Sinonasal T-cell expression of cytotoxic mediators granzyme B and perforin is reduced in patients with chronic rhinosinusitis

Abstract

Background: CD8+ T cells and natural killer (NK) cells are cytotoxic cells that use granzyme B (GrB) and perforin. Defective cytotoxic function is known to play a role in dysregulated immune response as seen in chronic sinusitis, also referred to as chronic rhinosinusitis (CRS). However, to our knowledge, in the United States, neither GrB or perforin expression has been reported in patients with CRS.

Objective: The aim of this study was to investigate sinonasal cytotoxic cells, their mediators, and cell-specific distribution of these mediators in patients with CRS with nasal polyp (CRSwNP) and in patients with CRS without nasal polyp (CRSsNP).

Methods: Blood and sinus tissue samples were taken from patients with CRSsNP (n = 8) and CRSwNP (n = 8) at the time of surgery. Control subjects (n = 8) underwent surgery for cerebrospinal fluid leak repair or to remove non-hormone-secreting pituitary tumors. The cells were analyzed via flow cytometry by using CD8 expression to identify cytotoxic T cells and CD56 expression to identify NK cells. Intracellular GrB and perforin expression were analyzed with flow cytometry.

Results: We observed no significant differences in plasma or peripheral blood immune cell numbers or specific levels of GrB or perforin among the groups. In the sinonasal mucosa of the patients with CRSsNP and the patients with CRSwNP, there was a significant decrease in GrB and perforin levels (p < 0.05) despite similar or increased numbers of cytotoxic cells when compared with the controls. The overall decrease in GrB and perforin in the sinonasal mucosa of the patients with CRSsNP and the patients with CRSwNP was due to decreased T cell production. There was no difference in total NK cell count or expression of perforin or GrB among all the groups.

Conclusion: Total levels of sinonasal GrB and perforin were decreased in the sinonasal mucosa of both the patients with CRSwNP and the patients with CRSsNP compared with the controls, whereas sinonasal CD8+ T cells, (but not NK cells,), intracellular stores of GrB and perforin were reduced in the patients with CRSwNP compared with the controls.

Figure 1.

Cell-specific distribution of granzyme B (GrB) (A) and perforin (B) in sinonasal tissue. CD8⁺ T cells are the predominate source of GrB (except in patients with chronic rhinosinusitis with nasal polyp [CRSwNP]) and perforin in all the groups. Natural killer (NK) cells are the next most abundant source of GrB and perforin. Another unknown cell type accounts for 5–10% of sinonasal GrB and perforin in all the groups.

Figure 2.

The percentage of cells positive for granzyme B (GrB) (A) and perforin (B), and quantified levels of GrB (C) and perforin (D) in the sinonasal mucosa. Note that the total percentage of cells and the amount of GrB and perforin present, as quantified mean fluorescent intensity (MFI), was reduced in both chronic rhinosinusitis (CRS) groups. *p < 0.05 versus control.

Figure 3.

The percentage of CD8⁺ T cells and CD56⁺ natural killer (NK) cells present in the sinonasal mucosa. (A) Patients with chronic rhinosinusitis with nasal polyp (CRSwNP) had increased numbers of sinonasal CD8⁺ T cells compared with the controls. (B) There was no significant difference in the number of sinonasal NK cells between the groups. *p < 0.05 versus control.

Figure 4.

T-cell production of granzyme B (GrB) and perforin was reduced in patients with chronic rhinosinusitis without nasal polyp (CRSsNP) and patients with chronic rhinosinusitis with nasal polyp (CRSwNP). The percentage of sinonasal cells that stained positively for both CD8 and GrB (A) or perforin (B). To correct for the difference in cell infiltrate, GrB (C) and perforin (D) staining was examined as a percentage of total CD8⁺ T cells present. *p < 0.05 versus control.

Figure 5.

Natural killer (NK) cell production of granzyme B (GrB) and perforin was not altered in patients with chronic rhinosinusitis without nasal polyp (CRSsNP) or chronic rhinosinusitis with nasal polyp (CRSwNP). The percentage of sinonasal cells that stained positively for both the NK cell marker CD56 and GrB (A) or perforin (B). To correct for differences in cell infiltrate, GrB (C), and perforin (D) staining was examined as a percentage of total CD56⁺ NK cells present. *p < 0.05 versus control.