Downregulation of Leukocyte Migration After Treatment With CCR5 Antagonist Maraviroc

To the Editors: Clinical studies indicate that treatment with CCR5 antagonist maraviroc (MVC) induces a greater immunologic benefit that is independent of HIV load suppression.1,2 Various mechanisms could be involved in this phenomenon, such as downregulation of inappropriate immune activation by CCR5 blockade, reduction of T-cell apoptosis, and cytokine expression.3 Thus, whether maraviroc can have immunologic properties beyond its capacity to inhibit virus entry is an area of active investigation. Given the central role of CCR5 in governing both trafficking and recruitment of leukocytes into inflamed tissue, the analysis of the effect of CCR5 antagonists on the modulation of cell migration.4 Here, we studied the chemotactic activity of peripheral blood mononuclear cells (PBMC) from HIV-infected patients undergoing a MVC-based regimen. In addition, the direct effect of MVC on leukocyte migration toward CCR5-using chemokines was also assessed in vitro. We analyzed 3 viremic patients who received a regimen including MVC + optimized background therapy because of a history of multiple treatment failure. Blood samples were collected at the beginning of MVC-based regimen and at months 1 and 6. The chemotactic activity of PBMC (1 × 106 cells/mL) was carried out by using Boyden method with blind-well chambers with polyvinylpyrrolidone-free polycarbonate filter 8 mm (Neuroprobe, Cabin John, MD). The synthetic peptide Formyl-Methionyl-Leucyl-Phenylalanine (FMLP) (Sigma, St. Louis, MO) at concentration of 10−5 and recombinant human chemokine RANTES (100 ng/mL) were used as chemo-attractants. The migration was assessed by counting cells in the bottom chamber by flow cytometry (FACS Calibur using CellQuest software) using TrucountTM tubes (BD Biosciences, Pont-de-Claix, France). To eliminate cellular debris, R1 gate was defined in a dot plot of Forward Scatter Channel versus Side Scatter Channel. The absolute number of migrated cells was calculated as number (n) of cells/beads counted × total number of beads. The percentage of migrated cells was calculated as the mean of 3 n values using the following formula: % = n of migrated cells/n of 100% controls × 100. In another set of experiments, PBMCs isolated from healthy donors buffy coat were pretreated in vitro with MVC (200 nM) (Celsentri; Pfizer, Inc, New York, NY). Drug concentration was chosen on the basis of data of pharmacokinetic parameters reported in MVC-treated patients.5,6 After 18 hours of incubation at 37°C under 5% CO2 atmosphere, PBMCs were evaluated for chemotaxis. All experiments were performed in triplicate. One-way analysis of variance was used for statistical analysis. The results showed that, after starting MVC-based regimen, the 3 patients achieved undetectable plasma HIV RNA (<20 copies/mL) with a mean increase in CD4 count of 210 cells per milliliter. The pre-MVC mean CD4 was 369 cells per milliliter. The study of ex vivo chemotaxis showed that there was a mean reduction in migrated cells toward FMLP of 53% and 60.3% after 1 and 6 months of MVC treatment, respectively (Fig. 1A). In the experiments assessing the direct effect of MVC on cell migration, we found that the chemotactic activity of PBMCs toward both RANTES and FMLP was significantly inhibited by in vitro drug treatment (mean ± SD, 42 ± 6% versus 8 ± 3% for FMLP and 56 ± 3% versus 8 ± 2% for RANTES) (P < 0.001 for both) (Fig. 1B).FIGURE 1: Effect of maraviroc treatment on peripheral cell chemotaxis. A, The ex vivo migration in response to FMLP was assessed in PBMCs from 3 HIV-infected patients before initiation of and during a maraviroc-based regimen. B, The migration (mean ± SD) in response to FMLP and RANTES was assessed in PBMCs from healthy donors after in vitro treatment with MVC (200 nM). *Represent values statistically significant (P < 0.001) in comparison with controls (1-way analysis of variance).The virological and immunologic benefits of MVC, the first CCR5 antagonist approved for treatment of HIV infection, are well known. However, what are the in vitro and in vivo immunologic consequences of pharmacologic inhibition of CCR5 remain to be investigated.7 In the present study, we demonstrated that PBMCs from patients undergoing an antiretroviral regimen containing MVC revealed diminished migratory responses. To investigate if this phenomenon could be related to a direct effect of the drug, we analyzed cell chemotactic activity after in vitro treatment with MVC. We found that MVC, used at concentrations which are compatible with those achieved in vivo in the serum of treated subjects, exhibited the ability of inhibiting chemotactic activity of PBMCs in response to FMLP and to CCR5-binding chemokine RANTES. The present findings are intriguing and support the additional immunologic effects of MVC beyond the pure anti-HIV effect. The CCR5 inhibitors might have a potential role in the downregulation of HIV-associated chronic inflammation by blocking the recirculation and trafficking of infected cells in inflamed tissue.8 The antichemotactic activity of CCR5 antagonist could have also potential therapeutic implication for the management of inflammatory conditions other than HIV.9 A critical issue associated with the block of cellular migration induced by CCR5 antagonist could be the impairment of the innate immune response against pathogens, although no evidence of increase in infectious complications infections has been reported yet in MVC-treated patients. The study of CCR5 expression on cell surface and the measurement of cell calcium flux could contribute in better understanding the mechanisms of MVC antichemotactic effect. Raffaella Rossi, PhD* Miriam Lichtner, MD, PhD† Ilaria Sauzullo, PhD* Fabio Mengoni, PhD* Raffaella Marocco, MD† Anna Paola Massetti, MD, PhD* Claudio Maria Mastroianni, MD, PhD† Vincenzo Vullo, MD* *Department of Infectious and Tropical Diseases, Sapienza University, Rome, Italy †Department of Infectious Diseases, Sapienza University, Polo Pontino, Latina, Italy