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In this paper we demonstrate that the cells which initiate replication of Epstein-Barr virus (EBV) in the tonsils of healthy carriers are plasma cells (CD38 hi , CD10 − , CD19 + , CD20 lo , surface immunoglobulin negative, and cytoplasmic immunoglobulin positive). We further conclude that differentiation into plasma cells, and not the signals that induce differentiation, initiates viral replication. This was confirmed by in vitro studies showing that the promoter for BZLF1, the gene that begins viral replication, becomes active only after memory cells differentiate into plasma cells and is also active in plasma cell lines. This differs from the reactivation of BZLF1 in vitro, which occurs acutely and is associated with apoptosis and not with differentiation. We suggest that differentiation and acute stress represent two distinct pathways of EBV reactivation in vivo. The fraction of cells replicating the virus decreases as the cells progress through the lytic cycle such that only a tiny fraction actually release infectious virus. This may reflect abortive replication or elimination of cells by the cellular immune response. Consistent with the later conclusion, the cells did not down regulate major histocompatibility complex class I molecules, suggesting that this is not an immune evasion tactic used by EBV and that the cells remain vulnerable to cytotoxic-T-lymphocyte attack.

We have used latent infection with the human herpesvirus Epstein-Barr virus to track the dispersal of memory B cells from the mucosal lymphoid tissue of Waldeyer's ring (tonsils/adenoids). EBV is evenly distributed between the memory compartments of Waldeyer's ring and the peripheral blood. However, it has an approximately 20-fold higher preference for Waldeyer's ring over the spleen or mesenteric lymph nodes. These observations are consistent with a model whereby the virus preferentially establishes persistent infection within memory B cells from Waldeyer's ring. The virus then colonizes the entire peripheral lymphoid system, at a low level, by trafficking with these memory B cells as they circulate through the body and back to Waldeyer's ring. This pathway may reflect that of normal memory B cells derived from nasopharyngeal and other mucosal lymph nodes.

An effective host defense against lung bacterial infection is dependent primarily upon the rapid clearance of the organism from the respiratory tract, which is mediated by the influx and/or activation of phagocytic cells, including neutrophils (polymorphonuclear leukocytes [PMN]) and macrophages (27). The recruitment and activation of leukocytes in the setting of bacterial challenge is a complex and dynamic process which involves the coordinated expression of both pro- and anti-inflammatory cytokines (10–12, 14, 15). Tumor necrosis factor alpha (TNF) is a 17-kDa cytokine which is a critical component of an effective antibacterial host defense (2, 9, 13, 16). Specifically, TNF is a potent activator of both PMN and macrophages, leading to enhancement of protease release, stimulation of the respiratory burst, and induction of leukocyte and vascular adhesion molecule expression, which are essential for transmigration of these cells into sites of infection (7, 19, 24). Moreover, PMN and macrophage microbicidal activity is augmented by endogenous or exogenous TNF (22, 26). Finally, TNF is expressed in increased amounts in the airspaces of humans with bacterial pneumonia (21) and in the lungs of mice challenged with bacterial pathogens (9, 18). The inhibition of TNF has been shown to impair lung bacterial clearance and in some but not all studies to attenuate lung PMN influx in response to aerosolized or intratracheally (i.t.) administered Pseudomonas aeruginosa and Klebsiella pneumoniae (2, 9, 15, 18), resulting in markedly decreased survival in animals with bacterial pneumonia (18). However, TNF can mimic many of the detrimental pathophysiologic events that occur in sepsis and sepsis syndrome (1, 28), making it difficult to obtain the beneficial therapeutic effects of TNF without its cytotoxic consequences. Recently, a TNF agonist peptide composed of the 11 amino acids that constitute the site of binding of native human TNF to its receptors (referred to as TNF70-80) has been characterized (17, 23). Binding of TNF70-80 to TNF receptors (both p55 and p75) has been shown to mediate many leukocyte-activating effects of native TNF. Specifically, this peptide directly stimulates and primes neutrophils for enhanced protease release and respiratory burst, enhances neutrophil phagocytic activity, and augments neutrophil killing of Plasmodium falciparum in vitro and clearance of Plasmodium chabaudi in mice in vivo (17). In that study, TNF70-80 was associated with minimal toxicity when administered systemically, due in part to the fact that this peptide did not alter adhesive properties of the endothelium (17). The emergence of multidrug-resistant microbes in the immunocompromised host has made the treatment of bacterial infections of the lung increasingly difficult (3, 4, 6, 20, 25), underscoring the importance of the immune host defense in determining the eventual outcome of severe pneumonia. Given these clinical dilemmas, we performed this study to determine if the intrapulmonary and/or systemic administration of TNF70-80 was capable of augmenting host innate immunity in the setting of murine gram-negative pneumonia.

Tumor necrosis factor (TNF) is a proinflammatory cytokine which has recently been shown to have beneficial effects in the setting of acquired host immunity. However, the role of TNF in innate immune responses, as in the setting of bacterial pneumonia, has been incompletely characterized. To determine the role of TNF in gram-negative bacterial pneumonia, CBA/J mice were challenged with 10(2) CFU of Klebsiella pneumoniae intratracheally, resulting in the time-dependent expression of TNF MRNA and protein within the lung. Passive immunization of animals with a soluble TNF receptor-immunoglobulin (Ig) construct (sTNFR:Fc) intraperitoneally 2 h prior to K. pneumoniae inoculation resulted in a significant reduction in bronchoalveolar lavage neutrophils, but not macrophages, at 48 h, as compared with animals receiving control IgG1. Furthermore, treatment with sTNFR:Fc resulted in 19.6- and 13.5-fold increases in K. pneumoniae CFU in lung homogenates and plasma, respectively, as compared with animals receiving control IgG1. Finally, treatment of Klebsiella-infected mice with sTNFR:Fc markedly decreased both short- and long-term survival of these animals. In conclusion, our studies indicate that endogenous TNF is a critical component of antibacterial host defense in murine Klebsiella pneumonia.

Effective host defense against bacterial invasion is characterized by the vigorous recruitment and activation of inflammatory cells, which is dependent upon the coordinated expression of both pro- and anti-inflammatory cytokines. Interleukin-10 (IL-10) is a recently described cytokine with potent anti-inflammatory properties in vivo and in vitro. In this study we investigated whether IL-10 could directly regulate the ability of neutrophils (PMN) to phagocytose and kill bacteria. Initial studies demonstrated that human recombinant IL-10 (hrIL-10) inhibited the ability of PMN to phagocytose Escherichia coli in vitro. Inhibition of phagocytosis occurred in the absence of changes in CR1 (C3b) or Fc receptor expression, as treatment of PMN with IL-10 failed to induce significant changes in Fc gamma IIR, Fc gamma IIIR or CR1 cell surface expression. However, incubation of PMN with IL-10 resulted in a dose-dependent decrease in CDIIb (Mac-1) expression. In addition to effects on PMN phagocytosis, hrIL-10 significantly attenuated PMN microbicidal activity, as bactericidal assays revealed that co-incubation of PMN with hrIL-10 resulted in a marked decrease in killing of phagocytosed bacteria. Furthermore, IL-10 inhibited the production of superoxide from PMA-stimulated PMN, suggesting that the detrimental effects of IL-10 on PMN microbicidal activity were due, in part, to suppression of respiratory burst. In summary, our studies indicate that IL-10 inhibits PMN-dependent phagocytosis and killing of E. coli in vitro, and suggest that this cytokine may impair effective antibacterial host defense in vivo.

Effective host defense against bacterial invasion is characterized by the vigorous recruitment and activation of inflammatory cells, which is dependent on the coordinated expression of both pro-and anti-inflammatory cytokines. In this review, we present evidence indicating that both C-X-C and C-C chemokines are integral components of antibacterial host defense. Specifically, in vitro studies indicate that C-X-C chemokines [interleukin-8 (IL-8) and macrophage inflammatory protein 2 (MIP-2)] and the C-C chemokine macrophage inflammatory protein 1 alpha (MIP-1α) augment the ability of polymorphonuclear leukocytes (PMNs) and alveolar macrophages, respectively, to phagocytose and kill Escherichia coli. In addition, the intratracheal instillation of Klebsiella pneumoniae in CD-I mice results in time-dependent production of MIP-2 and MIP-1α, and the inhibition of MIP-2 bioactivity in vivo results in decreases in lung PMN influx, impaired bacterial clearance, and early mortality. Finally, the anti-inflammatory cytokine interleukin-10 (IL-10) is also expressed within the lung during the evolution of Klebsiella pneumonia, and neutralization of IL-10 in vivo results in enhanced proinflammatory cytokine production, bacterial clearance, and increases in both short- and long-term survival. In conclusion, our studies indicate that specific chemokines are important mediators of leukocyte recruitment and/or activation in bacterial pneumonia and that the expression of these chemokines is regulated by endogenously produced IL-10.

We have used latent infection with the human herpesvirus Epstein-Barr virus to track the dispersal of memory B cells from the mucosal lymphoid tissue of Waldeyer's ring (tonsils/adenoids). EBV is evenly distributed between the memory compartments of Waldeyer's ring and the peripheral blood. However, it has an approximately 20-fold higher preference for Waldeyer's ring over the spleen or mesenteric lymph nodes. These observations are consistent with a model whereby the virus preferentially establishes persistent infection within memory B cells from Waldeyer's ring. The virus then colonizes the entire peripheral lymphoid system, at a low level, by trafficking with these memory B cells as they circulate through the body and back to Waldeyer's ring. This pathway may reflect that of normal memory B cells derived from nasopharyngeal and other mucosal lymph nodes.

The role of macrophage inflammatory protein-2 (MIP-2) in bacterial pneumonia was characterized. Mice were challenged with Klebsiella pneumoniae intratracheally, and organs were harvested at 8, 24, and 48 h. Inoculation with K. pneumoniae resulted in the time-dependent expression of MIP-2 mRNA and protein within the lung, which was maximal 48 h after inoculation. Mice were then passively immunized with rabbit anti-murine MIP-2 serum intraperitoneally 2 h before administration of K. pneumoniae. Treatment with anti-MIP-2 serum resulted in a 60% decrease in lung neutrophil (PMNL) influx and a significant increase in K. pneumoniae colony-forming units in both lung and liver homogenates. Finally, treatment with anti-MIP-2 serum decreased early (48-72 h) but not late (after 72 h) survival in animals with Klebsiella pneumonia. This study indicates that MIP-2 is produced during Klebsiella pneumonia and inhibition of MIP-2 bioactivity in vivo results in decreased PMNL influx and lung bacterial clearance in murine Klebsiella pneumonia. MIP-2 is produced during Klebsiella pneumonia and inhibition of MIP-2 bioactivity in vivo results in decreased PMNL influx and lung bacterial clearance in murine Klebsiella pneumonia.