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. 1999 May;58(5):297–302. doi: 10.1136/ard.58.5.297

Macrophage inflammatory protein 1 alpha expression by synovial fluid neutrophils in rheumatoid arthritis

Y Hatano 1, T Kasama 1, H Iwabuchi 1, R Hanaoka 1, H Takeuchi 1, L Jing 1, Y Mori 1, K Kobayashi 1, M Negishi 1, H Ide 1, M Adachi 1
PMCID: PMC1752877  PMID: 10225815

Abstract

OBJECTIVE—To determine the contribution made by synovial fluid (SF) neutrophils to the augmented expression of macrophage inflammatory protein 1 α (MIP-1α) in rheumatoid arthritis (RA).
METHODS—Neutrophils were isolated from samples of SF from RA patients and peripheral blood (PB) samples from RA patients and healthy controls. Cell associated MIP-1α was visualised immunohistochemically, and cell associated MIP-1α as well as MIP-1α secreted into the SF was assayed by ELISA. Steady state expression of MIP-1α mRNA was assessed by reverse transcription polymerase chain reaction (RT-PCR).
RESULTS—Freshly isolated SF neutrophils contained significantly higher concentrations of both MIP-1α protein and its transcript than PB neutrophils from either RA patients or healthy controls; incubation in the absence or presence of tumour necrosis factor α for 24 hours resulted in a significant increase in MIP-1α secretion by RA SF neutrophils compared with neutrophils obtained from either normal PB or RA PB; and expression of MIP-1α by SF neutrophils was well correlated with both RA disease activity and SF mononuclear cell (MNC) counts.
CONCLUSION—Expression and secretion of MIP-1α by SF neutrophils may be indicative of local and systemic inflammation in RA. Moreover, this C-C chemokine may contribute to the recruitment of MNCs from the bloodstream into synovial joints and tissues.



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Figure 1  .

Figure 1  

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Concentrations of MIP-1α in lysates of freshly isolated neutrophils or in synovial fluids. (A) Neutrophils were obtained from PB of patients with RA (n=8), or healthy normal controls (n=10), and from SF of patients with RA (n=15), then cell associated MIP-1α (pg/million cells) was assessed by ELISA. (B) SF was obtained from patients with RA (n=15) or OA (n=8), then MIP-1α in SF was assessed by ELISA. Data are expressed as the mean (SEM). * p < 0.05 v RA PB or normal PB (A), or OA (B).

Figure 2  .

Figure 2  

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Photomicrograph of the immunohistochemical localisation of antigenic MIP-1α in freshly isolated RA SF neutrophils. (A) Stained by control IgG. (B) Stained by anti-MIP-1α antibody demonstrating the presence of neutrophil associated MIP-1α antigen.

Figure 3  .

Figure 3  

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Analysis of MIP-1α mRNA in freshly isolated neutrophils obtained from RA SF, RA PB or normal PB using RT-PCR. (A) Representative neutrophil derived MIP-1α mRNA. GAPDH primers were used as an internal control. M shows molecular weight markers (100 bp ladder); (B) correlation between MIP-1α mRNA concentrations and neutrophil associated MIP-1α protein. MIP-1α mRNA expression was semiquantitated and normalised to GAPDH as the MIP-1α/ GAPDH mRNA ratio. Each point represents an individual patient.

Figure 4  .

Figure 4  

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Correlation between SF neutrophil associated (A), or SF MIP-1α (B) and serum CRP concentration. Each point represents an individual RA patient.

Figure 5  .

Figure 5  

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Basal and TNF-α induced MIP-1α secretion by neutrophils obtained from RA SF, RA PB, or normal PB. Neutrophils were incubated for 24 hours in the absence (medium) or presence of TNF-α (10 ng/ml), after which MIP-1α in the supernatants was assessed by ELISA. Data are expressed as the mean (SEM) of triplicate cultures of three independent experiments. * p values < 0.05 v medium alone.

Figure 6  .

Figure 6  

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Correlation between SF neutrophil associated (A) or SF MIP-1α (B) and SF MNC counts. Each point represents an individual RA patient.

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