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Prevalence of Mixed Cryoglobulins in Relation to CD4 Cell Count among Patients Coinfected with HIV and Hepatitis C Virus

  1. Laurent Aaron1,
  2. Pascal Lebray2,
  3. Marie-Alexandra Alyanakian3,
  4. Laurent Roudière1,
  5. Audrey Therby1,
  6. Marie-Laure Chaix4,
  7. Bertrand Dupont1,
  8. Stanislas Pol2, and
  9. Jean-Paul Viard1
  1. 1Service des Maladies Infectieuses et Tropicales, Paris, France
  2. 2Service d'Hépatologie, Paris, France
  3. 3Laboratoire d'Immunologie, Paris, France
  4. 4Laboratoire de Virologie, Assistance Publique-Hôpitaux de Paris, Centre Hospitalier Universitaire Necker Enfants-Malades, Paris, France
  1. Reprints or correspondence: Dr. Jean-Paul Viard, Service des Maladies Infectieuses et Tropicales, Hôpital Necker Enfants-Malades, 149 rue de Sèvres, 75743 Paris Cedex 15, France (jean-paul.viard{at}nck.ap-hop-paris.fr).
 
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Abstract

Cryoglobulinemia was studied in human immunodeficiency virus—positive, hepatitis C virus (HCV)—positive patients in relation to their CD4 cell count. Cryoglobulinemia was found in 18 (31.6%) of 57 patients: 17 (44.7%) of 38 patients with a CD4 cell count of ⩾200 cells/µL versus 1 (5.3%) of 19 patients with a CD4 cell count of <200 cells/µL (P = .0064). Cell-mediated immunity could, therefore, contribute to the production of HCV-associated cryoglobulins.

Cryoglobulins are circulating immunoglobulins that undergo reversible insolubilization at low temperatures. Type I cryoglobulins are single monoclonal immunoglobulins, and types II and III (mixed cryoglobulins) are monoclonal and polyclonal immunoglobulins, respectively, that have rheumatoid factor activity [1]. Mixed cryoglobulins are found in viral infections. Reported prevalences vary from 40% to 56% in hepatitis C virus (HCV) infection [2, 3] and from 34.5% to 81% in HIV/HCV coinfection [2, 4–7], but the association with the CD4 cell count is unclear. Therefore, we studied the prevalence of cryoglobulins in HIV-1/HCV-coinfected patients in relation to the CD4 cell count, which provides the unique opportunity to assess the influence of cell-mediated immunity on the production of HCV-associated cryoglobulinemia

Patients and methods. From November 2000 through May 2003, consecutive HIV/HCV—positive patients attending the infectious diseases and hepatology departments of Hôpital Necker (Paris, France) were evaluated for the presence of cryoglobulins. The exclusion criteria were as follows: discordant HCV markers (i.e., negative serological test results and positive HCV RNA test results, or the reverse); the presence of chronic hepatitis B virus infection or other chronic infections, acute infection (during the 3 months after resolution), or neoplastic or autoimmune diseases; and a history of treatment with IFN-α or IL-2. Clinical, epidemiological, virological, and biochemical data, as well as data obtained from biopsy, were collected. Each patient was examined for cryoglobulin-associated symptoms. Urinalysis and liver and kidney function tests were performed. Sixty-two HIV-infected, HCV-negative patients were also studied, as a control group. The duration of HIV/HCV coinfection was estimated on the basis of the time that the first positive test results were obtained. Overall, 75% of the patients were receiving antiretroviral drugs at the time of enrollment.

For the detection of cryoglobulin, samples of blood were collected in prewarmed tubes and were immediately transferred to the laboratory at 37°C and allowed to clot. Serum was separated by centrifugation at 37°C and was stored at 4°C for 7 days. Visible precipitates were washed 5 times at 4°C, and total protein levels were measured by ultraviolet absorption at 280 nm and 260 nm. Serological tests for HCV were performed with ELISA and RIBA3 tests (Ortho Diagnostic Systems). Plasma HCV RNA levels were quantified with the Amplicor-Monitor assay, version 1.0 (Roche). Plasma HIV-1 RNA levels were measured with the Amplicor HIV Monitor assay (Roche). Absolute CD4 cell counts were determined by flow cytometry with a FACScan equipped with CellQuest software (both Becton-Dickinson). Statistical analysis was performed with Statview software (Abacus Concepts). The Mann-Whitney U test and the χ2 test were used, as appropriate. All patients gave informed consent.

Results. Fifty-seven HIV/HCV-coinfected patients were included in the present study (table 1). Most patients had been infected with HIV and HCV via exposure to blood. A total of 53.2% of patients were infected with HCV genotype 1; 29.8% were infected with genotype 3; 17% were infected with genotype 4; 4.3% were infected with genotype 2; and a few patients were infected with 2 genotypes. The levels of HCV viremia and the prevalence of cirrhosis were significantly greater among patients with a CD4 cell count of <200 cells/µL than among patients with a CD4 cell count of ⩾200 cells/µL. The results of standard biological investigations were not different between the 2 groups (data not shown).

Table 1
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Table 1

Demographic and clinical characteristics of HIV-1/hepatitis C virus (HCV)—coinfected and HIV-1—infected, HCV-negative patients in a study of cryoglobulin production.

In accordance with the findings of previous reports [4–7], cryoglobulins were detected in 18 (31.6%) of 57 patients, but the prevalence of cryoglobulins was significantly higher among patients with a CD4 cell count of ⩾200 cells/µL than among patients with a CD4 cell count of <200/µL (17 [44.7%] of 38 vs. 1 [5.3%] of 19; P = .0064). In other words, HCV-associated cryoglobulins were virtually absent among patients with a CD4 cell count of <200 cells/µL (cryoglobulins were detected in only 1 patient, who had a CD4 cell count of 189 cells/µL), whereas, among patients with a CD4 cell count of ⩾200 cells/µL, the prevalence was similar to that observed among patients who are not infected with HIV.

As expected on the basis of other studies [4, 5, 8], only 3 patients—all with a CD4 count of ⩾200/µL—presented with cryoglobulin-related complications (1 had nephropathy and 2 had peripheral neuropathy). Fourteen patients underwent repeated testing for cryoglobulinemia: the presence or absence of cryoglobulinemia was constant in most cases, but mixed cryoglobulins appeared in 1 of 3 patients who were observed during immune restoration while receiving HAART. Cryoglobulins were detected in only 4.9% of HIV-positive, HCV-negative patients (table 1).

Discussion. The relationships between cryoglobulinemia and T cell immunity are poorly understood, and the interactions between T cell—mediated and B cell—mediated responses in HCV infection are complex. A good specific immune response limits the evolution of HCV, particularly in HIV-infected patients. Thus, as expected, we found higher levels of HCV viremia and higher fibrosis scores in patients with a CD4 cell count of <200 cells/µL than in patients with a CD4 cell count of ⩾200 cells/µL. On the other hand, hepatic inflammation is secondary to T cell—mediated immune response, and the release of factors from inflammatory cells and hepatocytes induces hepatic stellate cells to initiate fibrogenesis and, ultimately, cirrhosis [9].

In addition, cryoglobulinemia may result from B cell activation by HCV infection and may represent an attempt to prevent the persisting infection of hepatocytes with HCV [10]. Indeed, one study found that, during the treatment of cryoglobulinemia with anti-CD20 monoclonal antibody, the levels of HCV RNA increased, but the levels of cryoglobulins decreased [11].

HIV infection leads to a profound immune dysregulation involving CD4 cells, CD8 cells, and B cells [12]: the susceptibility of B cells to apoptosis is increased, and, because of CD4 cell defects, B cells show poor responses to mitogenic and antigenic stimuli in vitro and poor antibody responses to T cell—dependent and independent antigens in vivo. Therefore, cell-mediated immunity could play a role in the secretion of cryoglobulins by B cells.

At variance with the results of the present study, the presence of cryoglobulinemia was not correlated with the CD4 cell count in 2 studies of coinfected patients with milder immunosuppression (mean CD4 cell counts, 453 cells/µL and 333 cells/µL) [5, 13]. The present study included patients who had a wider range of CD4 cell count values, with a stronger representation of significantly immunosuppressed individuals, which may explain these apparently discrepant findings. In addition, the mean cryoglobulin concentration has been found to be lower in HIV/HCV coinfection than in HCV monoinfection (268 mg/L vs. 585 mg/L; P < .03) [4]. Antinori et al. [14] also reported the remission of symptoms and the disappearance of HCV-associated cryoglobulinemia, despite detectable HCV viremia, in a patient who became HIV-infected and developed immunosuppression (the CD4 cell count decreased from 337 cells/µL to 21 cells/µL). Conversely, Monsuez et al. [15] described the appearance of cryoglobulin-associated polyarthralgia in one HIV/HCV-coinfected patient at the time of immune restoration while the patient was receiving HAART (the CD4 cell count increased from 70 cells/µL to 567 cells/µL). In the present study, one patient developed cryoglobulinemia during immune restoration.

Further evidence that T cells help in the production of cryoglobulins is the influence of HLA type II polymorphism on the production of HCV-related cryoglobulins [16]. The presence of HLA-DR11 increased the risk of HCV-associated type II cryoglobulinemia and was associated with spontaneous viral clearance, sustained response to IFN-α, and a low risk of fibrosis during chronic infection. Conversely, HLA-DR7 protected against the production of cryoglobulins but was associated with a high risk of chronic infection and fibrosis.

At variance with the findings of previous studies [4, 8, 13] but in accordance with those of a recent report [5], HIV-positive, HCV-negative patients in the present study did not have a high prevalence of production of cryoglobulins. This may be explained by the fact that most of the patients in the present study and those in the study by Fabris et al. [5] were receiving antiretroviral therapy, and a high proportion of patients (60%) had an undetectable plasma HIV RNA load.

In conclusion, cell-mediated immunity seems to play a role in the production of cryoglobulins by B cells. HCV-associated cryoglobulins could be an indirect marker of an immune response against HCV, because the cryoglobulins disappear during HIV-induced cellular immunosuppression. The disappearance of immunity to HCV—which is in part reflected by the absence of cryoglobulins—could be related to the higher levels of viremia that may contribute to the more-severe liver disease observed in severely immunocompromised patients.

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Acknowledgments

We thank Aline Maignan for data management.

Potential conflicts of interest. All authors: no conflicts.

  • Received July 7, 2004.
  • Accepted September 6, 2004.
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  1. Clin Infect Dis. (2005) 40 (2): 306-308. doi: 10.1086/427029
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