CD4+ T cells enhance the unloaded shortening velocity of airway smooth muscle by altering the contractile protein expression

Abstract

Abundant data indicate that pathogenesis in allergic airways disease is orchestrated by an aberrant T-helper 2 (Th2) inflammatory response. CD4(+) T cells have been localized to airway smooth muscle (ASM) in both human asthmatics and in rodent models of allergic airways disease, where they have been implicated in proliferative responses of ASM. Whether CD4(+) T cells also alter ASM contractility has not been addressed. We established an in vitro system to assess the ability of antigen-stimulated CD4(+) T cells to modify contractile responses of the Brown Norway rat trachealis muscle. Our data demonstrated that the unloaded velocity of shortening (Vmax) of ASM was significantly increased upon 24 h co-incubation with antigen-stimulated CD4(+) T cells, while stress did not change. Enhanced Vmax was dependent upon contact between the CD4(+) T cells and the ASM and correlated with increased levels of the fast (+)insert smooth muscle myosin heavy chain isoform. The levels of myosin light chain kinase and myosin light chain phosphorylation were also increased within the muscle. The alterations in mechanics and in the levels of contractile proteins were transient, both declining to control levels after 48 h of co-incubation. More permanent alterations in muscle phenotype might be attainable when several inflammatory cells and mediators interact together or after repeated antigenic challenges. Further studies will await new tissue culture methodologies that preserve the muscle properties over longer periods of time. In conclusion, our data suggest that inflammatory cells promote ASM hypercontractility in airway hyper-responsiveness and asthma.

Figure 1. CD4⁺ T cell purity

A, flow cytometric quantification of the purity of the CD4⁺ T cells (>95%) after magnetic selection. CD4⁺ T cells viewed by bright field imaging (B) and by fluorescence microscopy after treatment with CD4 Alexa488 antibody (C).

Figure 2. Mechanical properties of airway smooth muscle (ASM) incubated with ovalbumin (OVA)-stimulated splenocytes

A, unloaded velocity of shortening (V_max) of ASM co-cultured in the absence (open columns) or in the presence of total splenocytes (filled columns) for 24 or 48 h (n = 5 rats per group). B, stress generated by ASM incubated in the absence (open squares) or in the presence of total splenocytes (filled columns) for 24 or 48 h (n = 6 rats per group). C, representative force–velocity curves of ASM co-cultured in the absence (open circles) or in the presence of total splenocytes (filled circles). F/Fmax: force normalized to the maximal force. The force–velocity relationships were accurately fitted by the Hill hyperbolic model, r² > 0.96.

Figure 3. Western blot analysis of myosin light chain kinase (MLCK; A), 7-amino acid insert in N-terminal part of smooth muscle myosin [(+)insert SMMHC; B], total SMMHC isoform (C), α-smooth muscle actin (SM actin; D) and (+)insert to total SMMHC ratio (E) from ASM strips incubated in the absence (CTR; open columns) or in the presence of OVA-stimulated splenocytes (SPL; filled columns)

n = 3 rats per group. Representative Western blots are shown. *P < 0.05.

Figure 4. Mechanical properties of ASM incubated with OVA-stimulated CD4⁺ T cells

A, V_max of ASM incubated in the absence (open columns) or presence of OVA-stimulated CD4⁺ T cells (filled columns) for 24 or 48 h. B, stress generated by ASM incubated in the absence (open columns) or presence of OVA-stimulated CD4⁺ T cells (filled columns) for 24 or 48 h. C, representative force–velocity curves of BN ASM co-incubated in the absence (open circles) or in the presence of CD4⁺ T cells (filled circles) for 24 h. F/Fmax: Force normalized to the maximal Force. Force –velocity relationships were accurately fitted by the Hill hyperbolic model, r² > 0.96. n = 5 rats per group. *P < 0.05.

Figure 5. Western blot analysis of MLCK (A; n = 7 rats per group for 24 h and n = 3 rats per group for 48 h), (+)insert SMMHC isoform (B; n = 6 rats per group for 24 h and n = 3 rats per group for 48 h), SMMHC (C; n = 7 rats per group for 24 h and n = 3 rats per group for 48 h), α-smooth muscle actin (D; n = 6 rats per group for 24 h and n = 3 rats per group for 48 h) and ratio of (+)insert SMMHC to total SMMHC (E; n = 6 rats per group for 24 h and n = 3 rats per group for 48 h) for ASM incubated in the absence (open columns) or in the presence of OVA-stimulated CD4⁺ T cells (filled columns)

Representative Western blots are shown. *P < 0.05; **P < 0.01.

Figure 6. Phosphorylation of myosin LC₂₀

A, response to acetyl-β-methylcholine chloride (MCh) stimulation in ASM incubated for 24 h in the absence (open columns) or in the presence of OVA-stimulated CD4⁺ T cells (filled columns). B, a representative Western blot of phos-tag gel of phosphorylated (p-LC₂₀) and total LC₂₀ (LC₂₀) after MCh stimulation of a muscle strip incubated in the absence (control) or in the presence of OVA-stimulated CD4⁺ T cells (T cells). *P < 0.05. n = 6 rats per group.

Figure 7. Mechanical effect of contact between OVA-stimulated CD4⁺ T cells and ASM strips

The V_max (A) and stress (B) measured for ASM strips incubated for 24 h in the absence of CD4⁺ T cells (open columns) or in the presence of CD4⁺ T cells either in contact (filled columns) or in Transwells (hatched columns). n = 7 rats per group. *P < 0.05.