process of proliferation, differentiation and migration. Thus, decreased expression levels of calpain may lead to inefficient immune function, which may be an explanation for the hypoimmunity of -the elderly.

Traditional inflammation, caused by infection or injury, consequently results in resolution, due to restoration of the structure and function of affected tissues (44). Infection and injury may activate calpains in these types of resolving inflammation. The Shigella virulence effector, VirA may stimulate calpain activation by promoting Ca²⁺ influx. The VirA-induced calpain activity may promote the polymerization events of actin, a cytoskeletal protein, driving bacterial entry. However, in capn4 (the encoding gene of calpain)-deficient cells, there was increased proliferation of bacteria, indicating that calpain performs a dual function during infection. Calpain activation may ultimately lead to necrotic cell death by degradation of p53, thereby restricting Shigella intracellular proliferation (45). This may be a protective mechanism of the host to defend against infection. By contrast, calpain activity is involved in the apoptotic cell death of lung epithelia and aggravates the dysfunction of the epithelial barrier following Acinetobacter baumannii infection (46). An in vitro study has shown that coxsackievirus B3 (CVB3)-induced calpain activation may enhance autophagy in the early phase of infection in H9c2 cardiomyocytes, and this process may be beneficial to the replication of CVB3 in the host cells (47). In addition to infection, injury (such as stretching from mechanical ventilation) may induce the activation of calpain. The mechanical stretch-activated calpain was identified as an upstream regulator in ventilator-induced lung injury. It may upregulate NOS-3 activity, inducing ICAM-1 phosphorylation and polymorphonuclear neutrophil infiltration (48).

Increased expression of calpain-1 was observed in the cardiomyocytes obtained from mice with severe sepsis (49). Calpain-1 activity may be induced by the stimulus of LPS via the activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (50). The rapid cleavage of dystrophin by calpain disrupts the dystrophin-glycoprotein complex, aggravating the damage to the myocardium (49). The sarcomere may be disrupted by calpain and subsequently degraded by the ubiquitin-proteasome system, resulting in cardiac contractile dysfunction and skeletal muscle wasting (49,51). The activated calpain may induce the activation of caspase-3, leading to the apoptosis of cardiomyocytes (35) and pulmonary microvascular endothelial cells (52). Similarly, the calpain-induced caspase-3 signaling pathway also contributes to dysfunction of the diaphragm (53) and skeletal muscles (54). In addition to the dysfunction of organs, calpains are associated with the process of coagulation during sepsis by inducing the apoptosis of platelets (55) and promoting the release of the procoagulant microparticle (56). The above-mentioned findings imply that calpain participates in the pathogenesis of sepsis and may present as a therapeutic target in future.

Although the majority of cases indicate that the activation of calpain may promote tissue damage and that the inhibition of calpain may be beneficial, it should be noted that contradictory results have also been reported. Using an acute bacterial peritonitis model, Kumar et al (57) showed that calpain may serve a protective role during acute bacterial infection. Compared with the wide type mice, the capns1 knockout mice, in which the expression of calpain was deficient, showed impaired bacterial killing for the attenuation of IL-1α release and neutrophil recruitment, suggesting that calpains may be beneficial to bacteria clearance (57). The various roles of calpain may correlate with its differential involvement in different disease states, the variation of each isoform in the calpain family and the specificity of tissues.

Non-resolving inflammation, including diabetes, atherosclerosis, multiple sclerosis and certain types of cancer, is different from resolving inflammation due to the failure to recover from these types of diseases. The process of these diseases seems to be chronic from the outset without any signs of infection, however, with obvious signs of inflammation evidenced by the infiltration of the tissue by inflammatory cells (44). Studies have shown that activation of calpains is enhanced in these types of disease, suggesting that calpain may be involved in the pathogenesis. For example, evidence from the comparison of spinal cord tissue samples from Parkinson's disease patients and age-matched healthy subjects revealed that the upregulation of calpain may be associated with inflammatory response-mediated neuronal death for the elevation levels of inflammatory mediators and calpains (58).

Under the stimulation of hyperglycemia, the activity of calpain may be upregulated by increased Na⁺/H⁺ exchange and the protein kinase C (PKC) signaling pathway (43,59), leading to the alteration of vascular reactivity. Activated calpain may cleave the Ca²⁺-sensing receptor (CaSR), a G protein-coupled receptor, in the endothelia and smooth muscles, resulting in CaSR function loss and late complications in macrovascular disease (60). In addition, calpain may dissociate HSP90 from eNOS in the endothelia, leading to downregulation of the expression level of NO, which may increase reactive oxygen species production, thus aggravating endothelial dysfunction (43,61). In chronic hyperglycemia with insulin deficiency rats, it was shown that PKC-induced calpain-1 activation may upregulate ICAM-1 (59). The decreased NO level and increased level of adhesion molecules may contribute to endothelial dysfunction, promoting the inflammatory response in the microenviroment resulting from diabetes. In addition to microvascular dysfunction, calpains participate in the pathogenesis of diabetic cardiomyopathy under the stimulus of high glucose via the NADPH oxidase-dependent signaling pathway (62). Rac1, an essential subunit of NADPH oxidase, may be significant in the activation of calpain (63). The activated calpain-1 may induce the apoptosis of cardiomyocytes by downregulating Na⁺/K⁺ ATPase, contributing to the cardiac complications associated with diabetes (64).

Angiotensin II may activate calpains via the activation of angiotensin II type 1 receptor signaling (65) and the upregulation of epidermal growth factor receptor (37). The activated calpains may enhance the expression of inflammatory mediators by promoting the activation of NF-κB, and may increase the leukocyte-endothelium interaction and albumin permeability (65). These effects in the microcirculation may be involved in the process of cardiovascular remodeling. Similarly, certain reports have shown that calpain-1 and calpain-2 may be involved in left ventricular (LV) remodeling. A mouse model with cardiomyocyte-specific deletion of Capn4 demonstrated relatively improved cardiac function and reduced mortality following myocardial infarction (66). These results demonstrate the possible associations between the inhibition of NF-κB activation, decreased apoptosis of cardiomyocytes (66) and reduced myocardial hypertrophy and fibrosis, leading to the significant attenuation of LV remodeling (67).

Although many of the mechanisms of calpain activity remain unclear, it has been identified that calpains are involved in numerous types of non-resolving inflammation disease, including asthma, atherosclerosis, multiple sclerosis and rheumatoid arthritis (22,68–70). These diseases make up a high proportion of the disease spectrum, however, are associated with a low possibility of complete recovery. Calpains may present as a therapeutic target for their significant activity during the pathological process of these types of chronic diseases.