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. 2004 Nov;63(11):1470–1477. doi: 10.1136/ard.2003.013060

Anti-CD4 monoclonal antibody treatment in acute and early chronic antigen induced arthritis: influence on macrophage activation

K Nissler 1, D Pohlers 1, M Huckel 1, J Simon 1, R Brauer 1, R Kinne 1
PMCID: PMC1754787  PMID: 15479897

Abstract

Objective: To investigate the indirect effects of anti-CD4 treatment on the functions of macrophages (CD4– in mice) in the acute and early chronic phase of mouse antigen induced arthritis (AIA).

Methods: C57BL/6 mice with AIA were treated intraperitoneally with the anti-CD4 mAb GK1.5 or control rat IgG on days –1, 0, 1, 3, 5, and 7. Proinflammatory cytokines (IL1ß, IL6, and TNFα) were quantified by sandwich ELISA in joint extracts, serum, and supernatants of ex vivo stimulated spleen/lymph node cells or peritoneal macrophages (+LPS/IFNγ). Nitric oxide (NO) levels in supernatants of ex vivo stimulated peritoneal macrophages were measured by the Griess reaction. Proteolytic activity in joint homogenates was analysed by gelatin, casein, and elastin zymography, and substrate assays.

Results: Anti-CD4 treatment significantly reduced joint swelling in acute (days 3, 5) and early chronic AIA (day 7) and diminished inflammation and destruction scores in late chronic AIA (day 21). On day 3, anti-CD4 treatment significantly reduced IL6 levels in all compartments. IL1ß was reduced in joint extracts, unaffected in serum or cells from lymphoid organs, and increased in stimulated peritoneal macrophages. TNFα was significantly increased in the joints, decreased in serum, and otherwise unchanged. NO production by stimulated peritoneal macrophages was significantly reduced by anti-CD4 treatment. Lower activity of matrix metalloproteinases and neutrophil elastase was seen in joint extracts of anti-CD4 treated animals than in IgG treated AIA controls.

Conclusion: CD4+ T cell directed treatment had strong local and systemic effects on macrophages. These indirect effects may contribute to the reduction of destructive mediators/joint destruction in AIA.

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Figure 1.

Figure 1

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 Joint swelling and histology of AIA. (A) Time course of joint swelling in AIA after treatment with the anti-CD4 mAb GK1.5 or control rat IgG. Results are expressed as means (SEM) of 10 individual animals in each group. Arrows indicate the days of treatment (days –1, 0, 1, 3, 5, and 7). (B) Histological score of joint inflammation and joint destruction in the acute phase (day 3) and the chronic phase (day 21) of AIA. Results are expressed as means (SEM) of 10 individual animals in each group. *p⩽0.05, **p⩽0.01 in comparison with IgG treated controls.

Figure 2.

Figure 2

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 Cytokine levels on day 3 of AIA. Concentrations of IL1ß (A, B, C, D, E), IL6 (F, G, H, I, J), and TNFα (K, L, M, N, O) in joint extracts, supernatants of stimulated lymph node cells, spleen cells, or peritoneal macrophages, as well as serum of GK1.5 treated or rat IgG treated mice, as determined by sandwich ELISA. Results are expressed as means (SEM) (n = 6 for each group). *p⩽0.05, **p⩽0.01 in comparison with IgG treated controls.

Figure 3.

Figure 3

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 Correlations between cytokine levels in joint extracts and joint swelling on day 3 of AIA. Correlations (Spearman rank test) between joint IL1ß, IL6, and TNFα levels and joint swelling of individual mice treated with GK1.5 or rat IgG (total of n = 18 for each cytokine).

Figure 4.

Figure 4

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 NO levels on day 3 of AIA. Levels of NO in culture supernatants of LPS/IFNγ stimulated peritoneal macrophages from GK1.5 treated or rat IgG treated mice. Results are expressed as means (SEM) (n = 6 for each group). **p⩽0.01 in comparison with IgG treated controls.

Figure 5.

Figure 5

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 "Total" MMP activity in joint extracts on day 3 of AIA. Total activity of MMP was determined by cleavage of the peptide MCA-Pro-Leu-Gly-Leu-DAP (DNP-Ala-Arg-NH2), resulting in an increase of fluorescence. Results are expressed as means (SEM) (n = 8 for each group). *p⩽0.05 in comparison with IgG treated controls.

Figure 6.

Figure 6

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 Zymography of joint extracts on day 3 of AIA. Zymography of equal amounts (protein content) of joint extracts from GK1.5 treated or rat IgG treated mice using (A) gelatin, (B) casein, or (C) elastin as a substrate. Markers of molecular weight (Mol wt) or the positions of recombinant human MMP standards (Std) are indicated on the right. Elastase activity in (C) was blocked by the specific inhibitor MeOSuc-Ala-Ala-Pro-Val-CMK.

Figure 7.

Figure 7

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 Elastase activity in joint extracts on day 3 of AIA. Activity of neutrophil elastase was determined by cleavage of the elastase-specific, synthetic substrate MeOSuc-Ala-Ala-Pro-Val-pNA, resulting in an increase of absorbance. Results are expressed as means (SEM) (n = 8 for each group). *p⩽0.05 in comparison with IgG treated controls.

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