The role of substance P in microvascular responses in murine joint inflammation

Abstract

1. Rheumatoid arthritis is a serious, inflammatory disease of the distal joints that has a possible neurogenic component underlying its pathology. 2. Substance P (SP), an endogenous neuropeptide that acts upon the neurokinin 1 (NK(1)) receptor, is released from sensory nerves and is involved in neurogenic inflammation. 3. In this study, we have developed novel techniques to determine the contribution of SP to microvascular responses in a model of complete Freund's adjuvant (CFA)-induced arthritis in NK(1) knockout mice. 4. Detailed analysis in normal mice revealed that CFA (20 microg i.art.)-induced plasma extravasation was raised from 18 to 72 h, when compared with intravascular volume. By comparison, knee swelling was sustained for 3 weeks. Neutrophil accumulation mirrored plasma extravasation. SP (10 pmol i.art.) caused significant acute plasma extravasation, but not other parameters, in wild type (WT), but not NK(1) knockout mice. CFA (10 microg i.art.) induced a significantly decreased intravascular volume, presumably due to decreased blood flow, at early time points (5 and 7 h) in WT but not NK(1) knockouts. Otherwise, similar responses in WT and NK(1) knockout mice were observed. However, injection of SP into CFA-pretreated joints caused a significant enhancement of plasma extravasation and knee swelling in the WT but not NK(1) knockouts. 5. In conclusion, the present study has used novel techniques in WT and NK(1) knockout mice to show that SP can modulate vascular tone and permeability in the inflamed joint via activation of the NK(1) receptor and that SP-induced responses are more pronounced where pre-existing inflammation is present.

Figure 1

Time course of CFA-induced inflammation in the mouse knee joint. Effect of intra-articular injection of CFA (20 μg, 20 μl) and saline (contralateral joint, 20 μl) on (a) plasma extravasation (n=5–7) and intravascular volume (n=4–6), (b) knee swelling (n=9–12) and (c) neutrophil accumulation as synovial MPO activity (n=3–7) over a 3-week time course in CD1 mice. All results are expressed as mean±s.e.m. (a) Results are expressed as a ratio of counts detected in the CFA-treated compared to the saline-treated joint. Statistical analysis was performed to determine whether ¹²⁵I-albumin accumulation due to plasma extravasation was significantly greater than that due to increased intravascular volume using unpaired t-tests. ^*P<0.05; ^***P<0.001 vs intravascular volume data. (b) Statistical significance was shown by paired t-tests. ^***P<0.001 vs saline-treated joints. (c) Statistical significance was shown by unpaired t-tests. ^**P<0.01; ^***P<0.001 vs saline-treated joints.

Figure 2

Effect of exogenous SP on plasma extravasation and knee swelling in the naïve joint. Effect of SP (100 pmol, 10 μl) and saline (contralateral joint, 10 μl) on (a) intravascular volume and plasma extravasation (n=4–6) and (b) knee swelling (n=6–7) after a 30-min time course in wild type (WT) and NK₁ receptor knockout (KO) mice. (c) Effect of SP (1–1000 pmol, 10 μl) on neutrophil accumulation, assessed as MPO activity after a 5-h time course in WT mice. Results are mean±s.e.m. Statistical significance was evaluated using an unpaired t-test to compare WT vs NK₁ receptor KO mice. ^**P<0.01 vs WT mice; ^$P<0.01 vs intravascular volume data.

Figure 3

Role of NK₁ receptors in CFA-induced plasma extravasation and intravascular volume changes. The effect of intra-articular injection of CFA (10 μg, 10 μl) and saline (contralateral joint, 10 μl) on (a) intravascular volume and (b) plasma extravasation after a 3 h (n=4–5), 5 h (n=5–7), 7 h (n=5) or 18 h (n=3–5) time course in wild type (WT) and NK₁ receptor knockout (KO) mice. Results are expressed as mean±s.e.m. Statistical significance was evaluated using unpaired t-tests. ^*P<0.05; ^***P<0.001 vs intravascular volume data at the equivalent time point; ^$$P<0.01 vs WT mice.

Figure 4

Role of NK₁ receptors in CFA-induced knee swelling. Effect of intra-articular injection of CFA (10 μg, 10 μl) and saline (contralateral joint, 10 μl) on knee swelling after (a) a 5-h time course (n=13) and (b) an 18-h time course (n=7) in wild type (WT) and NK₁ receptor knockout (KO) mice. Results are expressed as mean±s.e.m. Statistical significance was evaluated using unpaired t-tests to compare WT vs NK₁ receptor KO mice and CFA-treated joints vs saline-treated joints. ^**P<0.01; ^***P<0.001 vs saline-treated joints.

Figure 5

Role of NK₁ receptors in CFA-induced neutrophil infiltration. Effect of intra-articular injection of CFA (10 μg, 10 μl) and saline (contralateral joint, 10 μl) on synovial MPO activity after (a) a 5-h time course (n=4–5) and (b) an 18-h time course (n=5) in wild type (WT) and NK₁ receptor knockout (KO) mice. Results are expressed as mean±s.e.m. Statistical significance was evaluated using unpaired t-tests to compare WT vs NK₁ receptor KO mice and CFA-treated joints vs saline-treated joints. ^*P<0.05; ^***P<0.001 vs saline-treated joints.

Figure 6

Histology of joint sections. Haemotoxylin and eosin staining of joint sections to show the effect of intra-articular injection of CFA (10 μg, 10 μl) on synovial neutrophil infiltration in (a) WT and (c) KO mice. Staining for neutrophils in the saline-treated joints of (b) WT and (d) KO mice is also shown. Arrows indicate areas of neutrophil accumulation.

Figure 7

Effect of exogenous SP on knee swelling, plasma extravasation and neutrophil infiltration in the CFA-pretreated joint. Effect of SP (100 pmol, 10 μl) and saline (contralateral joint, 10 μl) on (a) plasma extravasation (n=5–6), (b) knee swelling (n=6) and (c) synovial MPO activity (n=6–7) in CFA-pretreated joints of wild type (WT) and NK₁ receptor knockout (KO) mice. Results are mean±s.e.m. Statistical significance was evaluated using an unpaired t-test to compare WT vs NK₁ receptor KO mice. ^***P<0.001 vs WT mice; ^$P<0.05 vs saline-treated joint.