Thalidomide analogue CC-3052 reduces HIV+ neutrophil apoptosis in vitro

Abstract

Thalidomide has significant immunomodulatory properties and has been used successfully in the treatment of oral ulcers and wasting in HIV patients. However, its use is limited by its poor bioavailability due to low solubility and short half life in solution, and teratogenic and neurotoxic side-effects. Recently, water-soluble analogues of thalidomide with significantly greater immunomodulatory activity and reduced side-effects have become available. We examined the effect of thalidomide and one analogue, CC-3052, on neutrophil apoptosis following culture for 20 h in vitro. Apoptosis was assessed by reduced CD16 expression and Annexin V binding using flow cytometry. Thalidomide or CC-3052 alone had no effect on neutrophil apoptosis when used at physiological levels. However, when used together with prostaglandin E2 (10-7 M), a potent adenylate cyclase activator, CC-3052 but not thalidomide (both 10-5 M) reduced apoptosis in neutrophils from normal and HIV+ donors. The reduced apoptosis could not be attributed to the ability of CC-3052 to reduce tumour necrosis factor-alpha (TNF-alpha) production, but may be due to its PDE4 inhibitor properties, as it increased [cAMP]i, and mimicked the effect of increasing [cAMP]i using dibutryl cAMP, a membrane-permeable analogue of cAMP. The results suggest a role for thalidomide analogue CC-3052 in reducing persistent activation of the TNF-alpha system in HIV without markedly impairing neutrophil viability.

Fig 2

Neutrophils from HIV⁺ patients do not exhibit altered spontaneous apoptosis. Apoptosis of freshly isolated and 20 h cultured neutrophils from healthy controls (□) and HIV⁺ patients (▪) assessed by CD16^low expression. Results are mean ± s.e.m. of HIV⁺ (n = 10) and healthy control (n = 11) neutrophils.

Fig 1

Evaluation of neutrophil apoptosis by changes in expression of CD16 and Annexin V. Flow cytometric analysis of human neutrophils freshly isolated (left panel) and after culture for 20 h (right panel). Scatter plots show neutrophils labelled with monoclonal antibodies as follows: (a) isotype controls; (b) Annexin V–FITC alone; (c) CD16–PE alone; and (d) Annexin V–FITC and CD16–PE. Single-parameter histograms for CD16–PE staining are also shown (e) to illustrate CD16^high and CD16^low populations.

Fig 3

Thalidomide or analogue CC-3052 alone do not alter neutrophil apoptosis. Apoptosis of neutrophils (PMN) cultured 20 h with 10⁻⁵ m thalidomide or analogue CC-3052 assessed by CD16^low expression. Results are mean ± s.e.m. of 10 experiments unless indicated otherwise.

Fig 5

Neutrophil dose response to thalidomide analogue CC-3052 and prostaglandin E₂ (PGE₂). □, Control level of apoptosis as assessed by CD16^low expression after 20 h culture. (a) Increasing CC-3052 concentration with constant amount of PGE₂ (10⁻⁷ m) and (b) increasing PGE₂ in the presence of CC-3052 (10⁻⁵ m). Results are mean values ± s.e.m. of three separate experiments carried out using healthy control neutrophils.

Fig 4

Synergistic effect of prostaglandin E₂ (PGE₂) with CC-3052 on neutrophil apoptosis. Effect of PGE₂ (10⁻⁷ m) alone or co-cultured with either thalidomide (Thd) or CC-3052 (both at 10⁻⁵ m) on neutrophils (PMN) from healthy donors (□, n = 11) or HIV⁺ donors (▪, n = 10) in indicated number of experiments (n). Apoptosis was assessed by CD16^low expression after 20 h culture. *P < 0·05 (paired Students t-test).

Fig 6

Neutralizing tumour necrosis factor-alpha (TNF-α) increases neutrophil apoptosis in vitro. Effect of rhTNF-α (50 ng/ml) and anti-TNF-α (10 μg/ml) on neutrophil (PMN) apoptosis after 20 h in vitro culture. No marked difference (< 2%) was seen using an equivalent concentration of isotype control antibody in three experiments using normal control PMN (data not shown). Results are mean ± s.e.m. of 10 experiments. *P < 0·01 (paired Students t-test).

Fig 7

Altered expression of Annexin V and CD16 on addition of cAMP or a combination of CC-3052 + prostaglandin E₂ (PGE₂). Flow cytometry analysis of 20 h aged neutrophil population cultured with: (a) no addition; (b) db cAMP (10⁻⁴ m); (c) CC-3052 (10⁻⁵ m); (d) PGE₂ (10⁻⁷ m); (e) CC-3052 + PGE₂ (concentrations as above). Scatter plots illustrate two-colour fluorescence obtained when neutrophils are stained for Annexin V–FITC and CD16–PE. Representative data obtained from two experiments using normal healthy neutrophils are shown.

Fig 8

Effects of CC-3052 and thalidomide (both at 10⁻⁵ m) in the presence of prostaglandin E₂ (PGE₂) (10⁻⁷ m) on intracellular cAMP levels in normal human neutrophils (PMN). Two million PMN were cultured for 1 h with drug additions and cAMP content was determined by ELISA. Results expressed as picomols of cAMP/10⁶ cells representing mean ± s.e.m. of three separate experiments.