Use of intravenous immune globulin in the ICU: a retrospective review of prescribing practices and patient outcomes.

RATIONALE: Intravenous immune globulin (IVIG) is a pooled human blood product. Much of IVIG use in Canada is prescribed for 'unlabelled' or 'off-label' indications. Due to costs, risk of use and limited supply, knowledge about the use of IVIG is important. We collected data regarding the usage of IVIG and outcomes of patients receiving IVIG in the intensive care units (ICUs) of two community and three academic hospitals. METHODS: We reviewed the charts of adult patients who received IVIG in the five ICUs over a 5-year period. Data collection included demographics, severity of illness, indication for and dose of IVIG, mortality and adverse effects. On the basis of a classification developed by Canadian Blood Services, the indications for IVIG were then classified as 'appropriate' or 'inappropriate'. RESULTS: One hundred and forty-five patients received IVIG in the ICU. In all, 19% of IVIG prescriptions were for 'appropriate' indications and 7% were 'inappropriate'. The remaining 74% were prescribed for indications with some evidence to support their use. Three indications accounted for 50% of all IVIG prescribed: Guillain-Barre syndrome (GBS), necrotising fasciitis (NF) and toxic epidermal necrolysis (TEN). Both the community and academic centres prescribed IVIG for similar indications. Adverse effects associated with IVIG administration included deep vein thrombosis/pulmonary embolism, fever and renal failure, although direct causation related to IVIG could not be established. The overall mortality rate was 55%. CONCLUSIONS: IVIG is used relatively infrequently in the critical care setting. The most common indications were GBS, TEN and NF. Mortality was high. There was no difference between community and academic ICUs.

Lysis of human glial cells by major histocompatibility complex-unrestricted CD4+ cytotoxic lymphocytes.

We have investigated lysis of cultured human glial cells by non-major histocompatibility complex (MHC)-restricted or 'promiscuous' CD(4+)-T lymphocytes, activated either under relatively long-term limiting dilution culture conditions in the presence of phytohemagglutinin (PHA) and interleukin (IL)-2, or under short-term PHA-activated bulk culture conditions. Specific effector:target cell ratio-dependent lysis of oligodendrocytes (OGCs) by CD4+ T lymphocytes, generated under both of the above conditions, was observed in an 18-h 51Cr-release assay, but not in a 5-h assay. The extent of CD4 T-cell-mediated OGC lysis was less than for the tumor necrosis factor (TNF)-alpha-sensitive cell line U937, but greater than for TNF-resistant cell lines (K562, EL4). The effect could not be reproduced by T-cell culture supernatants or by high concentrations of recombinant TNF-alpha or beta. Anti-TNF-alpha antibody did not inhibit CD4-mediated lysis of OGC or U937 cells, but did inhibit U937 lysis induced by recombinant TNF-alpha, added in amounts exceeding those secreted by CD4 T cells. Human astrocytes and microglia were also susceptible to CD4+ T-cell-mediated lysis. Our results suggest that non-antigen non-MHC-restricted CD4+ T-cell-mediated injury of human glial cells can occur and may be dependent or enhanced by effector:target cell contact.

Differential expression of heat shock proteins by human glial cells.

Heat shock proteins (HSP) have been implicated in the interactions between the gamma delta T lymphocyte population and target tissues. gamma delta T cells are found in increased numbers in multiple sclerosis (MS) plaques compared to their proportion in peripheral blood, co-localizing with oligodendrocytes (OGC) expressing HSP. We have demonstrated that such gamma delta T cells can induce in vitro lysis of human adult-derived OGC. Using immunohistochemical and flow cytometry techniques, we examined the constitutive and/or inducible expression of HSP in or on adult human-derived glial cell cultures in vitro. HSP70 was expressed in OGC maintained at basal temperature, but the expression of the inducible HSP70 protein was upregulated by a prior 43 degrees C heat exposure. HSP70 could not be detected within astrocytes (GFAP+ cells), whether heat stress was applied or not. Constitutive expression of HSP60 could be discerned on the surface of all OGC under non-stressed culture conditions. Only some astrocytes demonstrated minor punctate surface HSP60 staining, whereas the remainder did not express HSP60 constitutively. These observations raise the possibility that OGC, by virtue of their differential expression of HSP compared to other glial cells, may be particularly prone to interaction with HSP-reactive gamma delta T cells. Such findings may further implicate gamma delta T cells in the pathogenesis of MS, a putative autoimmune disease in which immune-mediated injury is directed specifically against the oligodendrocyte-myelin unit within the central nervous system.

Peripheral blood gamma-delta T cells lyse fresh human brain-derived oligodendrocytes.

T cells are postulated to contribute to the injury of the oligodendrocyte-myelin complex underlying the demyelinating disease multiple sclerosis (MS). The apparent lack of class I or II major histocompatibility complex (MHC) expression in situ on human oligodendrocytes and the consistent failure to identify a universal myelin antigen in MS suggest that the immune damage might be mediated by effector T cells that are capable of reacting in an antigen-nonspecific and possibly MHC-unrestricted manner, such as T cells expressing the gamma-delta T-cell receptor. Since gamma-delta T cells are reported to be present in MS plaques and an increased number are found in the cerebrospinal fluid of patients with MS, we directly examined whether gamma-delta T cells are capable of inducing injury to human oligodendrocytes. We found, using a 6-hour 51Cr release assay, that oligodendrocytes cultured from surgically resected human brain specimens were effectively lysed in a dose-dependent manner by human gamma-delta T cells (28 +/- 5% mean specific lysis, n = 6, at an effector-target ratio of 20:1). Although heat shock protein HSP72, a putative gamma-delta T-cell recognition molecule, could be induced in vitro in our oligodendrocytes, an antibody to HSP72 did not inhibit gamma-delta T cell-mediated lysis of oligodendrocytes. These results suggest that gamma-delta T cells gaining entry into the central nervous system may be deleterious to oligodendrocytes and thus may contribute to the pathogenesis of MS.

Gamma-interferon promotes proliferation of adult human astrocytes in vitro and reactive gliosis in the adult mouse brain in vivo.

Reactive gliosis is a characteristic response of astrocytes to inflammation and trauma of the central nervous system. To investigate whether soluble factors (cytokines) from inflammatory mononuclear cells that accumulate at lesion sites can provide the cellular signals to initiate gliosis and to identify such cytokines, we have tested and found that supernatants derived from subsets of activated human T lymphocytes (CD8+ or CD4+) are potent mitogens for cultured human adult astrocytes. This effect is blocked by a neutralizing antibody to gamma-interferon (IFN). Recombinant IFN alone can induce proliferation of human adult astrocytes in vitro and increase the extent of trauma-initiated gliosis in the adult mouse brain. The astrocyte proliferation-inducing activity of supernatants of glial cultures treated with IFN can be completely blocked with IFN-neutralizing antibody, suggesting that the proliferative effect does not require intermediary cytokines or cells. These results implicate IFN as an important mediator of the gliosis observed in pathologic conditions of the adult central nervous system associated with infiltrating lymphocytes.

Phenotypic and functional characteristics of activated CD8+ cells: a CD11b-CD28- subset mediates noncytolytic functional suppression.

Freshly isolated human CD8+ cells can be divided into mutually exclusive subsets bearing the phenotypes CD11b+(CD28-) or CD28+(CD11b-). We found that activation of CD8+ cells with anti-CD3 mAb and IL-2 preferentially expanded the CD11b-(CD28+) subset. This subset, when separated and activated independently, mediated both functional suppression and lectin-dependent cell cytotoxicity (LDCC). CD28- cells, prepared by elimination of the CD 28+ cells from expanded unfractionated CD8+ cell cultures, retained functional suppressor activity but demonstrated reduced LDCC compared to either the CD28+(CD11b-)-enriched fraction or the unfractionated CD8+ population. The majority of the CD28- cells were also CD11b-, reflecting the observation that initially CD11b+ cells lose CD11b expression following activation with anti-CD3 mAb and IL-2. Our results therefore indicate that CD8+ cells deriving from the CD11b+CD28- subset, but expressing neither CD11b nor CD28 after activation, represent the main noncytotoxic functional suppressor cell in the mitogen "activated" suppressor assay. The preferential expansion of CD8+CD28+ cells relative to CD8+CD28- cells, if occurring in vivo in the central nervous system (CNS) compartment, would be consistent with observed phenotypic analysis of cerebrospinal fluid-derived T cells and might contribute to the reduced functional suppressor activity previously found for CNS compared to peripheral blood-derived lymphocytes.

Expression and modulation of HLA-DR on cultured human adult astrocytes.

Expression of Class II major histocompatibility complex (MHC) antigens on astrocytes has been implicated as contributing to the immune responses characteristic of chronic autoimmune diseases of the central nervous system. We examined the properties and regulation of HLA-DR on cultured human adult astrocytes. We found that a proportion of human astrocytes from each of fifteen individual donors expressed HLA-DR under basal culture condition; while this proportion differed among the human subjects (range 3-65%), the results for each individual remained relatively constant when analyzed at several time points (up to 125 days in vitro). Attempts to modulate HLA-DR expression by a variety of cytokines likely to be present in inflammatory infiltrates in the brain showed that only gamma-interferon could increase the proportion of human astrocytes that expressed HLA-DR. Whether the variability of HLA-DR expression on astrocytes between different individuals reflects a genetic trait which can influence susceptibility to autoimmune central nervous system diseases remains to be determined.

Serum cytotoxicity to human and rat oligodendrocytes in culture.

Serum from multiple sclerosis (MS) patients can cause demyelination in rat CNS explant cultures and induce cytotoxicity to rat oligodendrocytes in culture. The interpretation of these findings for MS is complicated by the fact that injury to myelin and oligodendrocytes can also be induced with normal human serum. In this study, we confirmed that serum from MS patients and healthy control subjects can cause in vitro toxicity to rat oligodendrocytes, as established by a 51Cr release assay, but we did not detect toxicity to human cultured oligodendrocytes. Morphologic changes after 5-6 h incubation with the sera were also extensive in the rat oligodendrocyte cultures. No morphologic changes or changes in cell numbers could be detected in the human cultures upon examination by light microscopy and by immunofluorescent staining with anti-GalC antibody.

Human oligodendrocytes are susceptible to cytolysis by major histocompatibility complex class I-restricted lymphocytes.

The majority of human oligodendrocytes in enriched glial cell cultures expresses class I major histocompatibility complex (MHC) antigens. We used a 51Cr release assay to study the susceptibility of oligodendrocyte-enriched glial cells to MHC-restricted and non-restricted immune-mediated cytolysis. Mitogen-activated mononuclear cells induced significant lysis in a lectin-dependent cytotoxicity assay. Mononuclear cells allo-activated in a one-way mixed lymphocyte culture with E- cells from the glial cell donor induced a significantly higher degree of oligodendrocyte cytolysis than mononuclear cells activated with E- cells bearing MHC-class I antigens discordant with the glia. Cytolysis by alloactivated unfractionated lymphocytes and by purified CD8+ lymphocytes was reduced by an anti-class I antibody (W6/32). Our findings suggest that human oligodendrocytes can be susceptible targets for MHC class I-restricted lysis.

DNA synthesis by young adult human-derived astrocytes in vitro.

We have assessed whether adult human non-neoplastic astrocytes exhibit DNA synthesis in vitro, as measured using a double immunofluorescence technique to detect incorporation of 5-bromodeoxyuridine (BrdU) by nuclei of glial fibrillary acidic protein (GFAP)-containing cells. Dissociated cell cultures containing GFAP+ cells were established from surgically resected temporal lobe tissue from 3 young adult individuals operated upon to remove epileptogenic foci. In 5-18-day-old cultures from each of the 3 individuals, we observed GFAP+ cells whose nuclei had incorporated BrdU. BrdU nuclear staining was found in GFAP+ cells with either flat or process-bearing morphologies. The mean mitotic index for the GFAP+ cells was 6% (range 2-12%) as compared to a mean mitotic index of 29% for the GMK-7 glioma cell line. Our results do indicate that astrocytes derived from young adult donors, unlike such cells derived at autopsy from elderly adults, are capable of DNA synthesis in vitro, albeit to a markedly lesser extent than reported for fetal human astrocytes.

Immunohistochemical studies of adult human glial cells.

Using immunohistochemical techniques, we examined major histocompatibility complex (MHC) antigen expression on astrocytes, oligodendrocytes, and macrophages-microglia derived from surgically resected tissue from young adults and maintained in dissociated cell cultures supplemented with either fetal calf or human AB serum. The majority of these cells in culture expressed class I MHC antigens. MHC class II expression was observed on only a restricted proportion of astrocytes either under basal or induction conditions (gamma-interferon, activated lymphocyte supernatants), on the majority of macrophages-microglia under inducing conditions, and not on oligodendrocytes. MHC class II expression on astrocytes in culture did not correlate with the extent of in situ gliosis or with in vitro cell morphology. MHC antigen expression was not detected in situ immunohistochemically. These data extend observations on the dissociation of in vivo and in vitro expression of MHC antigens on glial cells. The apparent greater expression of MHC class II antigens on macrophages-microglia compared to astrocytes raises the issue of the relative roles of each of these cell types in promoting immune reactivity under pathologic conditions.