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Active Infection by Variant A of Human Herpesvirus Six Causes Destruction of Lymphoid Tissues in HIV Infected Individuals 

Konstance Kehl Knox, Ph.D. and Donald R. Carrigan Ph.D.
Institute for Viral Pathogenesis, 10437 Innovation Drive, Milwaukee, Wisconsin

Presented at the 40th Interscience Conference on Antimicrobial Agents and Chemotherapy
Toronto, Canada; September 18, 2000

Abstract

Background

The pathogenesis of AIDS centers upon the progressive destruction of the lymphoid tissues of patients infected with HIV.  Mechanisms of this lymphoid tissue involution are poorly understood, but direct tissue destruction by HIV is unlikely to be involved.  Previous work by our laboratory has demonstrated that the A variant of human herpesvirus six (HHV-6A) reactivates to productive infection in the lymph nodes of virtually every HIV infected individual when their absolute CD4+ lymphocyte counts are greater than 300 per mm3. 

Methods

Lymph node biopsies from 12 patients with progressive HIV disease and from 4 individuals with nonprogressive HIV disease were immunohistochemically stained with a monoclonal antibody specific for a structural glycoprotein of HHV-6A.  

Results

All 16 lymph node biopsies examined contained cells actively infected with HHV-6A.  However, the densities of the HHV-6A infections in the lymph nodes of patients with nonprogressive HIV disease were significantly (p < 0.02) lower than in lymph nodes from patients with progressive HIV disease.  Further, a significant (p < 0.035) inverse correlation was found between the absolute CD4+ lymphocyte counts of the patients and the density of the HHV-6A infection in their lymph nodes.  Finally, the densities of HHV-6A infections were significantly (p < 0.016) higher in lymph node areas undergoing active lymphocyte depletion than in areas free of destructive pathological changes.  

Conclusion

HHV-6A is the cause of the lymphoid tissue destruction that occurs in patients infected with HIV, probably through a synergistic interaction between it and HIV.  Nonprogressive HIV disease is associated with low level, and apparently well controlled, HHV-6A infection.  It can be postulated that early intervention with effective antiviral agents, such as ganciclovir, may slow or even stop the HHV-6 mediated destruction of lymphoid tissues in HIV infected individuals.  

Introduction

Tissue involution with lymphocyte depletion and histiocytosis are the major histopathological correlates of the destruction of lymphoid tissues in HIV infected.
The basic cause of these pathological processes remains unclear, but direct effects by HIV are unlikely to underlie these pathologies.
Previous work from our laboratory has shown that:

• HHV-6 reactivates to active infection early in the course of HIV infection (absolute CD4 counts > 300/mm3) .

• Lymph node infections at those early time points are predominantly due to the A variant of HHV-6 (HHV-6A).

• 100% (21/21) of patients with symptomatic HIV disease have active HHV-6A infections in most, if not all, of their tissues.

We have now extended these studies by exploring the relationships between HHV-6A infection of the lymph nodes of HIV infected individuals and the histopathological changes present in the tissues.

Methods

Patient Tissues

• Lymph node biopsies from HIV infected individuals available in the Department of Pathology of the Medical College of Wisconsin were studied.  All of these patients  eventually died of AIDS.

• Lymph node biopsies from four HIV infected individuals with "non-progressive" HIV disease were obtained from Drs. Giuseppe Pantaleo and Oren Cohen of the Laboratory of Immunoregulation of the National Institutes of Health.

Immunohistochemical Staining (IHC)

• HHV-6A and HHV-6B infected cells were identified by staining with variant specific murine monoclonal antibodies.

• Densities of HHV-6A infected cells in the lymph node tissues was determined by direct counting.

Histopathological and quantitative virological analyses of lymph node biopsies

• Consecutive sections of the lymph node biopsies were stained  with hematoxylin/eosin, for HHV-6A infected cells and for HHV-6B infected cells by IHC.

• Identical grids consisting of 2 millimeter by 2 millimeter squares were then drawn on each of the slides.

• Each grid on the H&E stained slides was scored for the degree of lymphocyte depletion present using the following system: score 1: less than 10% depletion, score 2: 10% to 50% depletion, score 3: 50% to 75% depletion, and score 4: greater than 75% depletion (Figure 1).

•  Slides stained for HHV-6A and HHV-6B infected cells were evaluated by direct counting of the cells within each grid on the slide.

•  Pooled data were then statistically analyzed for relationships among lymphocyte depletion scores and HHV-6A and HHV-6B infected cell densities.

Control Tissues

Lymph nodes from 4 HIV negative normal individuals and from 3 lymph nodes from HIV negative patients with follicular hyperplasia served as control materials.

Results

• HHV-6A Infection in Lymph Nodes of HIV Infected Individuals

• All sixteen of the lymph node biopsies studied contained cells actively infected with HHV-6A

• No cells actively infected with HHV-6A were detected in any of the control tissues (p < 0.0001 by Fisher's Exact Test).

• The densities of HHV-6A infected cells in the lymph node biopsies were inversely related to the absolute CD4+ lymphocytes counts of the patients at the time of the biopsies (Spearman Rank Correlation: r = ‑0.588; p < 0.035).

• The median HHV-6A infected cell density of the patients with progressive HIV disease (717/cm2)  was significantly (Figure 2; p < 0.02 by two-tailed Mann‑Whitney Test) higher than that of patients with nonprogressive HIV disease (170/cm2).
  

Click picture to view larger version of figure.

Figure 1. Histopathological grading system used for evaluation of lymph node biopsies. Panels A - D show tissue sections stained with hematoxylin/eosin that respectively exemplify lymphocyte depletion scores of 1: less than 10% depletion; score 2: 10% to 50% depletion; score 3: 50% to 75% depletion; and score 4: greater than 75% depletion. Arrowheads indicate infiltrating histiocytes and fibrosis. 

Click thumbnail to view larger version of figure.

Figure 2. Association between type of HIV disease and density of HHV-6A infection in the patients' lymph nodes. Long horizontal lines indicate mean value. Short horizontal lines denote the 95% confidence intervals (p < 0.02 by two-tailed Mann-Whitney Test). 

Histopathological Correlates of HHV-6A Infections 

Only low levels of HHV-6A infection were found in lymph node areas with either low or no depletion of lymphocytes and in areas with essentially complete replacement of the lymphoid tissue by histiocytes and fibroblasts (Figure 3).  In contrast, very high densities of HHV-6A infected cells were present in lymph node areas undergoing active lymphocyte depletion [Figure 4].  The differences between the HHV-6A infected cell densities of lymph node areas with lymphocyte depletion scores of 1 or 4 and those of areas with lymphocyte depletion scores of 2 or 3 were significant (p < 0.016 by Kruskal-Wallis Nonparametric ANOVA test).  

Click thumbnail to view larger version of figure.

Figure 3.  Comparison of HHV-6A infected cell density and lymphocyte depletion scores for the entire data set for lymph node biopsies from HIV infected patients.  Long horizontal bars indicate the mean values.  Short horizontal bars indicate the 95% confidence intervals for the HHV-6A infected cell densities for each lymphocyte depletion score. Differences between the infected cell densities for the lymphocyte depletion scores was significant (p < 0.016).  

Click picture to view larger version of figure.

Figure 4. IHC staining of a section of lymph node from an HIV infected patient for cells actively infected with HHV-6A.  Panel A: Low magnification view showing a large area of active lymphocyte depletion in the upper right side of the tissue section.  Arrowheads indicate the intact periphery of a follicle undergoing follicular lysis (upper right of follicle). Panel B: Higher magnification view of the area of follicular lysis shown in panel A.  Some of the cells actively infected with HHV-6A are indicated by arrowheads.

Conclusions

Lymph nodes from nonprogressive HIV disease patients contained significantly lower densities of HHV-6A infected cells compared with progressive HIV disease patients.

•  The density of HHV-6A infected cells was inversely related to the absolute CD4+ lymphocyte count at the time of the biopsy.

•  Only low levels of HHV-6A infection were present in lymphoid tissue areas with no destructive pathological changes

•  Very high levels of HHV-6A infection were seen in tissue areas undergoing active destruction

These results have implications concerning other established aspects of the pathogenesis of HIV-1 disease:

•  HHV-6A infection of lymphocytes and macrophages induces the production of high levels of TNFa

•  HHV-6A infected T lymphocytes produce a soluble substance that dramatically upregulates HIV-1 replication

•  HIV encoded tat protein upregulates the replication of HHV-6A

Thus we propose that HHV-6A and HIV become locked in a synergistic cycle such as the one illustrated in Figure 5.  The ultimate result of this cycle is the progressive destruction of lymphoid tissues in patients infected with HIV.

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