The breakdown of the cytokine network subsequent to human immunodeficiency virus infection

The acquired immunodeflciency syndrome (AIDS) is a clinically multifaceted disease induced by infection with the human immunodeficiency virus (HIV). HIV infection results in a complex pattern of immunologic alterations that leads to the development of AIDS in the majority of HIV seropositive (HIV+) individuals. The reduction in CD4 T lymphocyte counts is the hallmark of HIV infection; nevertheless, long before the reduction in CD4 counts reaches critical levels, a series of profound and complex defects that impair the function of CD4 T lymphocytes can be detected. Thus, HIV infection is characterized by quantitative and qualitative defects affecting CD4 T lymphocytes. It was suggested recently that programmed cell death (PCD) is an important mechanism leading to CD4 depletion in HIV infection, and that susceptibility of peripheral lymphocytes to PCD is differentially regulated by diverse cytokines. Thus, type 1 cytokines would protect CD4 lymphocytes against PCD, whereas type 2 cytokines would not protect against, and could augment, PCD. We suggest that the qualitative alterations of the immune response provoke the CD4 depletion characteristic of HIV disease via type 2 cytokinemediated augmentation of PCD, and are therefore ultimately responsible for the progression of HIV infection. Finally, we summarize recent data showing that three correlates of disease progression: emergence of HIV strains with syncitium-inducing ability (SI), type 1-to-type 2 cytokine shift, and CD4 depletion, are significantly associated, suggesting a complex interconnected virologic-immunologic pathogenesis of HIV infection.

The breakdown of the cytokine network subsequent to human immunodeficiency virus infection M. Clerici Defective interleukin-2 production as the hallmark of qualitative lymphocyte defects in HIV infection Early and complex T helper (Th) cells defects have been described in HW infected individuals. [1][2][3][4][5][6] We have analysed the qualitative defects of HW infection by examining in vitro antigenand mitogen-stimulated interleukin (IL)-2 production by PBMC of H1V+ individuals. PBMC were stimulated in vitro with recall antigens (influenza virus, tetanus toxoid, or HIV peptides); HLA alloantigens (ALLO); or phytohaemagglutinin (PHA). These stimuli were chosen because they activate different Th-antigen presenting cell (APC) pathways, 7 allowing for a more complete evaluation of the immune system. Thus, recall antigens are exclusively presented by self-APC to autologous CD4; HLA alloantigens can be presented by self-APC to autologous CD4 or they can be directly recognized by autologous CD4 and CD8 T lymphocytes on the surface of allogeneic APC; whereas PHA stimulation of T lymphocytes is only marginally dependent on processation and presentation by APC (see Table  1). 7 The stimulation of PBMC with this panel of antigens allowed us to recognize that only a minority (40%) of HIV+ asymptomatic patients can respond by IL-2 production to recall, ALLO and PHA. 5 Thus, a complex pattern of defects in IL-2 production was observed in the majority of HIV+, asymptomatic individuals. Indeed, approximately 40% of these individuals showed a selective defect in IL-2 production in response to recall antigens, whereas 10% of them could only respond to PHA, or could not produce IL-2 in response to any stimulation ( 10%). 5 Because all these patients were in the same clinical stage (Walter-Reed stage 1), and  [4][5][6][7] These two sub-populaobserved functional defects were not secondary tions, named T helper 1 (TH1) and T helper 2 to a decrease in CD4 counts. 5 These experi-(TH2), respectively, were subsequently described ments allowed us to conclude that: (1) defective in humans by Romagnani. 8'9 It was suggested ability to produce IL-2 can be detected even in recently that some phenotypic markers may allow the earlier, asymptomatic phases of H1V infecus to differentiate TH1 and TH2 lymphocytes. tion; and (2) these defects are not secondary to Thus, it was reported that TH2 lymphocytes may either a decrease in CD4 counts or a clinical express higher quantities of CD30,iOas well as of progression in H1V infection. Similar analyses in a particular isoform of BB-7 (BB-7.2). A third vertically transmitted paediatric H1V infection group has observed that CD4+CD7+ T lymshowed that: (1) defective IL-2 production is phocytes preferentially secrete TH1 cytokines, observed also in the majority of H1V+ paedi-whereas CD4+CD7--T lymphocytes preatric patients; 8 and (2) defective in vitro ferentially produce TH2 cytokines. Nevertheless, antigen-and mitogen-stimulated IL-2 producno marker has yet been identified that exclusively tion is associated in paediatric HIV disease with clusters on TH1 or TH2, and the definition of an augmented incidence of opportunistic and TH1 and TH2 lymphocytes is still essentially bacterial infections. 8 In the attempt to analyse based on the different cytokines that are prowhether the defective element is the T lymphoduced by these cells. Thus, TH1 responses are cyte or the antigen presenting cell, we performed characterized by secre{ion of interferon (IFN)q, experiments in which cells of HIV-discordant and IL-2 and subsequent promotion of cell medimonozygotic twins were matched. The results ated immunity (CMI), whereas TH2 responses repeatedly indicated the absence of defects in the are characterized by secretion of IL-4 and IL-5, ability of H1V+ APC to process and present with subsequent activation of humoral immunity antigens. 9'm and generation of antibodies. Interestingly, TH1 We subsequently verified whether defects in and TH2 are cross-regulating as IFN-, suppresses IL-2 production could be reverted by treatment the activation of TH2 lymphocytes, whereas IL-4 with antiretroviral drugs. Thus, we measured in suppresses the production of TH1 cytokines. vitro stimulated T lymphocyte proliferation and IL-2 is a prototypical product of TH1 lympho-IL-2 production in two cohorts of HIV+ adults cytes, and IL-2 production can be down-regulated and one cohort of paediatric patients, treated by TH2 cytokines (classically IL-4). Thus, we with zidovudine, sCD4-IgG or ddI respectively, began exploring the possibility that the defective We observed that all three compounds were IL-2 production observed in HIV+ asymptomatic capable of temporarily restoring IL-2 producinfection could be accompanied by augmented tion independently of any variation in CD4 IL-4 production. We decided to analyse cytokine counts. -That this is not an aspecific effect of production by whole PBMC and not by CD4 + T all antiretroviral drags was shown by a fourth cell clones because we believe cytokine produccompound, which was not able to restore T tion by whole PBMC to be a more important 22 231. lymphocyte proliferation or IL-2 production (M. approximation to the in vivo situation. In fact, Clerici et al., unpublished observations). Addieven if T cell clones are an important experitionally, the in vitro restoration of IL-2 producmental tool, we were concerned about the artetion by antigen-or mitogen-stimulated peripheral facts present in the cloning methodology. Our blood mononuclear cells provoked by ddI was concerns about clones can be summarised as associated in paediatric HIV infection with a follows: (1) Th cell cloning results in the loss significant reduction of o2POrtunistic infections (during the cloning process) of important, non-T during clinical follow-up. Again, the positive accessory cells that produce cytokines with changes in clinical parameters was associated immunoregulatory properties, (2) cloning may The breakdown of the ytokine network subset of HIV+ asymptomatic patients showing defective IL-2 generation in response to recall antigens. 24 Similarly, IL-4 mRNA was detected in PHA stimulated PBMC of HIV+ individuals, but not in unstimulated PBMC of the same individuals, or in HIV--controls. 24 Even more important, IL-4 neutralizing antibodies were capable to restore in vitro antigen-stimulated proliferation and IL-2 production in the majority of patients secreting high amounts of IL-4. 24 Because IL-10 was described as able to suppress the secretion of type 1 cytokines and CMI even more powerfully than IL-4, we next decided to measure IL-10 production in HIV+ asymptomatic patients. Similarly to the methods used to select for populations of T cells, as the technigenerate IL-4, we stimulated PBMC in vitro with ques involve the selection and expansion of cells PHA for 48 h; the amount of IL-10 produced was in relatively high concentrations of IL-2; and (3) measured using commercially available ELISA kits, the investigation of PBMC tests for the effects of and IL-10 mRNA was quantified using PCR cytokines produced by multiple cell types on Th methods. We observed that IL-10 production was cell function, and includes both autocrine and augmented in H1V+ asymptomatic patients paracrine regulation whereas in cloned T cell showing defective IL-2 production, and that the tests for the cytokines produced by these clones patients with the most severe defective IL-2 prois limited to autocrine regulation. Because our duction (i.e. inability to generate IL-2 even in obseeeations were based on cytokine production response to PHA) generated the highest quanby all the cells circulating in the peripheral blood tities of IL-10. 25 By analogy to what was obseeeed and not on clonal isolation, we decided to for neutralizing antibodies to IL-4, in vitro identi the cytokine patterns obseeeed as type 1 antigen-stimulated proliferation and IL-2 produc-(mainly CMI-inducing), and type 2 (mainly tion could be restored in the majority of these humoral immunity-inducing). 22'2-3-Because we patients by neutralizing antibodies to IL-10. 25 defined cytokines on a functional basis, we could Similarly, we could restore in vitro antigen-stimuenlarge the group of type 1 cytokines to include lated proliferation and IL-2 production, as well as It-12 and, probably, IL-15 (all cytokines that mitogen-stimulated IFNq, production, by IL-12, 26 mainly stimulate CMI, but are not mainly or a prototypical type-1 cytokine described by exclusively produced by clones of CD4 + T lym-Chehimi et al. as defective, even in the earlier phocytes) in addition to IL-2 and IFNq,. Similarly, phases of HIV infection, iv Thus, we proposed IL-6, IL-10 and probably IL-13 were considered to that HIV infection is associated with the probe type 2 cytokines even if they are produced by gressive impairment of type 1 cytokine produccells other than CD4 + T lymphocytes, as their tion, and the progressive augmentation of type 2 main effect is that of stimulating B cell activity cytokine secretion. 22'23 It is important to underand antibody generation (see Table 2).
line that these events are observed in the asymp-To measure IL-4, we stimulated PBMC of tomatic phase of HIV infection, before the HIV+ asymptomatic donors in vitro with PHA development of full blown disease. These data, for 48 to 72h, as suggested by kinetic studies, confirmed by numerous other authors, 28-37 are and we evaluated the amount of IL-4 present in the experimental basis of the type-I/type-2 the supernatants using a cell line (a kind gift of hypothesis of HIV infection. Dr William Paul, NIAID, NIH) whose growth is Because cytokine secretion is associated with a dependent on IL-4. (To simplify the assay, we are quantity of diverse biological effects which are now measuring IL-4 using commercially available readily measurable, we suggest that the list of ELISA kits.) To analyse the production of IL-4 at these effects, which are frequently observed in the molecular level, we also quantified (in colla-HIV+ patients and are presented in Table 3, boration with Dr Thomas Wynn, NIAID, NIH) the strongly support the hypothesis of a profound expression of IL-4 mRNA in the PBMC of the cytokine imbalance in HIV infection. Thus, the same patients. We observed that IL-4 production impairment of delayed type hypersensitivity reacwas greatly augmented in PHA-stimulated supertion is secondary to defective IFN-,, IL-2 and ILnatants of HIV+ individuals as compared to 12 production, whereas hyper-IgE is secondary to HIV-controls, and that the increased IL-4 prothe augmented production of IL-4, and hyperduction was most likely to be observed in the eosinophilia is secondary to the augmented pro- Table 3. Unfavourable prognostic signs in HIV infection Decreased production of type cytokines (IFN-7, IL-2, IL-12) Increased production of type 2 cytokines (IL-4, IL-5, IL-6, IL-10) Reduced delayed type hypersensitivity (DTH) reactions IL-4-driven hyper-lgE IL-5-driven hypereosinophilia duction of IL-5. All these parameters are oredictors of poor prognosis in HIV infection. 3"8-46 Interestingly, it has recently become evident that defective in vitro antigen-and mitogen-stimulated IL-2 production is predictive for subsequent reduction in CD4 counts, the time to acquired immunodeficiency syndrome (AIDS), and time to death. 47

Different patterns of disease progression in HIV infection
Higher CD4 counts, better preserved IL-2 secretion, and the absence of the clinical signs of type 1/type 2 cytokine imbalance listed in Table   3 are associated with a lack of progression of HIV infection. It has recently become evident that, although the vast majority of HIV+ individuals progress to AIDS, a minority of HIV+ patients exists that does not develop AIDs or show a critical reduction in CD4 T cells, despite long-lasting infection with HIV. 48 These patients have recently been defined long-term non-progressors (LTNP). Different groups have focused on diverse aspects of this phenomenon, the solution of which holds the possible key to a cure for AIDS. Two biological interpretations can be offered in the attempt to explain long-lasting HIV infection without AIDS. Thus, these patients may have been infected by a defective, less pathogenic HIV variant, or these patients may have a stronger immune response, capable of keeping HIV under control. More likely, a strong immune response is preventing HIV from becoming frankly virulent in these individuals. In support of this hypothesis are the findings by Ho and colleagues, and Fauci and colleagues indicating that strong H1V-specific CTL were observed in two groups of LTNP. 49'5 Additionally, it was recently shown that: (1) preserved T cell function correlates with stable CD4 counts; 51 (2) disease-free survival correlates with gag-specific cytotoxic T lymphocyte activity; 52'53 and (3) HIV infected chimpanzees become seropositive but do not progress toward AIDS. 54 Finally, it has recently been verified that neither a type 1-to-type 2 cytokine shift nor programmed cell death are present 55 56 in HW+ chimpanzees.
We have recently analysed (in collaboration with the Clinica delle Malattie Infettive, Universitt di Milano) cytokine production in a group of LTNP, and compared the cytokine profile with that observed in a group of patients with progressive HIV infection. As we expected, a strong type 1/weak type 2 cytokine profile was observed in the LTNP, whereas a symmetrically opposite weak type 1/strong type 2 cytokine profile was observed in patients with progressive HW infection. 57 Interestingly, a significantly increased percentage of CD4+/CD7--T lymphocytes (as remembered above, CD4 +/CD7--T lymphocytes predominantly secrete type 2 cytokines) was observed in the individuals with progressive HIV infection, but not in the LTNP. 57 We have performed (in collaboration with the Cattedra di Pediatria IV, Universitt di Milano)a second study in a cohort of vertically infected children with different patterns of disease progression. In fact, HIV vertical infection follows a bimodal pattern according to which 20% of vertically infected children develop MDS and eventually die within the first year of life, whereas the remainder develop MDS at a constant rate per year, reaching the median at about 5 years after birth. 58 We have thus studied a group of vertically infected HW+ children who did not develop AIDS within the first year of life, and we identified two subsets of children of comparable age, the first one of which was asymptomatic, while the second one showed severe signs of H1V disease. The results indicated that, despite the presence of severe defects in type 1 cytokine production in both groups of children, a greatly increased type 2 cytokine secretion was characteristic of the symptomatic, but not of the 43 t n asymptomatic children.
In eresti gly, a significant association with hyper-IgE (the production of which is stimulated by IL-4 secretion) was observed in the paediatric cohort. 43 Thus, we suggest that a strong type 1/weak type 2 cytokine production is associated with delayed (or absent) progression of H1V infection to MDS.
Susceptibility of T lymphocytes to programmed cell death (PCD)is differentially regulated by type 1 and type 2 cytokines It has been observed that PCD is increased in HIV infection, and it was thus suggested that PCD could be one of the mechanism(s) primarily responsible for CD4 depletion in the progression tO AIDS. 59'6 HIV + lymphocytes undergo PCD in unstimulated conditions, and much more so 6O 61 when stimulated with mitogens.
One of the major differences between H1V+ and H1V--lymtropism for different cell lines, and the capacity phocytes is that whereas only mitogen-activated to induce the formation of syncytia in vitro.  HIVlymphocytes (blasts) will undergo PCD To verify whether the isolation of syncytiumupon a second mitogenic restimulation, even inducing H1V is correlated with the type 1-to-type resting (unstimulated) HIV + PBMC will undergo 2 cytokine shift, we have analysed virologic and PCD upon stimulation. Therefore, two serial in immunologic parameters in two groups of HIV vitro stimuli are needed to induce PCD in HIV-vertically infected children of comparable age lymphocytes, whereas a single in vitro stimulation who have or have not progressed to AIDS. We will provoke PCD in HIV+ lymphocytes, suggest-observed that progression to AIDS in paediatric ing that in H1V infection lymphocytes are pre-H1V infection is associated with isolation of HIV activated in vivo to undergo PCD upon in vitro SI variants and increased production of the type restimulation. ' 2 cytokines IL-4 and IL-10. Additionally, we Mitogenic stimulation will activate CD4 and observed that these two parameters are statisti-CD8 lymphocytes via T cell receptor-mediated cally associated and that extensive CD4 loss is stimulation, inducing PCD in both subsets of T associated both with the isolation of SI variants lymphocytes. It was recently obseeeed that PCD and increased IL-4 production (M. Clerici et al., is differentially regulated by type 1 and type 2 submitted). These recent data indicate that the cytokines. Thus, PCD was prevented in vitro by virologic and immunologic parameters character-IFN-7, IL-2 and IL-12 whereas PCD was not preistic of advanced H1V infection are strictly assovented, or was augmented, by IL-4 and IL- 10. ciated, and strongly support a virologic-Even more relevant was the observation that PCD immunologic pathogenesis leading the appearcould be prevented by neutralizing antibodies to ance of AIDS. type 2 cytokines, but could not be prevented, or was even raised by neutralization of type 1 cytokines. 2 We have recently obseeeed that the Cnluin selective stimulation of CD4+ lymphocytes by recall antigens will induce PCD exclusively in the We suggest that the complex qualitative altera-CD4 subset, a situation that more closely resemtions observed in HW infection are responsible bles that observed in viva Even in this situation, for the progression of HIV disease to AIDS, and PCD was oppositely modulated by type 1 and that the dramatic reduction of CD4 T lymphotype 2 cytokines, or by the neutralization of type cytes which is the hallmark of this disease is 1 or type 2 cytokines. Additionally, we verified mainly secondary to phenomena of PCD that is that PCD is effected by lymphotoxin, and that increased by a type 1/type 2 cytokine imbalance. lymphotoxin is responsible for a soluble-factor Thus, we suggest that every therapeutic mediated amplifying loop which causes PCD in approach to HIV infection should consider the innocent-bystander lymphocytes. Finally, the necessity to restore the normal functionality of PCD-inducing effect of lymphotoxin is differen-the immune system. These approaches could at tially influenced by type 1 and type 2 cytokines least theoretically be based on the utilization of: (m. Clerici et al., submitted). We suggest that the (1) type 1 cytokines; (2) antibodies neutralizing impaired production of type 1 cytokines and the type 2 cytokines; or (3) pharmacological comaugmented generation of type 2 cytokines charpounds aimed at the selective stimulation of acteristic of H1V infection results in the destructype 1 cytokine secretion and subsequent augtion of CD4 lymphocytes, which is increased by mentation of CMI. It is important to notice that type 2 cytokines and mediated by lymphotoxin, the results of a first clinical trial based on the Thus, antigen stimulation of H1V+ lymphocytes utilization of IL-2 have shown significant in the presence of abnormally low concentraimprovement in CD4 counts (possibly via the tions of IFN-7, IL-2 and IL-12, and of abnormally prevention of IL-2-induced PCD). Thus, we elevated concentrations of IL-4 and IL-10 results strongly favour approaches based on the in the induction of PCD, instead of the induction restoration of normal cytokine production in of T cell proliferation, the therapy for HIV infection, as we believe that a strong CMI is associated with better prognosis, and will ultimately be more capable of Virologic and immunologic correlates of controlling HIV replication and disease progrespoor prognosis are associated sion.
Finally, it was suggested that the development ACKNOWLEDGEMENTS. This work was supported by of AIDS is secondary to the emergence of an HIV grants Istituto Superiore di Sanirk n. 9204-31 and phenotype with a rapid/high replication rate, a 9304-40.