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From the Tulane National Primate Research Center,* Tulane University, Covington, Louisiana; and the New England Primate Research Center, Harvard Medical School, Southborough, Massachusetts
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Abstract |
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Molecularly cloned SIVmac239 causes acquired immune deficiency syndrome (AIDS) and death in 
25% of inoculated rhesus monkeys within 6 months of inoculation with 50% mortality in a little more than 1 year.12
The remaining animals develop a more protracted course. Although SIVmac239, the prototypic pathogenic molecular clone, does not replicate efficiently within macrophages in culture, it does infect macrophages in vivo as early as 21 days after infection.13-15
Furthermore, 40% of rhesus macaques that die with AIDS after infection with SIVmac239 display lesions associated with infection of macrophages such as encephalitis (SIVE) and giant cell pneumonia.1,12,16
The acquired ability of SIVmac239 to replicate efficiently in monocytes/macrophages in culture was originally mapped to nine amino acid changes in envelope.16-19 The variant of SIVmac239 with these amino acid changes in env was designated SIVmac239/316 env,17 hereafter referred to as SIVmac239/316. Further work showed that of these nine amino acid changes four (V to M at residue 67, K to E at residue 176, G to R at residue 382, and K to T at residue 573) were strongly associated with the ability of the parental virus, SIVmac239 to replicate well in cultured macrophages.17,19 In addition to conferring macrophage tropism these changes were found to result in a significant decrease in the requirement for CD4 before CCR5 binding and to render the virus susceptible to antibody neutralization.20-25
These observations led to speculation that SIVmac239/316 would have a broader tropism than the parental SIVmac239 with a particular preference for CCR5high CD4low populations such as alveolar macrophages, parenchymal microglia in brain and other cell types, particularly those in immunologically privileged sites such as the brain. Contrary to this prediction is the observation that although animals infected with SIVmac239/316 do develop AIDS, macrophage-associated lesions such as SIVE have not been observed (Table 1)
. To try and resolve these observations we sought to determine the cell types infected in vivo in multiple tissues during both acute and chronic SIVmac239/316 infection. To our surprise, not only did we not find evidence of a broadened cell tropism for SIVmac239/316 compared toSIVmac239 but the only cell type we found infected in vivo were T lymphocytes. This raises interesting questions as to the applicability of in vitro studies that have defined the cell tropism of SIV and HIV to what is occurring in vivo.
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Materials and Methods |
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The viruses used for animal infection in this study were molecularly cloned SIVmac239 and SIVmac239/316.17,18,26,27 SIVmac239 is the prototypic pathogenic molecular clone of SIV. SIVmac239/316env (hereafter SIVmac239/316) was derived from SIVmac239 by exchanging the envelope gene of SIVmac316 into SIVmac239.17 SIVmac316 was obtained from an animal inoculated with SIVmac239 that died of AIDS and disseminated giant cell disease including SIVE and giant cell pneumonia.16 The differences between the clones of SIVmac239 and SIVmac239/316 used in this study are limited to nine amino acids in env.17,18 Therefore differences in tropism and disease pathogenesis can be attributed to the env protein. The stop codons in nef and transmembrane were removed in both the parental SIVmac239 and SIVmac239/316.
Animals and Tissues
Tissues from 24 SIV-infected and 2 uninfected rhesus macaques (Macaca mulatta) were obtained from the pathology archives of the New England and Tulane National Primate Research Centers. This included 15 animals infected with SIVmac239/316 and 9 infected with the parental virus SIVmac239. All animals were infected with the same dose of SIV (50 ng of p27) and the same route (intravenous). Of the 15 animals infected with SIVmac239/316, 8 were euthanized within 50 days of infection as part of a serial sacrifice study and 7 were followed long term. Details on the animals including time after inoculation and major pathological findings are provided in Table 1
. Matched tissues from nine macaques infected with the parental SIVmac239 and two uninfected macaques, were examined in parallel. Data from numerous additional historical controls infected with SIVmac239 were also available.13-15,28-31
All animals received a complete necropsy and histopathological examination. Tissues selected for examination included brain, heart, lung, lymph nodes, spleen, thymus, and small and large intestine. All tissues had been collected immediately after euthanasia with an intravenous overdose of pentobarbital. Tissues were fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned at 6 µm, and stained with hematoxylin and eosin. Adjacent tissues were also embedded in optimal cutting temperature compound (O.C.T.; Miles Inc., Elkhart, IN) and snap-frozen by immersion in 2-metylbutane in dry-ice and cut into sections 6 to 8 µm thick.
Viral Load
Viral RNA loads in plasma of SIV-infected rhesus macaques were measured by quantitative real-time polymerase chain reaction as previously described.32-34
Localization of SIV-Infected Cells
In situ hybridization for SIV was performed using both riboprobes and random primed DNA probes as described previously.35 Briefly, for RNA in situ hybridization formalin-fixed, paraffin-embedded tissue sections were pretreated in a microwave with citrate buffer (antigen unmasking solution; Vector Laboratories, Burlingame, CA) for 20 minutes at high power according to the manufacturer’s instructions. Thereafter, sections were thoroughly washed, placed in a humidified chamber, and prehybridized at 45°C with hybridization buffer (containing 50% of formamide with denatured herring sperm DNA and yeast tRNA at 10 mg/ml each). SIV-digoxigenin-labeled anti-sense riboprobes (provided by Drs. V. Hirsch and C. Brown, National Institutes of Health, Rockville, MD) were used at a concentration of 10 ng/slide in hybridization buffer and hybridized overnight at 45°C. After hybridization slides were washed with 2x standard saline citrate, 1x standard saline citrate, 0.1x standard saline citrate, and blocking solution was applied. Fab fragments from an anti-digoxigenin antibody from sheep, conjugated with alkaline phosphatase (Roche, Penzberg, Germany), were used to detect digoxigenin-labeled probes. Controls included matched tissues from known positives and negatives and hybridization with digoxigenin-labeled sense RNA labeled with digoxigenin.
For DNA in situ hybridization the DNA probe used was a combination of two plasmids: a subclone of p239SpE3' in phosphate-buffered saline (PBS), which contains tat, rev, env, nef, and a small part of the 3'LTR, and p239SpSp5', which contains gag, pol, vif, vpx, vpr, and the 5'LTR in PBS. This combination provides essentially the entire SIVmac239 genome. The probes were labeled with digoxigenin-11-dUTP by random priming (Boehringer-Mannheim, Indianapolis, IN) as described previously.36,37 Hybridization was performed under denaturing conditions to detect both viral DNA and RNA. Formalin-fixed, paraffin-embedded tissue sections were pretreated in a microwave with antigen unmasking solution (Vector Laboratories) for 20 minutes at high power and according to the manufacturer’s instructions. Thereafter, sections were thoroughly sequentially washed, placed in a humidified chamber, and prehybridized at 37°C with hybridization buffer (containing 50% formamide with denatured herring sperm DNA and yeast tRNA at 10 mg/ml each) and washed with 2x standard saline citrate. SIV digoxigenin-labeled DNA probes were used at 0.5 ng/µl in hybridization buffer and hybridized overnight at 37°C. After hybridization, sections were treated as previously described above for RNA in situ hybridization. Selected positive tissues and serial sections of the test tissues hybridized with plasmid pUC19 labeled with digoxigenin were used as controls.
When immunohistochemistry followed in situ hybridization the slides were developed using NBT/BCIP stock solution (Roche, Mannheim, Germany) or Vector blue (Vector Laboratories) as chromogen. If immunofluorescence followed the in situ hybridization, 2-hydroxy-3-naphtoic acid-2'-phenylaniide phosphate (HNPP) fluorescent detection system (Boehringer Mannheim) was used. Briefly, slides were rinsed in detection buffer (0.1 mol/L Tris base, 0.1 mol/L NaCl, 0.01 mol/L MgCl2) and then 200 µl of HNPP/Fast Red TR (10 µg of HNPP in 1 ml of detection buffer plus 10 µl of Fast Red TR solution) was applied. This solution was filtered through a 0.2-µm nylon filter immediately before use, and then the slides were coverslipped and incubated for 30 minutes at room temperature in the dark.
Immunophenotype of Infected Cells
To define the immunophenotype of infected cells we performed combined in situ hybridization/immunohistochemistry as described previously.13-15,31
After in situ hybridization for viral nucleic acid as described above, single- or double-label immunohistochemistry or immunofluorescence was performed using a variety macrophage- and T lymphocyte-specific markers (Table 2)
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To more rigorously examine the cell types infected we performed additional multilabel techniques combining in situ hybridization with immunohistochemistry or immunofluorescence for two cell-type-specific markers as described previously14,31
using combinations of antibodies as shown in Table 3
. For these multilabel techniques, in situ hybridization was performed as described above except that the results of in situ hybridization were visualized with HNPP/Fast Red, which fluoresces intensely red. In addition to CD68, HAM56, and CD3 we used several additional T cell- and macrophage-specific antibodies (Tables 2 and 3)
. The cell type-specific antibodies of differing isotypes or species origin were applied sequentially followed by isotype-specific anti-mouse or species-specific secondary antibodies applied simultaneously. The secondary antibodies were coupled to either Alexa 488 (green) or Alexa 633 (far red) (Molecular Probes, Eugene, OR) as shown in Table 3
. After antibody treatment, sections were washed twice for 15 minutes in PBS with 0.2% fish skin gelatin. Finally, the sections were rinsed in doubly distilled H2O and mounted with aqueous mounting medium.
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Confocal microscopy was performed using a Leica TCS SP2 confocal microscope equipped with three lasers (Leica Microsystems, Exton, PA). Individual optical slices represent 0.2 µm and 32 to 62 optical slices were collected at 512 x 512 pixel resolution. NIH Image (version 1.62) and Adobe Photoshop (version 7.0) were used to assign colors to the four channels collected: HNPP/Fast Red substrate that fluoresces red when exposed to a 568-nm wavelength laser appears red, Alexa 488 (Molecular Probes) is green, Alexa 633 (Molecular Probes) appears blue, and the differential interference contrast (DIC) image is gray scale. The four channels were collected simultaneously. In some tissues and to differentiate between individual cells, To-pro3 (nuclear marker, Molecular Probes) was used at 1 µg/ml, incubated for 5 minutes, and tissues were washed in PBS. Co-localization of antigens is demonstrated by the addition of colors as indicated in the figure legends.
Quantitation of SIV-Infected Cells in Situ
Both semiquantitative and quantitative means were used to quantify the total number of infected cells and the number of infected macrophages. For subjective quantitation (Table 4)
sections were scored from negative (–) to ++++ as follows: absence of positive cells per section, –; 1 to 10 positive cells per x10 field, +; 10 to 30 positive cells per x10 field, ++; 30 to 100 positive cells per x10 field, +++; greater than 100 positive cells per x10 field, ++++.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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In acute infection the principal lesions in macaques infected with SIVmac239/316 were in the lymphoid tissues with follicular hyperplasia and dysplasia the most common finding (Figure 1)
. In long-term SIVmac239/316-infected macaques, five of seven animals developed AIDS at different times after SIV inoculation (Table 1)
. These animals had generalized lymphoid depletion and follicular involution characterized by enlarged hypocellular germinal centers containing amorphous eosinophilic material without discernible mantle zones (Figure 1, C and E)
. These changes are consistent with pathogenic SIV infection and AIDS.3-5,11,28
In addition, several chronically infected animals had extensive myocardial fibrosis, arterial thrombi, and arteriopathy involving small- to medium-sized vessels in heart, lung, brain, and kidney (Figure 1; B, D, and F)
. These vascular lesions were previously described as unique to macaques infected with SIV.11,39
Three of the five animals that developed AIDS also had a variety of opportunistic infections including SV40, adenovirus, lymphocryptovirus, Pneumocystis carinii, and Mycobacterium avium complex (Table 1)
. With the exception of inflammation associated with these opportunistic infections, SIVmac239/316-infected animals did not have typical primary SIV-associated inflammatory lesions such as giant cell pneumonia and SIVE.
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Viral loads in the animals infected with SIVmac239/316 have previously been reported32
as have viral loads in numerous animals infected with SIVmac239.32,33,40,41
Consistent with in vitro data17,42
early replication of SIVmac239/316 is similar to that of the parental SIVmac239 from which it was derived (Table 4)
. Viral loads at peak height (week 2) and at set point (weeks 20 to 50) were averaged and compared between 6 of the animals infected with SIVmac239/316 for which such data were available and 10 animals infected with SIVmac239 (Table 5)
. Although peak viral loads were similar, SIVmac239/316 infection is rapidly controlled with much lower viral loads at set point. Consistent with this observation, animals infected with SIVmac239/316 tend to have a prolonged survival32
but ultimately progress to AIDS as indicated in Table 1
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Localization of SIV-infected cells in tissues was examined by in situ hybridization using both riboprobes and random-primed DNA probes to detect viral RNA and/or DNA. Both techniques gave similar results, which are summarized in Table 4
. In animals infected with either SIVmac239 or SIVmac239/316, lymphoid tissues (lymph nodes, spleen, and gut-associated lymphoid tissue) harbored the majority of infected cells. In SIVmac239/316-infected animals, chronically infected animals generally had fewer SIV-positive cells compared to animals in the acute phase of infection consistent with viral load data (Tables 4 and 5)
. In acutely infected animals the pattern of viral infection was similar to what has been described in animals infected with the parental molecular clone SIVmac239.15,28,43
In addition, by 21 days after infection, a diffuse staining pattern was noted over germinal centers consistent with trapping of viral particles (antigen/antibody complexes) on follicular dendritic cells (Figure 2A)
. Although there were marked similarities between the tissue distribution of SIVmac239/316 and SIVmac239, animals infected with SIVmac239/316 had relatively less virus in tissues by in situ hybridization at later time points consistent with lower viral loads in these animals (Tables 4 and 5
and reference 32
). In addition, little if any infection of the central nervous system was detected (Table 4)
and the morphology of infected cells was more consistent with lymphocytes than macrophages (Figure 2)
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To examine the immunophenotype of infected cells we performed combined in situ hybridization for SIV followed by immunohistochemistry for macrophage or T-lymphocyte markers individually and in combination (Tables 2 and 3)
. Initially we performed routine in situ hybridization for SIV followed by immunohistochemistry for CD68 (a macrophage marker) in acute and long-term SIVmac239/316-infected animals using different chromogenic substrates. This technique revealed few if any SIV-infected macrophages (Figure 2C)
. To more rigorously examine the immunophenotype of the infected cells and avoid potential problems with color mixing when using chromogenic substrates we performed fluorescent in situ hybridization for SIV followed by immunofluorescence for macrophage- and T cell-specific markers individually and in combination (Table 3)
. Tissues examined included spleen, lymph node, and small and large intestine from animals infected with SIVmac239/316 and SIVmac239.
In animals infected with SIVmac239/316 the majority of the infected cells were T cells (positive for CD3, CD5, CD7) (Figure 3)
. In similar experiments with macrophage markers (CD68, HAM56, MAC387, and MRP8) we were able to find only a single SIV-infected macrophage in one tissue from one of the animals infected with SIVmac239/316. This lone infected macrophage was present in the subcapsular sinus of the axillary lymph node (Figure 3C)
. Thus macrophage infection in vivo by SIV mac239/316 is very rare. In contrast, in acute and long-term SIVmac239 infection many SIV-infected macrophages were found in lymph node, spleen, lamina propria of small and large intestine, and Peyer’s patches as early as 21 days after infection (Figure 4; A to C)
consistent with previous published reports.13-15
Giant cell pneumonia, enteritis, and SIVE with SIV infection of macrophages and multinucleated giant cells was a common finding in terminal SIVmac239 infection (Figures 4 and 5)
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Although macrophage infection by SIVmac239/316 was rare, the total number of infected cells/mm2 in these animals was less than in SIVmac239-infected animals (Figure 6)
. Therefore we also quantitated the number of infected macrophages in animals infected with each virus. As can be seen in Figure 6
the number of infected macrophages in lymph node and spleen of animals infected with SIVmac239/316 is much lower than in animals infected with SIVmac239 (ninefold less at the closest point, 50 days after infection) and cannot be explained simply on the basis of differences in the frequency of infected cells.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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The explanation for this observation is not immediately obvious but is likely related to the fact that these animals, as opposed to animals from which macrophage tropic viruses have been derived, were not also infected with virulent T-cell tropic viruses such as SIVmac239 from which they evolved. It is important to remember that the changes in SIVmac239/316 envelope occurred naturally and that uncloned SIVmac316 (from which the env in SIVmac239/316 was obtained) was isolated from lung macrophages in an animal infected with SIVmac239. This animal had AIDS and wide spread infection of macrophages manifest as SIVE and giant cell pneumonia.16,17 The development of macrophage tropism is also associated with a decreased dependence on CD4. This likely reflects an evolutionary process to adapt to infection of macrophages, some of which express little CD4.19 An additional consequence of the amino acid changes that confer macrophage tropism is that the virus is rendered much more susceptible to antibody neutralization.23,25,32,47,48 This is reflected in the longer survival and lower viral set points in animals infected with SIVmac239/31632 as compared to SIVmac239 but similar peak viral loads. This suggests that macrophage tropic viruses such as SIVmac239/316 are relatively easy to control in the absence of a more virulent virus such as SIVmac239 and that viral replication may be insufficient to cause severe giant cell disease. In this scenario, macrophage tropic viruses such as SIVmac239/316 are conceptually opportunists that require destruction of the immune system to have a significant impact.
Although SIVmac239/316 may indeed be easier to control than SIVmac239 this does not explain why infection of monocyte/macrophages was essentially nonexistent. Based on in vitro data, robust infection of monocyte/macrophage lineage cells would have been expected at least during the peak of viremia, 7 to 14 days after infection. Although it is possible that SIVmac239/316 may evolve away from macrophage tropism to escape neutralizing antibodies25
this would not be an issue during the early time points examined. We initially considered that perhaps the infection was altering expression of selected macrophage or T cell-specific molecules used in immunohistochemistry but after examining a series of macrophage and T-cell-specific markers (Table 2)
, two different in situ hybridization probes and performing multilabel confocal microscopy we are confident that the in vivo lack of macrophage infection is real. This leads us to believe that the conditions under which macrophage infection has been examined in vitro are not reflective of macrophages in vivo. From work by Becher and Antel50
and Williams and colleagues49
it has been shown that tissue macrophages such as brain microglia rapidly change their phenotype within hours of isolation from tissue becoming more activated with up regulation of a variety of immunologically important molecules such as MHCII, CD4, and so forth. This leads us to speculate that the immune dysfunction caused by a virulent virus such as SIVmac239 manifests as increased cytokine production,29,51,52
up-regulation of adhesion molecules, and may cause activation of tissue macrophages making them more like macrophages in vitro and susceptible to infection by SIVmac239/316. Although SIV rarely uses co-receptors other than CCR5 is it possible that altered co-receptor usage could contribute to macrophage tropism and differences in pathogenesis. In studies by Puffer and colleagues23
using envs from SIVmac239, SIVmac239/316 and other macrophage tropic viruses no evidence that co-receptor choice was responsible for altered tropisms was found: all used CCR5.
In contrast to the unexpected in vivo cell tropism of SIVmac239/316, the tropism of SIVmac239 for T cells and macrophages was in agreement with previously published work.11,14,15,53,54 It is worth noting however that infection of macrophages by SIVmac239 has been consistently observed in vivo by 21 days after infection, before any evidence of SIVE or other pathology associated with macrophage infection. This has been presumed to reflect the evolution of variant strains of SIVmac239 that had acquired SIVmac239/316-like changes in envelope and the ability to replicate efficiently in macrophages. However, it is possible that as was proposed above for SIVmac239/316, the in vitro culture conditions used to define the inability of SIVmac239 to replicate in macrophages do not adequately reflect in vivo conditions. In support of this, using microdissection techniques it has been recently shown that SIVmac239 env RNA is present in individual SIVE giant cells although this was interpreted as reactivation of latent infection of microglia by fusion into multinucleated giant cells.30 Nevertheless, the in vivo data examining cell tropism of SIV suggests that further study examining the timing of acquisition of macrophage-tropism in vivo and how, or if, those changes affect disease pathogenesis is warranted.
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Acknowledgements |
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Footnotes |
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Supported in part by the Public Health Service (grants RR00164, RR00168, NS30769, MH61192).
Accepted for publication September 2, 2004.
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